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Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.

Permanent Link: http://ufdc.ufl.edu/UFE0021556/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Proneth, Bettina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Bettina Proneth.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Haskell-Luevano, Carrie.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021556:00001

Permanent Link: http://ufdc.ufl.edu/UFE0021556/00001

Material Information

Title: Record for a UF thesis. Title & abstract won't display until thesis is accessible after 2009-12-31.
Physical Description: Book
Language: english
Creator: Proneth, Bettina
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2007

Subjects

Subjects / Keywords: Medicinal Chemistry -- Dissertations, Academic -- UF
Genre: Pharmaceutical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Statement of Responsibility: by Bettina Proneth.
Thesis: Thesis (Ph.D.)--University of Florida, 2007.
Local: Adviser: Haskell-Luevano, Carrie.
Electronic Access: INACCESSIBLE UNTIL 2009-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2007
System ID: UFE0021556:00001


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1 RATIONAL DRUG DESIGN APPROACHES TARGETING THE BRAIN MELANOCORTIN RECEPTORS By BETTINA PRONETH A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Bettina Proneth

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3 To my family for their love and support

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4 ACKNOWLEDGMENTS First of all I want to expre ss greatest gratitude to my a dvisor Dr. Carrie Haskell-Luevano, whose support, guidance and special dedication to science has helped me during the past years with my research and always created an inspir ing working atmosphere. Second I want to thank all former and current lab members of the Hask ell-Luevano laboratory for making my time here enjoyable, especially Dr. Zhimin Xiang who ta ught me the principles of receptor mutagenesis and molecular biology techniques and Krista W ilson and Dr. Andrzej Wilczynski who trained me in peptide synthesis. I also want to th ank Dr. Sally Litherland (Department of Pathology, Immunology and Laboratory Medicine, Universtiy of Florida) for her collaboration on flow cytometry and deconvolution microscopy, Dr. Will iam Millard for his collaboration on peptide iodination (Department of Pharmacodynamics, Univ ersity of Florida) an d Drs. Henry Mosberg and Irina Pogozheva (Department of Medicinal Chemistry, University of Michigan) for their collaboration on receptor homology modeling. Last but not least I want to thank my family for their unlimited love, sup port and encouragement.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .......10 ABSTRACT....................................................................................................................... ............18 CHAPTER 1 INTRODUCTION..................................................................................................................20 Obesity........................................................................................................................ ............20 Overview of the Melanocortin System...................................................................................21 Melanocortin Receptors...................................................................................................22 Endogenous Melanocortin Agonists................................................................................23 Endogenous Melanocortin Antagonists...........................................................................23 Melanocortin System and Obesity...................................................................................24 Contribution of the Research in this Dissertation towards Rational Drug Design.................25 2 METHODOLOGIES..............................................................................................................31 General Strategies and Concepts in Peptide Synthesis...........................................................31 Solid-Phase Peptide Synthesis.........................................................................................31 Fmoc Solid Phase Peptide Synthesis...............................................................................32 General Concepts and Strategies in Receptor Mutagenesis and Pharmacology.....................34 Generation of Epitope Tagged Receptors........................................................................34 Site-directed Receptor Mutagenesis................................................................................36 Cassette Mutagenesis.......................................................................................................39 Flow Cytometry...............................................................................................................40 Functional Reporter Gene Assay.....................................................................................41 Nonlinear Regression Analysis.......................................................................................43 Schild Analysis................................................................................................................44 Competitive Binding Assay.............................................................................................45 Experimental Procedures........................................................................................................46 Peptide Synthesis and Func tional Characterization........................................................46 Synthesis of tetrapeptide analogues.........................................................................46 Synthesis of alpha-MSH analogues..........................................................................48 Functional characterization at th e mouse Melanocortin receptors...........................50 Receptor Site-directed Mutagenesis................................................................................52 Residue numbering schemes....................................................................................52 Mouse MC3R epitope tagging.................................................................................52 Generation of receptor mutations.............................................................................53 Generation of stable cell lines..................................................................................54

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6 Functional bioassay..................................................................................................54 NDP-MSH iodination...............................................................................................55 Competitive displacement binding assays................................................................56 Flow cytometry........................................................................................................57 Transient transfection...............................................................................................57 Intracellular Expression of an Endogenous Agonist.......................................................58 Plasmid construction................................................................................................58 Flow cytometry........................................................................................................58 Flow cytometry and using live cells.........................................................................59 Deconvolution microscopy......................................................................................60 3 STRUCTURE-ACTIVITY RELATIONS HIP STUDIES OF TETRAPEPTIDE LIGANDS AT THE MOUSE MELANOCORTIN RECEPTORS........................................75 Introduction................................................................................................................... ..........75 Results........................................................................................................................ .............77 Discussion..................................................................................................................... ..........80 Substitution with Hydrophilic Groups.............................................................................81 Substitution with Hydrophobic Aliphatic and Aromatic Groups....................................82 Substitution with Halogens..............................................................................................83 Receptor Selectivity.........................................................................................................90 Conclusion..................................................................................................................... .........92 4 SITE-DIRECTED MUTAGENESI S STUDIES AT THE MOUSE MELANOCORTIN-3 RECEPTOR......................................................................................100 Introduction................................................................................................................... ........100 Results........................................................................................................................ ...........101 Functional Characterization of Wild-type and Mutant Flag-mMC3 Receptors............102 Synthetic Melanocortin Agonists...........................................................................103 Endogenous Agonists.............................................................................................105 Endogenous and Synthetic Antagonists.................................................................106 Flag-mMC3R Mutations resulting in a Loss of Functional Activity.....................108 Flag-mMC3R Mutations Causing Partial Agonism...............................................109 Cell Surface Expression of Flag-mMC3 Receptors......................................................109 Competitive Displacement Binding Studies..........................................................110 Discussion..................................................................................................................... ........111 AgonistReceptor Interactions : The Hydrophilic Region.............................................112 Agonist Receptor Interacti ons: The Hydrophobic Region..........................................118 Residues Important for Receptor Signal Transduction and Structure...........................121 Differentiation of SHU9119 Antagoni st versus Agonist activity..................................131 Determinants for AGRP Antagonist Activity................................................................138 Residues Involved In Differentia tion between MC3R and MC4R...............................140 Conclusion.....................................................................................................................142 5 A COMBINATION OF TETRAPEPTIDE SAR AND RECEPTOR MUTAGENESIS.....170

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7 Introduction................................................................................................................... ........170 Results........................................................................................................................ ...........171 Functional Characterizati on of MC3R Agonists...........................................................172 Functional Characterizati on of MC3R Antagonists......................................................173 Discussion..................................................................................................................... ........177 Conclusion..................................................................................................................... .......185 6 MOLECULAR MECHANISM OF CONSTI TUTIVE ACTIVATION OF A HUMAN MELANOCORTIN-4 RECEPTOR POLYMORPHISM.....................................................193 Introduction................................................................................................................... ........193 Results........................................................................................................................ ...........194 Competitive Binding Assay...........................................................................................195 Agonist Functional Assays............................................................................................196 Constitutive Activation..................................................................................................197 Receptor Cell Surface Expression.................................................................................197 Antagonist/Inverse Agonis t Functional Assay..............................................................198 Discussion..................................................................................................................... ........199 Conclusion..................................................................................................................... .......203 7 INTRACELLULAR EXPRESSION OF AN ENDOGENOUS AGONIST IN VITRO......216 Introduction................................................................................................................... ........216 Results and Discussion.........................................................................................................217 Future Directions.............................................................................................................. ....220 8 FUTURE DIRECTIONS......................................................................................................226 Experiment 1................................................................................................................... ......226 Experiment 2................................................................................................................... ......227 Experiment 3................................................................................................................... ......227 Experiment 4................................................................................................................... ......228 APPENDIX A SEQUENCING FILES.........................................................................................................235 B AGONIST AND ANTAGONIST PHARMA COLOGY CURVES AT THE WILDTYPE AND MUTANT MOUSE MELANOCORTIN-3 RECEPTORS...................324 LIST OF REFERENCES.............................................................................................................352 BIOGRAPHICAL SKETCH.......................................................................................................363

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8 LIST OF TABLES Table page 2-1 Properties of the flourescent dyes FITC and APC.............................................................67 2-2 Analytical data for peptides synthesized in this study.......................................................69 2-3 Sequences of primers used for the generation of FlagmMC3 receptor mutants................70 2-4 Sequences of primers used for the generation of the FlaghMC4 receptor mutants...........73 2-5 Concentration of DNA used in the transient tran sfection assay........................................74 2-6 Analytical data for peptides synthesized in this study.......................................................74 3-1 Common substituant constant s for aromatic sustituents....................................................93 3-2 Functional activities of th e DPhe modified tetrapeptides at the mouse melanocortin receptors...................................................................................................................... .......96 4-1 Primary sequence of the peptides used for pharmacological characterization of the wild-type and mutant Flag-mMC3 receptors...................................................................144 4-2 Functional activities of th e endogenous and synthetic a gonists at the wild-type and mutant Flag-mMC3 receptors..........................................................................................145 4-3 Functional antagonist pharmacology at the wild-type and mutant Flag-mMC3 receptors...................................................................................................................... .....149 4-4 Comparison of the wild-type and mutant mMC3R cell surface and total receptor expression levels usi ng Flow Cytometry.........................................................................154 4-5 Summary of the synthetic agonist NDP-MSH competitive displacement binding affinity studies (IC50) and functional (EC50) studies of the wild-type and mutant Flag-mMC3 receptors......................................................................................................157 5-1 Functional activities of th e ligands tested at the wild-type and mutant Flag-mMC3 receptors...................................................................................................................... .....189 6-1 Primary sequences of endogenous/synthet ic agonists and the endogenous antagonist/ inverse agonist hAGRP(87-132) used in this study.........................................................206 6-2 Competitive binding affinity IC50 results at the wild-type and mutant Flag-hMC4 receptors...................................................................................................................... .....207 6-3 Functional activity of mela nocortin ligands at the wild -type and mutant Flag-hMC4 receptors...................................................................................................................... .....208

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9 6-4 Comparison of receptor cell surface expres sion with the functi onal activity of the endogenous ligand -MSH and the level of constitu tive activation of mutant and wild-type Flag-hMC4 receptors.......................................................................................211 6-5 Functional activity of the antagonist hAGR P (87-132) at the wi ld-type and mutant Flag-hMC4 receptors.......................................................................................................212 7-1 Functional activity of -MSH, BP2-38 and BP2-67 at the wt Flag-hMC4R...................222 7-2 Functional activity of the melanocortin ligand -MSH at the wild -type and mutant Flag-hMC4 receptors.......................................................................................................223 8-1 Ala scan of NDP-MSH, -MSH and JRH887-9..............................................................230 8-2 Proposed Flag-mMC3R mutations for experiment 1.......................................................231 8-3 Proposed peptides for experiment 2.................................................................................232 8-4 Proposed Flag-mMC3R mutations for experiment 2.......................................................232 8-5 Proposed mutations for experiment 3..............................................................................233 8-6 Proposed mMC3R mutations for experiment 4...............................................................234

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10 LIST OF FIGURES Figure page 1-1 Overview of the melanocortin system...............................................................................28 1-2 Posttranslational processing of POMC re sulting in the generation of the endogenous melanocortin agonists........................................................................................................29 1-3 Primary sequences of the C-termini of the endogenous melanocortin antagonists...........30 1-4 Rational drug design as iterative process...........................................................................30 2-1 General strategy for Fmoc solid phase synthesis of peptides............................................61 2-2 Chemical structure of th e Wang and Rink amide resins....................................................62 2-3 Fmoc deprotection reaction................................................................................................62 2-4 Generation of an OBt ester using HBTU...........................................................................63 2-5 Ninhydrin test............................................................................................................. ........64 2-6 Schematic representation of the PCR st rategy used for epotipe tagging of the mouse MC3R with Flag................................................................................................................65 2-7 Verification of Flag -mMC3R cell expression....................................................................66 2-8 NDP-MSH agonist curve at the FlagmMC3R stably expressed in HEK293 cells............66 2-9 Potential pharmacological effects caus ed by introducing mutations into a GPCR...........67 2-10 Representation of the -MSH primer design.....................................................................67 2-11 Schematic representation of the -Galactosidase/CRE reporter gene assay......................68 2-12 Schematic representation of possible pharmacology curves.............................................68 3-1 Schematic representation of parameters that can be affected by substituents in a drug molecule....................................................................................................................... ......93 3-2 Graphic representation of the sustituent c onstants for para phenyl substituents used in this study..................................................................................................................... .......94 3-3 Schematic representation of the substituents in DPhe para position and their properties..................................................................................................................... .......94 3-4 Structures of amino acids used to replace DPhe in the peptide template Ac-DPhe-Xaa-Arg-Trp-NH2..............................................................................................95

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11 3-5 Pharmacology curves of the tetrapeptides substituted with halogens at the DPhe para position at the mMC3R......................................................................................................97 3-6 Antagonist pharmacology curves of BP7-26 at the mMC3R............................................97 3-7 Pharmacology curves of th e tetrapeptides substituted w ith chlorines and DNal2 at the mouse MC3R...............................................................................................................98 3-8 Pharmacology curves of the tetrapeptides substituted with halogens at the DPhe para position at the mMC3R......................................................................................................98 3-9 Illustration of possible geometri es of aromatic edge-to-face and stacking interactions................................................................................................................... ......99 4-1 Two-dimensional representation of the Flag-mMC3R, showing the residues that were mutated to Ala and Ser............................................................................................143 4-2 Proposed agonist residues DPhe-ArgTrp interactions with the mMC4R...................143 4-3 Pharmacology curves of agonists and antagonists at the w ild-type Flag-mMC3R.........153 4-4 Relative receptor cell surface and total expression of wild-type and mutant Flag-mMC3 receptors determin ted by flow cytometry...................................................156 4-5 Alignment of the mouse and human me lanocortin receptors and bovine rhodopsin.......161 4-6 Agonist pharmacology curves at mutant Flag-mMC3 receptors putatively involved in the hydrophilic binding pocket........................................................................................162 4-7 Explanation why no binding affinity va lue could be determined for the D121A............162 4-8 Pharmacology curves of TM6 residues putatively participa ting in the hydrophobic binding pocket of the Flag-mMC3R................................................................................163 4-9 Binding affinities to NDP-MSH of TM6 residues putatively pa rticipating in the hydrophobic binding pocket of the Flag-mMC3R...........................................................163 4-10 Schematic representation of the puta tive DPhe-Arg-Trp interaction with the mMC3R.......................................................................................................................... ..164 4-11 Schematic representation of possible side chain arrangements in the hydrophobic region around TM3 and TM6..........................................................................................164 4-12 Agonist and SHU9119 antagonist pharm acology curves at the T145A and T145S Flag-mMC3 receptors......................................................................................................165 4-13 Structure of the agonist MTII and the antagonist SHU9119...........................................165

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12 4-14 Antagonist pharmacology curves showi ng conversion of SHU9119 antagonist to agonism at TM3 and TM6 Flag-mMC3R residues..........................................................166 4-15 Antagonist pharmacology curves of SHU9119 resulting into conve rsion of antagonist to agonist activity at residue s from TM4, TM5, el3 and TM7........................................167 4-16 Antagonist pharmacology curves of S HU9119 at the mutant E94, D117 and D121 Flag-mMC3 receptors......................................................................................................168 4-17 Pharmacology curves of the antagonists AGRP and SHU9119 at the N91A Flag-mMC3 receptors......................................................................................................168 4-18 Comparison of agonist pharmacology cu rves of L247A Flag-mMC3R and L250A FlaghMC4R......................................................................................................................169 4-19 Transient transfection assay.............................................................................................169 5-1 Structures of the mMC3R agonists and antagonists used in this study...........................187 5-2 Two-dimensional represen tation of the Flag-mMC3R....................................................188 5-3 Antagonist pharmacology curves of te trapeptide ligands at the wild-type Flag-mMC3R...................................................................................................................190 5-4 Antagonist pharmacology curves of BP7-27 at the I132A Flag-mMC3R.......................191 5-5 Pharmacology curves of tetrapeptide ligands at the T270S Flag-mMC3R.....................191 5-6 Comparision of cAMP activities at 10-4M concentration of the tetrapeptide antagonists at the wild-type a nd F179A Flag-mMC3 receptors......................................192 6-1 Two-dimensional represen tation of the Flag-hMC4R.....................................................205 6-2 Agonist pharmacology curves at the wild -type and mutant Flag-hMC4 receptors.........209 6-3 Basal cAMP activity of the wild-type a nd the mutant Flag-hMC4 receptors stably expressed in HEK293 cells..............................................................................................210 6-4 Transient transfection assay.............................................................................................210 6-5 Relative cell surface a nd total cell expression of the wild-type and mutant Flag-hMC4 receptors.......................................................................................................211 6-6 Antagonist pharmacology curves of the antagonist/inverse agonist AGRP....................213 6-7 Possible physiological mechanism by wh ich AGRP might contribute to the obese L250Q phenotype through a regulatory feedback mechanism........................................214

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13 6-8 Comparison of wild-type hMC4R and L 250Q hMC4R in the putative inactive and active receptor states....................................................................................................215 7-1 Two-dimensional representation of the Flag-hMC4R showing the receptor mutations used for this study............................................................................................................222 7-2 Cell surface and total receptor expres sion of wild-type and mutant Flag-hMC4 receptor after transfection with pCDNA3 control and Met-MSH/pCDNA3.................223 7-3 Structure of DTAF-NDP-MSH compared to -MSH and NDP-MSH............................224 7-4 Live cell flow cytometry and deconvolution microscopy................................................225

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14 LIST OF ABBREVIATIONS Ala: Alanine Cys: Cysteine Asp: Aspartic Acid Glu: Glutamic Acid Phe: Phenylalanine Gly: Glycine His: Histidine Ile: Isoleucine Lys: Lysine Leu: Leucine Met: Methionine Asn: Asparagine Pro: Proline Gln: Glutamine Arg: Arginine Ser: Serine Thr: Threonine Val: Valine Trp: Tryptophan Tyr: Tyrosine A: Alanine C: Cysteine D: Aspartic Acid E: Glutamic Acid

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15 F: Phenylalanine G: Glycine H: Histidine I: Isoleucine K: Lysine L: Leucine M: Methionine N: Asparagine P: Proline Q: Glutamine R: Arginine S: Serine T: Threonine V: Valine W: Tryptophan Y: Tyrosine MC1R: Melanocortin-1 receptor MC2R: Melanocortin-2 receptor MC3R: Melanocortin-3 receptor MC4R: Melanocortin-4 receptor MC5R: Melanocortin-5 receptor GPCR: G-Protein coupled receptor SAR: Structure-activity relationship BMI: Body mass index WHO: World Health Organization

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16 MSH: Melanocyte stimulating hormone ACTH: Adrenocorticotropic hormone POMC: Proopiomelanocortin cAMP: Cyclic adenosine mono phosphate CRE: Cyclic adenosine mono phosphate response element IP: Inositol phosphate AGRP: Agouti-related protein 3D: 3-dimensional SPPS: Solid phase peptide synthesis TFA: Trifluoroacetic acid TM: Transmembrane Fmoc: 9-Fluorenylmethoxycarbonyl DMF: Dimethylformamide HBTU: 2-(1-H-benzotriazol1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HOBt: 1-Hydroxybenzotriazole DIEA: N, N-Diisopropylethylamine DCM: Dichloromethane HPLC: High performance liquid chromatography RP: Reverse phase UTR: Untranslated region mMC3R: Mouse melanocortin-3 receptor PCR: Polymerase chain reaction HEK: Human embryonic kidney FACS: Fluorescence activated cell sorting APC: Allophycocyanin

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17 FITC: Fluorescein isothiocyantate FSC: Forward scatter SSC: Side scatter ONPG: Ortho-nitrophenyl-D-galactopyranoside EC50: Half maximal effective concentration IC50: Half maximal inhibitory concentration Tis: Triisopropylsilane BSA: Bovine serum albumin DMEM: Dulbeccos modified eagle medium IBMX: Isobutylmethylxanthine hMC4R: Human melanocortin-4 receptor CB1: Cannabinoid-1 receptor 2AR: Beta-2 adrenergic receptor DTAF: 5-(4, 6-dichlorotriazinyl) aminofluorescein

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18 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy RATIONAL DRUG DESIGN APROACHES TARGETING THE BRAIN MELANOCORTIN RECEPTORS By Bettina Proneth December 2007 Chair: Carrie Haskell-Luevano, Ph.D. Major: Pharmaceutical Sciences Obesity is a rapidly growing h ealth problem in industrialize d countries in the world and represents a major risk factor for type II diab etes, cardiovascular disease and stroke. The brain Melanocortin-3 and -4 receptors (MC3R and MC4R) have been implicated in the regulation of obesity and energy homeostasis. Extensive studies ha ve been performed to understand the role of the MC4R in energy homeostasis, but little is known about the MC3R. One caveat is that compounds identified to date are not selec tive for the MC3R (i.e. > 500-fold). Ligands selectively targeting the MC3R can aid as in vivo tools to explore the phys iological functions of this receptor which may result in the discovery of new treatment options for obesity. Rational drug design targeting G-Protein c oupled receptors (GPCR) usually consists of an iterative process, combining ligand struct ure-activity relationship studies (SAR), site-directed receptor mutagenesis and GPCR homology modeling studies, which may ultimately result in the discovery of new selective ligands. Herein, tradi tional peptide SAR studies were undertaken that resulted in the identification of structural requirements underlying diffe rential MC3R and MC4R receptor activation and inhibition mechanisms. Fu rther, extensive site-directed mutagenesis studies of the MC3R led to the discovery of key receptor residues involved in ligand-receptor recognition and signaling processe s, differentiation of agonist versus antagonist activity and

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19 MC3R/MC4R subtype specificity. In a dual pronge d approach, tetrapeptide antagonists identified from above mentioned SAR studies were pharm acologically characteri zed at mutant MC3Rs identified from site-directed mutagenesis studies that switched antagonist into agonist function. These studies revealed important determinants suggesting distinct inhi bition mechanisms of tetrapeptide antagonist ligands at the MC3R. Finally, the molecu lar mechanism was investigated by which a polymorphic human MC4R, discovered in an extremely obese patient, causes constitutive receptor activity. Th is study resulted in a better understanding of the activation mechanism of the MC4R, valuable for modeling of GPCRs in the active state and docking of agonist ligands. In summary, this research contributed significantl y to the rational drug design of MC3R selective ligands potentially useful to unravel the myriad of physiological functions of this receptor subtype and to discover ne w agents for the treatment of obesity.

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20 CHAPTER 1 INTRODUCTION Obesity Obesity is a rapidly growing h ealth problem in the United States and other industrialized countries of the world. The rising prevalence of obesity might be due to multiple effects that result from a combination of high fat diet, lack of exercise and geneti c predisposition. The body mass index (BMI) is commonly used in classifyin g overweight and obesity in adult populations and individuals. It is defined as the weight in kilograms divided by the square of the height in meters (kg/m2). The World Health Organi zation (WHO) defines overw eight as a BMI equal or more than 25, and obesity as a BMI equal to or more than 30. The BMI provides a useful tool for the assessment of overweight and obesity but it should be consid ered as a rough guide because it may not correspond to the same degree of fatness in different individual s. The WHOs latest projections indicated that gl obally in 2005 approximately 1.5 b illion adults (age 15+) were overweight and at least 400 million were obese The WHO further projects that by 2015, approximately 2.3 billion adults will be overw eight and more than 700 million will be obese. Overweight and obesity lead to serious health consequences and are a major risk factor for chronic diseases such as cardiovascular diseas e (mainly heart disease and stroke), diabetes, musculoskeletal disorders and certain forms of ca ncer. Although the health benefits of weight reduction are well-recognized, weight-loss by diet and exercise fail in most patients, and the current marketed drugs have had limited success ( 1 ). Therefore, the development of agents that are directed towards inhibiting the caloric intake and/or increasing energy expenditure as well as the identification of new targets for physiological intervention represent an unmet medical need for effective weight loss therapies ( 1 ). The melanocortin system incl udes five genetic factors that play an important role in the control of food intake and body weight in humans and rodents.

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21 These targets include the brain melanocortin-3 (MC3R) and -4 (MC4R) receptors, Agouti Signaling Protein (ASP), Agouti-regulated prot ein (AGRP) and the melanocortin agonist precursor hormone proopi omelanocortin (POMC). Overview of the Melanocortin System The melanocortin system comprises fi ve melanocortin receptors (MCRs) ( 2-8 ) that belong to the superfamily of G-protein coupled recep tors (GPCRs) and are expressed differentially throughout the body ( 2-8 ). The physiological functions medi ated by the melanocortin receptors are regulated by endogenous agonists and an tagonists. The endogenous agonists are -, and melanocyte stimulating hormones (MSH) and ad renocorticotropic hormone (ACTH) and are derived from a common pr ecursor proopiomelanocorti n hormone (POMC) through posttranslational processing ( 9, 10 ). Upon agonist stimulation the melanocortin receptors undergo potential conformational changes allowing for interaction with he terotrimeric stimul atory G-Proteins (Gs). Further downstream signaling cascades in clude activation of adenylyl cyclase, production of cAMP, stimulation of protein kinase A and phosphorylation of cAMP response element binding protein (CREB) which will ultimately bind to cAMP response element (CRE) unit in the nucleus causing transcription and translation of effector pr oteins. MC3R signaling has been additionally associated with changes in intracellular calcium and Inositol Phosphate (IP3) levels and the protein kinase C pathway ( 11, 12 ). The endogenous antagonists Agout i and Agouti-related protein (AGRP) are believed to either block the effects caused by melanocortin agonists through competitive antagonism or even have physiological functions of their own through their inverse agonist activity ( 13-18 ). A schematic overview of the melanocortin sy stem is depicted in Figure 1-1.

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22 Melanocortin Receptors As mentioned above, five melanocortin recepto rs have been cloned to date, each with distinct tissue distribu tion, different pharmacological roles a nd differential affinities for their endogenous peptide ligands. The MC1R was the first subtype of the mela nocortin receptor family to be cloned. This receptor is most recognized for its expression on melanocytes, where it plays an important role in skin and hair pigmentation in mammals ( 3, 13, 19, 20 ). Additionally, the MC1R has been identified to mediate inflammatory reactions ( 21 ). The MC2R is expressed in the adrenal cortex and modulates adrenal glucocorticoid and aldosterone production as well as lipogenesis ( 3 ). Interestingly, it can be pharmacologically distinguished from other melanocortin receptor subt ypes in that it is only stimulated by ACTH and not by any of the other endogeno us melanocortin agonist ligands ( 3, 22 ). The MC3R is expressed in the brain and pe ripheral tissues such as the heart, kidney, gastrointestinal tract, and placenta ( 5, 6, 23, 24 ). This receptor subtype is involved in energy homeostasis ( 25, 26 ) as well as cardiovascular function ( 24, 27, 28 ), but the exact mechanisms remain to be identified. The MC4R, expressed in the brain ( 4, 7 ) has been demonstrated to play an important role in energy homeostasis, obesity and appe tite control in rodents and humans ( 29-31 ) and is also implicated in the modulation of er ectile function and sexual behavior ( 32, 33 ). The MC5R, expressed in the br ain and peripheral locations ( 8 ), has been implicated to be involved in the regulation of lipid secretion from exocrine glands ( 34 ). Targeted disruption of the MC5R resulted in a severe def ect in water repulsion and thermo regulation due to a decrease in sebaceous lipids ( 34 ). The physiological roles of the MC5R in other tissues in which it is expressed remain to be identified.

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23 Endogenous Melanocortin Agonists The endogenous melanocortin agonists are derive d through posttransla tional processing of their precursor proopiom elanocortin (POMC) ( 10, 35 ). POMC is expressed in the brain, skin, immune system and several other peripheral tissu es. In the brain POMC is localized to the pituitary gland, the arcuate nuc leus and the solitary tract ( 36 ). Posttranslational processing of POMC is tissue specific and result s into the production of various bioactive peptides, including -, and -MSH and ACTH by the prohormone convertases PC1 and PC2 ( 37, 38 ). In the anterior pituitary only prohormone convertase 1 (P C1) is expressed, but not PC2, resulting in the production of ACTH, and -lipotropin. In contrast, the expression of PC2 within the hypothalamus may lead to the production of -, -, and -MSH and ACTH. For -MSH Carboxypeptidase E further removes the C-termin al basic amino acids, followed by amidation and N-acetylation ( 39 ). All POMC derived melanocortin agonist peptides share a common message sequence of His-Phe-Arg-Trp which is postulated to be important for receptor recognition and activation ( 40, 41 ). Figure 1-2 illustrates the posttranslational processing of POMC as well as the primary sequences of the endogenous melanocortin agonists. Endogenous Melanocortin Antagonists The Arg-Phe-Phe residues, which are conserve d in both Agouti and AGRP, are believed to mimic the agonist Phe-Arg-Trp interactions with the receptors and are cr itical for antagonistic activity ( 42, 43 ). Pharmacological characterization studi es identified that Agouti antagonizes the skin MC1R and the brain MC3/4 receptors ( 13, 44 ), whereas AGRP antagonizes only the brain MC3 and MC4 receptors ( 14, 44 ). Further experimental eviden ce has been provided that Agouti and AGRP show inverse agonist activity in addition to competitive antagonism ( 15-17, 44, 45 ). Under physiological conditions, Agouti is expr essed in hair follicles of the skin ( 13 ), while

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24 AGRP is expressed primarily in the hypothalamu s of the brain and adrenal medulla ( 14 ). The primary structures of the endogenous an tagonists are illustra ted in Figure 1-3. Melanocortin System and Obesity The melanocortin system has been identified to participate in the regulation of feeding behavior, obesity, and ener gy homeostasis in rodents as well as in humans ( 26, 29-31, 46-49 ). It has been demonstrated that target ed disruption of the MC4R gene in mice results in hyperphagia, obesity, increased linear gr owth and hyperinsulinaemia ( 31 ). The MC3R knockout mice show increased fat mass and reduced lean ma ss while maintaining normal body weight ( 25, 26 ). POMC knockout mice which are lacking the coding region for melanocortin peptide agonists, are hyperphagic, obese, show defective adrenal de velopment and have altered pigmentation ( 50 ). When treated with daily injections of a mela nocortin agonist these mice showed substantial weight loss and darkening of the yellow fur color in the POMC knockout mouse ( 50 ). Pharmacological studies of intr acerebroventricular (icv) injecti on of the synthetic melanocortin agonist MTII ( 51, 52 ) inhibits feeding in mice, and this effect can be blocked by coadministration of the MC3R/MC4R antagonist SHU9119 ( 47 ). The endogenous MC3R and MC4R antagonist AGRP ( 14, 53 ) when administered centrally, produces an increase in food intake which is sustai ned for several days ( 54, 55 ). Ectopic expression of the AGRP protein in transgenic mice results in hyperphagia and obesity ( 14, 56 ). In humans, POMC polymorphisms resulted in early onset obesity, ad renal insufficiency and red hair ( 50, 57 ). In addition, more then 70 diffe rent single nucleotide polymorphisms (SNPs) of the MC4R gene have been discovered ( 29, 30, 48, 58-76 ), These polymorphisms have been associated with early and adult onset obesity. Polymorphisms in the MC3R coding and promoter region have been identi fied in severely obese humans ( 74-84 ), but the mechanisms by which these mutations lead to obe sity development remain to be identified. In summary, studies

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25 from genetic animal models, feeding studies and the discovery of polymorphisms in humans demonstrate the involvement of the melanocor tin system in obesity development. These observations clearly implicate the components of this system as potential dr ug targets. Contribution of the Research in this Di ssertation towards Rational Drug Design The MC3 and MC4 receptors are thought to be involved in energy and weight homeostasis, and have become potential drug targ ets for the treatment of obesity and its related diseases. Due to limited availability of GPCR biophysical (NMR and crystallogra phy) structural data, structure-based drug design ba sed on the 3D structur e of the target rece ptor is generally difficult. The design of ligands with specific activity and desi red pharmacological profile is a very complex task. Extensive knowledge about puta tive interactions betw een the ligand and the corresponding receptor will be highly valuable. This includes interactions crucial for both ligand binding and signal transd uction processes. Structureactivity relationship (SAR) studies of ligands usually involve systematic modification of a lead peptide a nd are designed to provide insight into potential interactions involved in the formation of the ligandreceptor co mplex. It is desirable to aquire knowledge about both favorable and unfavorable interac tions that may occur during potential ligand receptor interactions that result in either recepto r stimulation or inhibiti on. Modifications of the peptide ligand are performed to identify functionally important amino acid residues, residue positions that favor aromatic, constrained, hydr ophobic, hydrophilic or functional moieties, residues that enhance potency or receptor s ubtype selectivity, mini mal peptide length and potential amino acid functional moieties that may participate in the peptide pharmacophore ( 85 ). Part of the research presente d herein discusses structure-ac tivity relationship studies of a tetrapeptide ligand, where one postion will be system atically modified in attempts to gain insight

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26 into the requirements underlying agonist/antagonist activity at th e MC3R and subtype specificity of MC3R/MC4R. It is hypothesized that both residues of th e ligand and the receptors are important for ligand-receptor molecular recognition, receptor activation or inhibition and receptor subtype specificity. It is therefore important to gain knowledge about the recepto r residues involved in these processes. Structure-activity relationshi p studies of the receptor using site-directed mutagenesis are undertaken to identify the stru ctural determinants specifying high-affinity binding, subtype-selective binding, receptor activation mechanis ms and G-protein coupling. Research presented herein employs a dual-pronged approach of extensiv e site-directed receptor mutagenesis studies in addition to traditional ligand SAR studies. On the basis of the knowledge gained from lig and SAR, receptor site-directed mutagenesis and the crystal structure of bovine rhodopsin, a GPCR 3-dimensional (3D) receptor homology model can be generated. Information from site-d irected mutagenesis experiments are important to assist the docking of specific ligands into receptor models. Such receptor models have an essential role in providing new working hypothese s, which can be tested again using receptor site-directed mutagenesi s and ligand SAR. An iterative pro cedure involving molecular modeling, site-directed mutagenesis, ligand binding and SAR studies was employed to gradually improve the accuracy of receptor models. One caveat in using the crystal structures of bovine rhodopsin as template for modeling and ligang docking is that this model represents the receptor in its inactive or antagonist bound state conformation ( 86-88 ). It is commonly believed that GP CRs coexist in equilibrium between the inactive and active state and that receptor activation upon agoni st binding is accompanied by conformational changes ( 89 ). Thus, modeling of GPCRs in their active agonist bound

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27 conformation is a specia l challenge. It has been suggested that constitutivel y active receptors resemble the agonist bound receptor conformation ( 90 ). Herein, the molecular mechanism of a constitutively active mutant receptor will be inve stigated, by using site-directed mutagenesis, to gain insight into intra molecular interacti ons stabilizing the active and inactive receptor conformation, potentially useful for further stru cture-based drug design a pproaches of selective ligands. Polymorphic receptors may not respond to tr aditional drug therapy. It is therefore important to investigate the molecular mechanis m underlying these polymorphisms in attempts to identify compounds or treatment modalities that correct for the malfunction. Research will be discussed herein, focusing on the identification of new mechanisms that may be responsible for the impaired function of polymorphic receptors. Results from the research discussed in this study contribute essentially to future rational drug design approaches that target the melanoc ortin system and may ultimately lead to identification of new treatment options for obesity and related diseases. Fi gure 1-4 illustrates an overview of rational drug desi gn as iterative process.

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28 Figure 1-1. Overview of the melanocortin sy stem. When agonists bind to the receptor the following signal transduction cascade is init iated. Antagonists block these effects. The physiological functions of the melanocor tin receptors are depicted on the right. MSH, melanocyte stimulating hormone; ASP, agouti signaling protein; AGRP, agouti-related protein; Gs, stimulatory G-prot ein; AC, adenylyl cyclase; PKA, protein kinase A; cAMP, cyclic adenosine m onophosphate; CREB, cAMP response element binding protein; CRE, cAMP response element.

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29 Peptide Primary Sequence ACTH (1-24) SYSME HFRW GKPVGKKRRPVKVYP -MSH Ac-SYSME HFRW GKPV-NH2 -MSH ADKKDEGPYRME HFRW GSPPKD 2-MSH YVMG HFRW DRFG Figure 1-2. Posttranslational pr ocessing of POMC resulting in the generation of the endogenous melanocortin agonists. The endogenous agonist s are highlighted in red squares. The primary sequences of the melanocortin a gonists are shown below and their common core sequence His-Phe-Arg-Trp is highlighted in color.

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30 Figure 1-3. Primary sequences of the C-term ini of the endogenous melanocortin antagonists. The putative antagonist core sequence Arg -Phe-Phe is highlighted in color. Figure 1-4. Rational drug desi gn as iterative process.

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31 CHAPTER 2 METHODOLOGIES General Strategies and Concepts in Peptide Synthesis Solid-Phase Peptide Synthesis Solid-phase peptide synthesis (SPPS), was pioneered by Merrifields work in 1963 ( 91 ). This discovery resulted in a paradi gm shift of peptide synthesis, away from the classical solution phase approach. It has now become the method of choice to produce peptides and proteins. The use of a solid support has several advantages in peptide synthesi s. It is possible to use high excess of reagents at high concentrations to drive coupling reactions to completion. Excess reagents and side products can simply be separated from the growing peptide chain by filtration and washing, with savings in time and labor. Moreover, SPPS is amenable for automation and allows the synthesis of peptid es in high-throughput fashion. The general principles of SPPS, using Fmoc chem istry as example, are illustrated in Figure 2-1. All functional groups of th e amino acid side chains are pr otected by permanent protecting groups that are stable in the re action conditions that are used dur ing the peptide synthesis. The -amino group is protected by a temporary protec ting group and is removed after loading to the resin of the growing peptide chain. Unlike in natural protein synthesis, solid-phase peptide synthesis is carried out from the Cto N-termi nus. In the initial step, the first or C-terminal amino acid is attached to the resin via a linker. The next step involves removal of the N-terminal temporary protecting group and coupling of the second amino acid, which is usually pre-activated. This unit is th en deprotected again, re vealing a new N-terminal amine to which a further amino acid may be attached. This cycle is repeated until the desired peptide sequence is reached. The final step is the deprotection, wh ere the peptide is cleaved from the resin and permanent side chain protecting groups are removed ( 92 ).

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32 There are two main strategies of SPPS: Boc (t-butoxycarbonyl) ( 91 ) and Fmoc (9Fluorenylmethoxycarbonyl) ( 93 ) solid phase peptide synthesis. Boc chemistry is based on the differential acid lability of si de chain protecting groups. In th is approach the Boc group is removed by trifluoroacetic acid (TFA), and side chains protecting groups as well as peptide-resin linkages are removed at the end of the synthesi s by cleavage with hydrofluoric acid (HF). The use of the highly toxic and strong acid HF and the need for a special apparatus which is suitable for HF are limitations of this approach ( 92 ). Fmoc chemistry is an efficient alternative to the Boc strategy and was used for the pr eparation of the peptides de scribed in this disseration. Fmoc Solid Phase Peptide Synthesis The Fmoc method is based on an orthogonal protecting group strategy, using the base labile N-Fmoc group for protection of the -amino group and acid-labile side chain protecting groups and resin linke rs (Figure 2-1) ( 92 ). Examples for side chain protecting groups are t-butyl or trityl based moieties, and alkoxy based linkers are used for a ttachment of the peptide chain to the solid support. The N -Fmoc protecting group is removed prior to each coupling step with 20% piperidine in DMF. The coupling step is ca rried out with pre-formed activated esters in DMF. Cleavage of the peptide from the resin and side-chain deprotection is done in 95% TFA. The first step in Fmoc SPPS is the attachment of the C-terminal amio acid to the resin linker. This process is of special importance, since the extent of this reaction may determine the yield of the final product and th e amount of side products. Resins which were used in this dissertation, are the Wang and Rink amid resins (Figure 2-2). The Wang resin was used, when the free carboxylic acid at the peptide C-terminus was desired. This resin can be purchased with the first amino-acid already prel oaded. The Rink amide resin was used for peptides which are amidated at the C-terminal amino acid.

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33 The N -Fmoc removal is achieved by treating the resin with 20% piperi dine in DMF. The mechanism of the Fmoc deprotection reaction is illustrated in Figure 23. The key step is the deprotonation of the fluorene ring to generate the cyclopentadiene like intermediate, followed by elimination to form dibenzofulvene, which may be rapidly scavenged by piperidine. Coupling of N-protected amino acids requires activation of the carboxy component. The coupling reagent used for the generation of peptides synthesized herein is HBTU [2-(1 H benzotriazol-1-yl)-1,1,3,3-tetram ethyluronium hexafluorophosphate] (Figure 2-4) and HOBt (1hydroxybenzotriazole) was added as additive. Benzotriazole deriva tives form esters as highly reactive intermediates. These coupling reagents convert N-protected amino acids into their corresponding OBt esters. A tertiary amine, like DI EA is required to produce the carboxylate of N-protected amino acids which reacts with the coupling reagents (Figure 2-5). The coupling and deprotection reactions can be monitored using colorimetric tests. One of the most widely used tests to de tect the absence or pr esence of free amine groups is the Kaiser or Ninhydrin test (Figure 2-6) ( 94 ). The free primary amine is indicated by blue color change. For secondary amines, like proline, this test is not suitable. Herefore, the Chloranile test is recommended ( 92 ). Blue stained resin indicates the pres ence of free amines. After the successful assembly of a peptide chain on a solid support, the N-terminal amino group may be acetylated, using a 2:1 mixture of Acetic A nhydride and Pyridine. The resin is then washed several times with dichloromethane (DCM) and dried under vacuum over night. The final cleavage of the peptide from the resin and removal of side-chain protecting groups is done in concentrated trifluoroacetic ac id (HF). Savangers, like water, thiols or alkyl silanes are added to the cleavage cocktail to trap any carbocations that may be formed from sidechain protecting groups during these conditions. If the peptide contai ns Met, ethanedithiol has to

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34 be added to suppress methionine oxidation. Upon th e side-chain deprotection and cleavage of the peptide from the solid support, the peptide is pr ecipitated with anhydrous cold ethyl ether and centrifuged. This step is repeated twice. The precipitated peptide is then dried in vacuo overnight and will be further purified and analytically characterized. After synthesis and cleavage are complete, anal ytical characterization of the crude peptide is performed using reverse phase high performa nce liquid chromatography (RP-HPLC). Once the peak of interest is identified, purification is performed using RP-HPLC semi preparative chromatography (GRACE VYDAC C-18 column). The volume of the purified peptide samples is then reduced on a rotavap followed by l yophilization. The final peptide purity can be determined by analytical RP-HPLC in two different solvent systems (10% acetonitrile in 0.1% trifluoroacetic acid/water and a gradient to 90% acetonitrile over 35 min or 10% methanol in 0.1% trifluoroacetic acid/water and a gradient to 90% methanol over 35 min) and the correct molecular weight can be verified by using mass spectrometry (MS). General Concepts and Strategies in Receptor Mutagenesis and Pharmacology Generation of Epitope Tagged Receptors Epitope tagging of a receptor involves intr oducing a defined amino acid sequence into the primary amino acid sequence of the receptor. Using tags allows for the use of specific antibodies to characterize the receptor using either bioc hemical methods or immunocytochemical methods to localize the receptor w ithin cells. A tag which is commonly used for GPCRs in our laboratory is the Flag tag, with the am ino acid sequence DYKDDDDK. Epit ope tags can be placed at both, the Nand the C-termini of GPCRs. N-termin al tags have the advantage, that for GPCRs this receptor unit will be located outside the ce ll, and therefore fluorescent antibodies directed against the tag, allow for monitoring of recepto r cell surface expression. C-terminal tags have been widely used to monitor receptor regulation within cells. However this region of GPCRs is

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35 localized intracellularly and is thought to contain sites impor tant for receptor function, phosphorylation and internalizat ion and/or desensitization mechanisms. GPCRs, tagged with green fluorescent protein are widely used to study trafficking and localization of receptors within the cell. An advantage is that these constructs due to their auto fluorescence, can be used for studies in living cells. However, this protein contains 238 ami no acids and might more easily interfere with the receptor regulat ion mechanisms described above. The Flag-tag for the mouse MC3R was inserted at the N-terminus upstream of the methionine start codon of the open reading frame. It was extremely important to keep the mouse MC3R 5 UTR intact. Initi al insertion of the Flag tag without keeping th e 5UTR resulted in a nonfunctional receptor, unlike the plasmid for the human MC4R from our laboratory which does not contain 5UTR ( 18, 95 ). Subsequently, a new PCR strategy was developed that was modified from a method, previously described for the mGluR1a receptor ( 96 ). Two pairs of primers were used, one pair amplifying the vector sequence co ntaining the HindIII restriction site (Forward F1) and the N-terminus of the MC3R. The reverse prim er of this pair (R1) was extended at the 3 end with an additional sequence for the Flag ep itope. The second pair of primers F2 and R2 amplified the entire receptor sequence downstream the methi onine startcodon including the restriction site XbaI. Primer F2 contained residues at the 5 e nd encoding for the flag epitope. Both sets of primers were then used to amp lify their respective sequences in separate PCR reactions and the 2 products were purified by gel electrophoresis. The resulting products were mixed for a last round of PCR with the forwar d primer F1 and the reverse primer R2. The resulting PCR product contained the Flag epitop e inserted between the Methionine start codon and the first mMC3R amino acid. This was then pu rified and cut with the restriction enzymes HindIII and XbaI. The construct was then ligated into the pCDNA3 mammalian expression

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36 vector used for the generation of stable cell lines. The DNA seque nce was verified, cell surface expression was quantified using flow cytometry a nd the functional activity was tested compared to the untagged mMC3R. Figure 2-6 illustrates the PCR strategy used for the insertion of the Flag-tag at the Nterminus of the mouse MC3R. Figure 2-7 and 2-8 show verification of the receptor cell expression by flow cytometry and deconvolution mi croscopy and the functional activity of the FlagmMC3R. Site-directed Receptor Mutagenesis The cloning of the first GPCRs in the mid 1980s initiated mutagenesis studies in order to unravel the vast structural f eatures underlying receptor functi on and the formation of ligandreceptor complexes ( 97 ). Although, immense progress has b een made in the understanding how GPCRs function, signal and exert their physiological mechanisms, direct structural data of GPCRs is still limited to the crystal inac tive state structures of bovine rhodopsin ( 86-88 ). Sitedirected mutagenesis is an indi rect method to study the direct involvement of particular amino acid residues and/or receptor domai ns in specific functions, such as direct interaction with ligands, receptor activation, signa l transduction, receptor regulati on and also receptor expression on the cell surface ( 98 ). To test for these receptor functions, the w ild-type and mutant receptors are generally expressed in cell systems where they are test ed for ligand binding, re ceptor activation or inhibition, phosphorylation states and/or localizati on in the cell, just to name a few ( 98 ). A couple of guidelines for the selection of amino acids have been proposed. The most straightforward strategy might be choosing residues, based upon previous mutagenesis studies of the receptor family of interest or other GPCRs fr om different families. Another possibility is the use of sequence analyses of receptors that iden tified conserved residues in the majority of

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37 GPCRs. These conserved amino acids are thought to be important for receptor structure and function whereas residues conser ved in one receptor family are proposed to be important for ligand specificity. The residues that differ between receptor subtypes within one family may be participating in complementary ligand-receptor in teractions and may be important for receptor selectivity ( 85 ). Investigation of single nucleotide polymo rphisms of GPCRs causing specific phenotypes or diseases in animals and humans may be anot her interesting approach in selecting amino acid residues for site-directed mutagenesis experiments. Examination of the molecular mechanisms of naturally occurring mutations that result in ei ther loss-of-function or constitutive activation (enhanced basal activity in the ab sence of agonist ligand) might gi ve new insights into structurefunction relationships and activation mechanisms of receptors as well as a better understanding of disease processes. Once a specific residue of interest is identif ied, the common step is mutation to Alanine (Ala). Due to its small size and influence on the ove rall receptor structure, Ala scanning is often the method of choice for mutagenesis studies to i nvestigate the role of receptor residue side chains. To test the importance of a specific rece ptor locus, multiple mutations can be introduced at this position to investigate influences on charge, hydrogen bonding potential, hydrophobicity, volume and/or shape of the side chain. To examine the contribution of intramolecular interactions, single and reciprocal or compensating mutations can be generated. In cases where a second mutation is able to restor e the function that was disrupted by the first mutation, this is an indication that these re sidues share a common microenvironmen t and they show related rather than an independent contribution ( 98 ).

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38 An important consideration which has to be ta ken into account is that a specific mutation can have either one or multiple of the following effects (Figure 2-9) ( 98 ). A mutation may have localized effects and perturb interactions nece ssary for ligand binding an d/or functional activity but it may also interfere with the overall structur al integrity, the biosynthe sis and the trafficking of the GPCR to the cell membrane ( 98 ). Therefore it is necessary to test mutant GPCRs in different assays (surface expres sion, binding and functional assays) in order to analyze the true nature of the effect. The objective of rational drug design of ligands for GPCR is to gain a precise knowledge about the ligand-receptor complex ( 98 ). It is desirable to not only characterize the ligand-binding pocket of the receptor but also use multiple selected receptor agonists and antagonists to test hypotheses about the molecula r basis and different modes of ligand-receptor interaction ( 98 ). Three dimensional homology models are he lpful tools in the design as well as in the interpretation of mutagenesis studies and ma y help in mapping the binding sites of ligands. Vice versa, mutagenesis studies help in th e fine tuning of com putational models ( 98 ). For the generation of mutant receptors a P CR strategy is used. This method involves two matching oligonucleotide primers (reverse and forward) that c ontain the desired mutation. The plasmid DNA is then amplified in a mutagenesi s reaction. Despite the fact that the PCR product is mainly composed of mutated plasmid, the te mplate DNA is typically eliminated by enzymatic digestion with Dpn1, a restriction enzyme whic h cleaves only methylated DNA. The template, which is derived from competent DH5 cells and therefore is methyl ated, is destroyed in this step, but the mutated plasmid is preserved because it was generated in vitro and is unmethylated as a result. Our laboratory uses the pBKS vect or from Stratagene for the PCR mutagenesis reaction. To minimize errors, that can occur duri ng the PCR, the receptor construct containing the desired mutation is cut with restricti on enzymes and subcloned into the mammalian

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39 expression vector pCDNA3. After verification of the correct receptor sequence this plasmid is then stably transfected into HEK293 cells, us ing G418 selection. The a dvantage of the use of stably transfected cell lines is that the recep tor can undergo natural processes involved in internalization and recycling, as opposed to transient transf ection. The advantage of using transient transfection would be that it is fast (g eneration of stable cell lines can take up to 6 weeks) and it allows for initially high receptor ex pression. The use of stably transfected cell lines is the method of choice in our laboratory and was used to characterize th e wild-type and mutant melanocortin receptors in the subsequent assays. Cassette Mutagenesis For the generation of the -MSH plasmid, a strategy called casette mutagenesis was used. This type of mutagenesis involve s the cleavage by a restriction en zyme at a site in the plasmid and subsequent ligation of an oligonucleotide co ntaining the sequence to be inserted into the plasmid. Usually the restriction enzymes cutti ng the plasmid and the oligonucleotide are the same, permitting sticky ends of both plasmid and in sert to ligate to one another. This method was used to insert an oligonucleotide, containing the coding sequence for -MSH into the HindIII and XbaI restriction sites of the mammalian expression vector pCDNA3. In vivo -MSH is generated through posttranslational proces sing of the precursor hormone POMC. The nucleotide sequence for -MSH was therefore derived from the human POMC mRNA sequence (Gene Bank NM000939). The Kozak sequence as well as the methionine (Met) startcodon had to be added upstream the first -MSH acid to initiate translation in vitro To further ensure correct orientation of the -MSH insert into the plasmid, HindIII/Xba I restriction sites were added to the oligonucleotide primer sequences. The sequences for -MSH primer design are shown in Figure 2-10.

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40 Flow Cytometry Flow cytometry or fluorescence activated cell sorting (FACS) is a powerful technique for making rapid measurements of cells suspended in a stream of fluid one by one through a sensing point. Each measurement is made separately on a cell by cell basis. Flow cytometers measure multiple cellular parameters based on light scatter and fluorescence. The conjugation of dyes to ligands and to antibodies has made it possible to study the distribution of ligands and receptors on the cell surface, in subcellular compartments as well as ligand-recepto r interactions. Many of the fluorescent dyes can be used in combination which allows for multiple correlated measurements. In this dissertation Allophycoc yanin (APC) conjugated Anti-Flag antibody was used to quantify receptor cell surface and tota l receptor expression (Chapters 4-6) and a Fluorescein (FITC) conjugated ligand was used to measure the kinetics of ligand-receptor interaction (Chapter 7). The cells are usually stained by incubation with a fluorescently conjugated antibody or a fluorescently labeled ligand. To stain proteins in intracellular compartments, the cell membrane needs to be permeabilized prior st aining, for example with saponin dete rgent. It is important that the staining is specific, and even monoclona l antibodies may bind non-specifically. Another potential problem is optical crosstalk which can occur, when combinations of fluorochromes with overlapping emission spectra are used in the same experiment. Problems in sample preparation and staining can be mini mized by using the right controls. In flow cytometers each particle passes th rough one or more beams of light. The light scattering or the fluorescence emissions provide information about the properties of the cell, being measured. Light, scattered in the forward direction is colle cted by a lens which is called forward scatter channel (FSC). Th e FSC provides information about the size of a cell or particle and can distinguish between a live cell and debr is. The side scatter ch annel (SSC) provides

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41 information about the granularity and texture of particles. A combination of FSC and SSC provides a unique measurement for each cell and can be used to different iate between different cell types. Fluorescence measurements, when take n at different wavelength, can be used to provide qualitative and quantitative results a bout fluorescently labele d proteins. Different fluorescence channels (FL) detect the emitted light in flow cytometers. Scattered and fluorescent light are collected by photodete ctors which convert the photon pulses into electronic signals. Electronic and computational processing is then performed by the flow cytometer which results into graphic display and st atistical analysis of the measurement being made. A consideration when performing dual labe l experiments is the choice of the right fluorochromes. Ideally, they should be chosen from different ends of the spectrum to avoid that their emission spectrum will overlap. The fluorescent dyes which were chosen throughout this dissertation are a FITC (Fluor escein isothiocyanate) deri vative and APC (Allophyocyanin). These fluorophochromes were chosen to mini mize spectral overlap. FITC has an excitation maximum of 495nm which is close to the emission line of an argon-ion lase r and emits light at 520nm (green). In contrast, APC has an excitati on maximum of 650nm and can only be used for instruments which contain a helium-neon laser (las er line 630nm). This dye emits light at 660nm which allows for good distinction of the light spectrum of FITC ( 99 ). The spectral properties of these two dyes are shown in Table 2-1 ( 99 ). Functional Reporter Gene Assay The purpose of the functional assay is to inves tigate the effect of a mutation on the ability of the receptor to generate second messenger re sponse upon ligand stimulation. In the case of the melanocortin receptors we measure the amount of cAMP produced after receptor stimulation with a ligand. The assay which is used is a CRE/ -galactosidase report er gene assay ( 100 ). The reporter gene plasmid contains five CRE units and the coding region of -galactosidase.

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42 Increasing concentrations of intr acellular cAMP result into phos phorylation of th e transcription factor CREB by protein kinase A wh ich activates the transcription factor. It is therefore possible to monitor Gs activation by monitoring CREB me diated gene expression. Beta-galactosidase activity increases in a li near relationship to in tracellular cAMP. The fold increase in -galactosidase activity directly can be measured using a colormetric assay. The substrate ortho-Nitrophenyl-galactoside (ONPG) is a colorime tric substrate for detection of galactosidase activity. This compound is normally colorless. However if -galactosidase is present, it hydrolyzes the ONPG molecule into galactose and orthonitrophenol. The latter compound has a yellow coloration th at can be used to check for enzyme activity by means of a colorimetric assay. This assay can be performed in 96-well plates and OD405 can be measured in a plate reader. 3-Isobutyl-1-methylxanthine (IBMX) is adde d to the stimulation media, since it inhibits phophodiesterases in the cell, which a llows for maximal cAMP accumulation. Forskolin is used as positive control because it causes r eceptor independent production of cAMP resulting in characterization of the maximal cAMP leve ls observed in the experiment. A schematic representation of the -Galactosidase reporter gene assay is illustrated in Figure 2-11. Different pharmacological profiles are possible. Agonists activate th e receptors and cause in the case of the melanocortin system, stim ulation of cAMP producti on. Different agonists may have different potencies, whic h are reflected in their EC50 values. Agonists which do not cause a full response and show less than 100% efficacy are termed partial agonists. Special cases are constitutively active receptors th at possess increased basal activity in the absence of an agonist ligand. An antagonist is a receptor ligand that binds to a receptor but fails to activate it. If an agonist competes with a competitive antagonist fo r the same binding site on the same receptor, the agonist will be displaced, wh ich is depicted by a right shift of the dose-response curves.

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43 Some antagonists have been obser ved to have negative intrinsic activity and behave as inverse agonists, by decreasing the basal (agonist-independent or constitutiv e) receptor activity. Other antagonists may still have some stimulatory res ponse and show partial agonism in addition to competitive antagonism. Possible pharamcology curv es which are encountered throughout this dissertation are illustrated in Figure 2-12. Another class of anta gonists mediates their effects by interacting with an allost eric site of the receptor or bind irre versible to the receptor. However, these latter types of ligands ha ve not been observed in the cont ext of the melanocortin system. Nonlinear Regression Analysis After obtaining the raw data (OD405 measurements) from the CRE/ -galactosidase reporter gene assays, the results are normalized to prot ein content and forskolin (maximal receptor independent cAMP generation). Hereby the maximu m cAMP stimulation by forskolin is defined as 1 (y=1). These data are then being used to obtain dose-response curves. On the x-axis the concentration of the peptide ligand is plotted on a logarithmic scale, while on the y-axis the cAMP response from the CRE/ -galactosidase reporter gene a ssay normalized to protein and forskolin is plotted in the range from 0-1. A standard dose-response curve is defined by four parameters: the basal response (min), the maximum response (max), the slope and th e ligand concentration that causes a response halfway between basal and maximum (EC50) response. Hereby, the EC50 value should not be misunderstood. It is defined as the concentratio n of agonist that elic its a response half way between the basal (min) and maximum response (m ax). However this might not be the same value as the concentration that provokes a response of y=50% or y=0.5. Usually, the data are normalized to maximum response (Forskolin: y= 1.0), without subtractin g the basal response (min).

PAGE 44

44 If the min for example is approximately 0.2 and the max is 1.0, then the EC50 might be the concentration of agonist that causes a res ponse of approximately y=0.6. To obtain the doseresponse curve, the following equation is used, where Y=min+(max-min)/(1+10^((LogEC50X))). This equation represents th e response as a function of the logarithm of the concentration. X represents the logarithm of agoni st concentration and y represents the response. The variable min is the y value at the bottom plateau; max is the y value at the top plateau, and LogEC50 is the x value when the response is halfway between min and max. LogEC50 is the logarithm of the EC50. The dose-response curves obtained from our assa ys resemble the standard Hill slope, which equals 1 ( 101 ). Schild Analysis The antagonist potency pA2 values are derived from Schild analysis ( 102, 103 ). Schild defined the dose ratio of the agoni st concentration that elicits an equal res ponse in the presence (A) or absence (A) of antagonist s (I) as x. He also defined pAx, as the negative logarithm of the antagonist concentration in mol that produces th e dose ratio x. Taking the logarithm of equation (A/A)-1=[I]/Ki and substituting x for A/A results into equation log(x-1)=log[I]-logKi. The definition of pAx as log[I] changes this equation to log(x-1)=-pAx-logKi. If x equals 2, then pA2=-logKi. To determine graphi cally the value of pA2 from dose-response curves, the log(x-1) is plotted versus the log[I]. After linear regression the pA2 value equals the intercept on the xaxis (y=0). To construct a Schild plot, the dose-response curves for an agonist are determined in the absence (control) and the presence of various concentrations of a competitive antagonist. Once the actual experiments are performed, a series of dose ratios are calculated. For example the dose ratio of the EC50 values of agonist (A') in the presence of the an tagonist to the EC50 value of the agonist alone (A) is calculated. This is determined for several concentrations of antagonist and

PAGE 45

45 then log ((A'/A) -1) or log (x-1) versus the negative log[I] is pl otted, followed by linear regression. If the regression of log (x-1) and -log [I] is linear with a sl ope of -1, then this indicates that the antagonism is competitive a nd by definition the agonist and antagonist act at the same recognition sites. The x-intercept of the fitted regression line is an estimate of the antagonist pA2 value. The correct use of the Schild plot to estimate pA2 requires the antagonist to be competitive ( 102-104 ). Competitive Binding Assay Once a mutant receptor is characterized for its functional activity it is important to determine its binding affinity for a given ligand. Different scenarios might be envisioned. (i) Functional activity and binding affinity are similar to that observed with the wild-type receptor, and then the residue which was mutated was not important for liga nd binding or functional activity of the receptor. (ii) If no or decreased functional act ivity is observed, while ligand binding affinity is similar to the wild-type recep tor, then this residue might be important for receptor activation mechanisms. (iii) In the case where no functional activity and binding affinity can be determined or both values are significan tly reduced then the mutated residue might be located in the putative ligand-receptor binding po cket. (iv) If loss of function and binding mutations are determined it is al so important to verify the re ceptor cell surface expression using flow cytometry, since this mutant receptor might be retained intracellularly due to improper folding or it might not be expressed at all, due to de fective protein synthesis. In competitive binding assays a single concentrat ion of radiolabeled (hot) peptide is used to competitively displace its non labeled counte rpart, in a dose-response fashion to measure ligand receptor affinity IC50 values at the mutant receptors. Dose-response curves are generated by nonlinear regression where the maximum specific binding is normalized to 100%. The isotope which is chosen for the radioligand is I-125. Iodinated ligands have a higher specific

PAGE 46

46 radioactivity (2200 Ci/mmol) compared to Tritium H3 (29 Ci/mmol) and are especially useful in assays where receptor quantity is limited ( 104 ). However, the half-life of I-125 is considerably lower than H3 (60 days versus 12 years). To incorporate I-125 into the ligand structure, a tyrosine (Tyr) residue is required. NDP-MSH contains Ty r at the 2 position, whic h is not required for ligand-receptor interactions. This reaction is performed according to the Chloramine T method ( 105 ). Hereby, NaI-125 is oxidized to the free iodi ne which leads to iodination of Tyr, through electrophilic substitution. Remain ing iodine is reduced by adding sodium metabisulfite to the reaction mixture. The radiolabeled I-125 NDP-MSH is then purified by using size exclusion chromatography, and tested for total and nonsp ecific binding at the known mouse MC3R, prior to use for competitive binding assays. Experimental Procedures Peptide Synthesis and Func tional Characterization Synthesis of tetrapeptide analogues Peptide synthesis was performed using standard Fmoc methodology ( 93 ) on a semi automated synthesizer (LabTech, Louisville, KY). The amino acids Fmoc-Tyr(tBu), FmocHis(Trt), Fmoc-DPhe, Fmoc-D-p-Cl-Phe-OH, Fmoc-D-p-F-Phe-OH, Fmoc-D-p-Phe(NO2) and Fmoc-Trp(Boc), were purchased from Peptides International (Louisville, KY). Fmoc-p-aminoD-Phe(Boc)-OH, Fmoc-4-bromo-D-Phe-OH, Fm oc-p-Me-D-Phe-OH, Fmoc-p-tBu-D-Phe-OH, Fmoc-4-cyano-D-Phe-OH, Fmoc-p-Bz-D-Phe-OH and Fmoc-(3,4diCl)DPhe-OH were purchased from Bachem (Torrance,CA). Fmoc-4-iodo-D-phenylalanine ( p -I-DPhe), Fmoc-D-3-Cl-Phe-OH and Fmoc-D-4-Trifluoromethylphe nylalanine were purchased fr om Synthetech (Albany,OR). The coupling reagents 2 -(1-H-benzotriazol-1-yl )-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzot riazole (HOBt) were purchased from Peptides International. Glacial acetic acid (HOA c), dichloromethane (DCM), methanol (MeOH),

PAGE 47

47 acetonitrile (ACN), and anhydrous ethyl ether were purchased from Fisher (Fair Lawn, NJ). N N Dimethylformamide (DMF) was purchased from Burdick and Jackson (McGaw Park, IL). Trifluoroacetic acid (TFA), pyridine, piperi dine, and acetic anhydride were purchased from Sigma (St. Louis, MO). N N -Diisopropylethylamine (DIEA) and triisopropylsilane (Tis) were purchased from Aldrich (Milwaukee, WI). All reagents and chemicals were ACS grade or better and were used without further purification. The peptides were assembled on rink-ami de-MBHA resin, purchased from Peptides International. The synthesis was performed usi ng a 16 well Teflon reaction block with a course Teflon frit. Approximately 200 mg of resin (0.1 mmol) was added to each reaction block well. The resin was allowed to swell for 2 h in DCM and deprotected using 20 % piperidine in DMF for 2 min followed by 18 min 20% piperidine inc ubation at 450 rpms. A positive Kaiser test was performed indicating free amine groups on the resin ( 94 ). The growing peptide chain was added to the amide-resin using the general amino acid cy cle as follows: A 3-fold excess of amino acid (0.1 mmol scale) starting from the C terminus was added, containing the coupling reagents HOBt (0.1 mmol) and HBTU (0.1 mmol) in DMF. Subsequently, 90 L of DIEA were added and the reaction well volume was brought up to 5 mL us ing DMF. The coupling reaction was mixed for 2 h at 450 rpms, followed by emptying of the reaction block by vacuum. After the coupling cycle, the reaction block was emptied and the N -Fmoc-protected peptide resin was washed with DMF (8 mL, three times). N -Fmoc deprotection was performed by the add ition of 8 mL of 20% piperidine in DMF and mixed for 2 min at 450 rpms followed by a 18 min deprotection at 450 rpms. The reaction well was washed with DMF (8 mL, three times), and the next coupling cycle was performed as described above. Following N -Fmoc deprotection of the final amino acid, acetylation of the N

PAGE 48

48 amine was performed by addition of 4 mL of acetic anhydride and 2 mL of pyridine to the reaction block wells and mixed fo r 30 min at 450 rpms. The acetyl ated peptide resin was washed with DCM (8 mL, five times) and dried t horoughly prior to cleavage from the resin. Deprotection of the amino acid side chains and cleavage of the acetylated-peptide-resin was performed with 8 ml of cl eavage cocktail (95% TFA, 2.5% wa ter, and 2.5% Tis) for 2 h at 450 rpms. The cleavage product was emptied from the reaction block into a cleavage block containing the collection vials. The resin was wash ed with 1.5 ml of cleavage cocktail for 5 min and 450 rpms and added to the previous cleavag e solution. The peptides was transferred to 50 mL conical tubes and precipita ted with cold (4C) anhydrous et hyl ether (up to 50 mL). The flocculent peptide was pelleted by centrifugati on (Sorval Super T21 high-speed centrifuge using the swinging bucket rotor) at 4000 rpm for 5 min, the ether was decanted off, and the peptide was washed one time with cold anhydrous ethyl ether and again pelleted. The crude peptide was dried in vacuum for 24 h. A 15-30 mg sample of crude peptide wa s purified by RP-HPLC using a Shimadzu chromatography system with a photodiode array detector and a semipreparative RP-HPLC C18 bonded silica column (Vydac 218TP1010, 1.0 cm x 25 cm) and lyophilized. The purified peptides were analytically characterized by RP-HPLC using a two solvent system and mass spectrometry (University of Florida protein core facility). Table 22 shows the analytical data of the tetrapeptides synt hesized in this study. Synthesis of alpha-MSH analogues Peptide Synthesis was performed using st andard 9-fluorenylmethoxycarbonyl (Fmoc) methodology ( 106 ) in a manual reaction vessel. Amino acids Fmoc-Met, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc -His(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-PheOH, Fmoc-Arg(Pbf)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Gly, Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH

PAGE 49

49 and Fmoc-Val-OH were purchase from Peptides International (Louisville, KY). All reagents were ACS grade or better and were used without further purification. Approximately 370 mg of Fmoc-Val-Wang re sin (0.27meq/g substitution) (Peptides International, Lousiville, KY) was used fo r BP2-67 and approximately 250 mg Rink-amideMBHA resin (0.50 meq/g substitution) (Peptides In ternational, Lousiville, KY) was used for BP2-38. The Fmoc protecting groups were removed using 20% piperidine (Sigma Aldrich) in N,N-dimethylformamide (DMF) for 2 min and fo r 18 min. Amino acid coupling (3-fold excess) was accomplished using 2-(1H-benzot riazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU, 3-fold excess), 1hydroxybenzotriazole anhydr ous (HOBt, 3-fold excess), and diisopropylethylamine (DIEA, 5.1-fold excess) for 1.5 h. A positive ninhydrin test ( 94 ) result indicated free amine groups on the resi n. The growing peptide chain was added to the amide-resin using the general amino acid cycle as follows: A 3-fold excess of amino acid (0.1 mmol scale) starting from the C terminus wa s added. The amino acid solution contained the coupling reagents HOBt (0.1 mmol) and HBTU ( 0.1 mmol) in DMF. Subsequently, 90 L of DIEA were added and the reaction well. The coupling reaction was mixed for 1.5 hrs under nitrogen gas, followed by emptying of the reaction block by vacuum. After the coupling cycle, the react ion block was emptied and the N -Fmoc-protected peptide resin was washed with DMF, three times. N -Fmoc deprotection was performed by the addition of 8 mL of 20% pipe ridine in DMF and mixed fo r 2 min, followed by an 18 min deprotection. The reaction well was washed with DMF, three times, and the next coupling cycle was performed as described above. The finished pe ptide resin was washed with DCM, five times and dried thoroughly prior to cleavage from the re sin. Deprotection of the amino acid side chains and cleavage of the peptide-resi n was performed with 10 mL of cleavage cocktail for sulfur-

PAGE 50

50 containing peptides (91% TFA, 3% Triisopropyls ilane, 3% Ethandithiole and 3% water) for 2 hrs under nitrogen. The cleavage product was emptied fr om the reaction vessel into a 50 mL conical tube by gravity. The resin was washed with 1.5 ml of cleavage cocktail. The peptides were precipitated with cold (4 C) anhydrous ethyl ether (up to 50 mL ). The flocculent peptide was pelleted by centrifugation (Sorval Super T21 high -speed centrifuge using the swinging bucket rotor) at 4000 rpm for 5 min, the ether was deca nted off, and the peptide was washed one time with cold anhydrous ethyl ether and again pelleted. The crude peptide was dried in vacuum for 24 h. A 15-30 mg sample of crude peptide was purified by RP-HPLC using a Shimadzu chro matography system with a photodiode array detector and a semipreparative RP-HPLC C 18 bonded silica column (Vydac 218TP1010, 1.0 cm x 25 cm) and lyophilized. The purified peptides were analytically ch aracterized by RP-HPLC using a two solvent system and ma ss spectrometry (University of Florida protein core facility). Functional characterization at the mouse Melanocortin receptors For the (CRE)/ -galactosidase reporter gene assa ys the mMC1 and MC3-5 receptor sequences were cloned into the pCDNA3 mammalian expression vector and stably expressed in HEK293 cells. Herby HEK293 cells were transfected with pCDNA3 expression vector containing the corresponding melanocortin receptor DNA (20 g) using the calcium phosphate method ( 107 ). Stable receptor populations were generated using G418 selection method (1 mg/mL) for the subsequent bioassay analysis. HEK-293 cells were maintained in Dulbeccos modified Eagles medium (DMEM) with 10% ne wborn calf serum and plated 1 day prior to transfection at 1 to 2x10-6 cells/100 mm dish. For the (CRE)/ -galactosidase reporter gene assay, HEK-293 cells stably expressing the mouse MC1, or MC3-MC5 receptors were transf ected with 4 g of cAMP response element (CRE)/ -galactosidase reporter gene as previously described ( 100, 108-111 ). Twenty-four hours

PAGE 51

51 after transfection 5000-15000 cells we re plated into collagen treat ed 96-well plates and incubated overnight. Forty-eight hours after transfection, the cells were stim ulated with 100 L of peptide synthesized (10-4-10-12 M) or forskolin (10-4 M) control in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM isobutylmet hylxanthine) for 6 h. The anta gonistic properties of these compounds were evaluated by the ability of thes e ligands to competitively displace the MTII agonist in a dose-dependent manner, at up to 10 M concentrations. After stimulation the assay media was aspira ted and 50 L of lysi s buffer (250 mM TrisHCl, pH 8.0, and 0.1% Triton X-100) was added. The plates were st ored at -80C overnight. The plates containing the cell lysates were th awed the following day. For relative protein determination, aliquots of 10 L were taken fr om each well and transfer red to another 96 well plate. The relative protein was determined by adding 200 L 1:5 dilutio n Bio-Rad G250 protein dye:water to the 10 L cell lysate sample, and the OD595 was measured on a 96 well plate reader (Molecular Devices). To the cell lysate plates, 40 L of phos phate-buffered saline with 0.5% BSA were added to each well. Next, 150 L of substrate buffer [60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM -mercaptoethanol, 2 mg/mL of o-Nitrophenyl-Dgalactopyranoside (ONPG)] were added to each well and the plates were incubated at 37C. The sample absorbance, OD405, was measured using a 96-well plat e reader (Molecular Devices). Data points were normalized both to the relative protein cont ent and receptor independent forskolin values. The assays were performed usi ng duplicate data points and repeated in at least three independent experime nts. Data analysis, EC50, pA2 estimates, and their associated standard errors of the mean ( 112 ), were determined using the PRISM program (v4.0, GraphPad Inc.). Antagonistic properties were determ ined by the ability of each of those peptides to competitively

PAGE 52

52 displace the MTII agonist in a dose-dependent manner. The pA2 values were generated using the Schild analysis method ( 103 ). Receptor Site-direct ed Mutagenesis Residue numbering schemes Important residues in the transmembrane domain of GPCRs are given two numbering schemes. First, residues are numbered according to their positions in the receptor amino acid sequence. Second, residues are numbered relative to the most conserved residue in the TM in which it is located (N1, N2). N1 refers to the TM number, and N2 refers to the position of the residue relative to the most c onserved one with numbers decreasi ng towards the Nterminus and increasing towards the C-terminus. The most cons erved residue is assigned the position number 50. This numbering scheme simplifies the identifica tion of aligned residues in different GPCRs ( 113 ). Mouse MC3R epitope tagging The Flag-tag for the mouse MC3R was inserted at the N-terminus upstream of the methionine start codon of the open reading fram e. A new PCR strategy was employed that was modified from a strategy, previously described for the mGluR1a receptor ( 98 ). Two pairs of primers were used, one pair amplifying the vector sequence containing the HindIII restriction site (Forward F1 5ATACGACTCACTATAGGGAGACC3 ) and the N-terminus of the MC3R. The reverse primer of this pair (R1 5CTTGTCGTCGTCGTCCTTGTAGTC CATGGTTAACAGC3) was ex tended at the 3 end with an additional sequence for the Flag epitope (underlined). The second pair of primers F2 (5GACTACAAGGACGACGACGACAAG AACTCTTCCTGCTG3) and R2 (5GCATTTAGGTGACACTATAGAATAG3) amplif ied the entire receptor sequence

PAGE 53

53 downstream the methionine startcodon including th e restriction site XbaI. Primer F2 contained residues at the 5 end encoding for the flag epitope (underlined). Both sets of primers were then used to amp lify their respective sequences in separate PCR reactions using 34 cycles (95 C 30 sec, 56 C 1 min, 72 C 2 min) and the Master Taq DNA Polymerase Kit (Eppendorf). The presence of the resulting two products wa s verified by agarose gel electrophoresis. Both products were then mi xed for a last round of PCR with the forward primer F1 and the reverse primer R2. The re sulting PCR product contained the Flag epitope inserted between the Methionine start codon an d the first mMC3R amino acid. This was then purified and cut with HindIII and XbaI. The construct was then cloned into the pCDNA3 mammalian expression vector used for the gene ration of stable cell lines. The DNA sequence was verified by DNA sequencing (Univers ity of Florida core facilities). Generation of receptor mutations The mouse wild-type N-terminal Flag tagge d MC3R cDNA or th e human wild-type Nterminal Flag tagged MC4R cDNA subcloned into the pBKS plasmi d (Stratagene) were used for mutagenesis. In vitro mMC3 and hMC4 receptor mutagenesis was performed as described previously ( 18, 114 ). Amino acid modifications were in troduced by applying a polymerase chain reaction (PCR) strategy using pf u turbo polymerase (Stratagene) and a complementary set of primers containing the nucleotide mutation(s) resulting in the desi red residue change. The primer sequences are illustrated in Table 2-3 or th e mMC3R and in Table 2-4 for the hMC4R. Conditions for the PCR reaction were 95C 30 sec, 12 cycles of 95C 30 sec, 55C 1 min, 68C 9 min. The resulting product was purified using Qiaquick PCR Purification Kit, Qiagen and eluted in water. To eliminate remaining met hylated wild-type DNA, the sample was cut with Dpn1 (Invitrogen) leaving only unmethylated mutant DNA. The mutant mMC3R and hMC4R DNA was then transformed into competent DH5 E. coli cells, single colonies were selected and

PAGE 54

54 the presence of the desired mutation was ve rified by DNA sequencing. The plasmid containing the desired mutation was cut with the restric tion enzymes HindIII/XbaI and ligated into the HindIII/XbaI restricti on sites of the pCDNA3 expression vector (Invitrogen). Complete FlagmMC3R and FlaghMC4R sequen ces were confirmed free of P CR nucleotide base errors by DNA sequencing (University of Flor ida sequencing core facilities). Generation of stable cell lines Human Embryonic Kidney-293 (HEK-293) cells we re maintained as described previously ( 18, 114 ) in Dulbeccos modified Eagles medium (DMEM) with 10% newborn calf serum and plated 1 day prior to transfection at 1 x 106 cells/100-mm dish. Wild-type and mutant DNA in the pCDNA3 expression vector (20 g) were transfect ed using the calcium phosphate method ( 107 ). Stable receptor populations were generated usin g G418 selection (0.7-1 mg /mL) for subsequent bioassay analysis. Functional bioassay HEK-293 cells stably expressing wild type and mutant recepto rs were transfected with 4 g of cAMP response element (CRE)/ -galactosidase reporter gene as previously described ( 100, 114 ). Twenty-four hours after transfection, 5000-15000 cells were plated into collagen treated 96-well plates and incubated overnight. For Flag-mMC3R mutagenesis st udies, the cells were stimul ated 48 h after transfection with 100 L of peptide (10-6-10-12M) for -MSH, NDP-MSH, -MSH, -MSH and AMW3-130 and 100 L of peptide (10-5-10-11M) for JRH887-9 or forskolin (10-4M) control in assay medium (DMEM containing 0.1 mg/mL BSA and 0.1 mM is obutylmethylxanthine) for 6 h. For FlaghMC4R mutagenesis studies, the cells were stim ulated 48 h after transf ection with 100 L of peptide (10-6-10-12M) for, NDP-MSH, -MSH, -MSH and ACTH(1-24) and 100 L of peptide (10-5-10-11M) for -MSH and JRH887-9 or forskolin (10-4M) control in assay medium (DMEM

PAGE 55

55 containing 0.1 mg/mL BSA and 0.1 mM isobutylme thylxanthine) for 6 h. Subsequently, the assay media was aspirated, 50 L of lysis buffer (250 mM Tris -HCl, pH 8.0, and 0.1% Triton X100) was added and the plates were stored at -8 0C overnight. The plates containing the cell lysates were then thawed on the following day. Aliquots of 10 L were taken from each well and transferred to another 96 well plate for relative protein determination. The relative protein was determ ined by adding 200 L 1:5 dilution Bio-Rad G250 protein dye:water to the 10 L cell lysate sample and the OD595 was measured on a 96 well plate reader (Molecular Devices). To the cell lysate plates, 40 L of phosphate-buffered saline with 0.5% BSA was added to each well. Next, 150 L of substrate buffer [60 mM sodium phosphate, 1 mM MgCl2, 10 mM KCl, 5 mM -mercaptoethanol, 2 mg/mL of o-Nitrophenyl-Dgalactopyranoside (ONPG)] was adde d to each well and the plates were incubated at 37C. The sample absorbance, OD405, was measured using a 96-well plate reader (Molecular Devices). Data points were normalized both to th e relative protein content and non-receptor dependent forskolin values. Assays were performed using duplicate data points and repeated in at least three independent experiments. Data analysis, EC50, pA2 estimates, and their associated st andard errors of the mean ( 112 ), were determined by fitting the data to a nonlinear least-squares analysis ( 101 ) using the PRISM program (v4.0, GraphPad Inc.). The antagonist ic properties of hAGRP (87-132), SHU9119 nad the tetrapeptides BP7-26, BP7-27, BP7-147 and JR H887-12 were determined by the ability of each of those peptides to competitively displ ace the MTII agonist in a dose-dependent manner. The pA2 values were generated using the Schild analysis method ( 103 ). NDP-MSH iodination 125I-NDP-MSH was prepared using a modified Chloramine-T method as previously described ( 105 ). Using 50 mM sodium phosphate bu ffer (pH 7.4) as the reaction buffer, 125I-Na

PAGE 56

56 (0.5 mCi; Amersham Life Sciences, Inc., Piscat away, NJ, USA) was added to 40 g of NDPMSH (Bachem) in 10 L reaction buffer. To initia te the reaction, 20 L of a 2.4 mg/mL solution of Chloramine-T (Sigma Chemical Co., St Loui s, MO, USA) was added for 25-30 seconds with gentle agitation. This reaction was terminated by the addition of 100 L of a 4.8 mg/mL solution of sodium metabisulfite (Sigma Chemical Co.) for 20 sec with gentle agitation. The reaction mixture was then diluted with 200 L 10% BSA and the resulting mixture layered on a Bio-Gel P2 (Bio-Rad Labs) column (1.0 x 30 cm Econoc olumn, Bio-Rad Labs) for separation by size exclusion chromatography using 50 mM sodium phos phate buffer, pH 7.4 as column eluant. Fifteen drop fractions (approxi mately 500 L) were collected into glass tubes containing 500 L of 1% BSA. Each fraction was then counted on the Apex Automatic Gamma Counter (ICN Micromedic Systems Model 28023, with RIA AID software; Robert Maciel Associates, Inc.) to determine peak 125I incorporation fractions. A Bio-Gel P6 (Bio-Rad Labs) column (1.0x50 cm Econocolumn, Bio-Rad Labs) was used for the generation of I-125 labeled AGRP. Competitive displacement binding assays Human embryonic kidney-293 cells stably expres sing the wild-type and mutant receptors were maintained as described above. Tw o days preceding the experiment, (0.6.8) x 106 cells per well were plated onto collagen coated 12-well plates for Flag-hMC4R experiments or three days preceding the experiment, (0.6.8) x 106 cells per well were plated onto collagen coated 6well plates, respectively for Flag-mMC3R expe riments. The peptide NDP-MSH was used to competitively displace the 125I-radiolabeled NDP-MSH (150 000 cpm per well). AGRP was used to competitively displace the 125I-radiolabeled AGRP (300 000 cpm per well). Doseresponse curves (10-6 to 10-11 M) and IC50 values were generated and analyzed by the PRISM program (version 4.0, GraphPad Inc.). The percentage tota l specific binding was determined based upon the non-specific values obtained using 10-6 M NDP-MSH or AGRP for

PAGE 57

57 the respective radiolabeled peptide. Each expe riment was performed using duplicate data points and repeated in at least two i ndependent experiments. The sta ndard deviation errors and the standard errors of the mean were derived from the average percentage specific binding values from at least two independent experiments (SD) or at least three independent experiments (SEM) and using the PRISM program (v4.0, GraphPad Inc.). Flow cytometry Flow cytometry analysis of N-terminally Flag-tagged wild-type and mutant mMC3 receptors and hMC4 receptors was performed as described previously ( 18 ). An Allophycocyanin (APC)-conjugated anti-FLAG mono clonal antibody (Prozyme, San Leandro, CA) was used for both, the cell surface as well as intracellula r detection of the FlagmMC3 receptors. To detect the cell surface expression of w ild-type and mutant FlagmMC3 receptors and FlaghMC4 receptors, the cells were incuba ted 45 min with APC-conjugated anti-Flag monoclonal antibody at a concentration of 1 g/m illion cells. To detect the total (surface and intracellular) receptor expressi on cells were subsequently perm eabilized with saponin buffer and stained an additional hour with the APCconjugated anti-FLAG monoclonal antibody. Cells treated with Isotype control were used to set the background fluorescence staining for these analyses. BD Biosciences FACS Calibur flow cyto meters were used to co llect both stained cell percentages (surface and total) and mean fluores cence data were measured from a minimum of 10,000 collected events per sample and each experime nt was independently repeated three times. Transient transfection HEK-293 cells were maintained in DMEM with 10% newborn calf serum and plated 1 day prior to transfection at 2 x 106 cells/10-cm dish. The mutant and wild-type plasmid DNA was transfected at different increasing concentratio ns, as shown in Table 2-4, using the calcium phosphate method (50). Cells were then incuba ted overnight at 35C and 3% CO2, and the

PAGE 58

58 colorimetric reporter gene bioassa ys were performed as described above, with the exception that dose-response curves of compounds were not generated and only ba sal and forskolin values were measured ( 95, 114 ). Intracellular Expression of an Endogenous Agonist Plasmid construction The nucleotide sequence for -MSH was derived from human POMC (Gene Bank NM000939). The Kozak sequence, the methionine startcodon and HindIII/XbaI were added to the oligonucleotide primer seque nces. The sequence for the forw ard primer aMSH-F and the reverse primer aMSH-R was 5AGCTTACCATGtcctactccatggagcact tccgctggggcaagccggtgTGAT3 and 3ATGGTACaggatgaggtacctcgtgaaggcgaccccg ttcggccacACTAGATC5, respectively ( -MSH coding sequence in small letters). Both primers were ordered in gelpurifie d and phosphorylated form from Invitrogen. Annealing of the primers was done using equal amounts in 10X annealing buffer (100 mM Tris, 100 mM MgCl2, pH 7) at 95C for 10 min. After cooli ng down to room temperature, the insert was sub cloned into the pCDNA3 vector at the HindIII and XbaI restriction sites. Presence of the insert and correct sequence was verified by DNA sequencing (Universit y of Florida core facilities). Flow cytometry HEK-293 cells stably expressing the Wt and mu tant FlaghMC4 receptors maintained in DMEM with 10% newborn calf serum and 1% Pen/ Strep and seeded 1 day prior to transfection at 2 x 106 cells/10 cm dish. Wt and mutant FlaghM C4 receptors stably expressed in HEK293 cells, were transiently transfected 48 hrs prior Flow Cytometry with 5 g Met-MSH/pCDNA3 or pCDNA3 (control) using the calcium phosphate method (50). Ce lls were incubated overnight

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59 at 35C and 3%CO2. After 24 h the media was changed and cel ls were incubated for 24 h at 37C and 5%CO2. Flow Cytometry analysis of N-terminally Flag-tagged wild type and mutant hMC4 receptors was performed as described previously ( 18 ). An allophycocyanin (APC)-conjugated anti-FLAG monoclonal antibody (Prozyme, San L eandro, CA) was used for both, cell surface and intracellular detection of th e FlaghMC4 receptors. To detect cell surface expression of Wt and mutant FlaghMC4 receptors, cells were incubated 45 min with APC-conjugated anti-FLAG monoclonal antibody at a concentrat ion of 1g/million cells. To de tect the total (surface and intracellular) receptor expressi on cells were subsequently perm eabilized with saponin buffer and stained an additional hour with the APC-conjugated anti-FLAG monoclonal antibody. Cells treated with Isotype control (BD Bios ciences) were used to set the background fluorescence staining for these analyses. BD Bios ciences FACS Calibur flow cytometers were used to collect both stained cell percentages (sur face and total) and mean fluorescence data were measured from a minimum of 10,000 collected ev ents per sample and each experiment was independently repeated three times. Flow cytometry and using live cells For live cell flow cytometry, FlaghMC4R cells transiently transfected with 5 g MetMSH/pCDNA3 or pCDNA3 (control), respectively, were releas ed from the surface with by 5 min incubation at RT in cell dissociation buffer ( 0.02% EDTA in DPBS), centrifuged for 5 min at 600xg and the cell pellet was resuspend in cell me dia (DMEM with 10% ne wborn calf serum and 1% Pen/Strep) and transferre d into polypropylene tubes. Anti-Flag-APC for surface staining was added to the cells and polypropylene tubes were incubated 45 min at incubator at 37C and 5% CO2. Subsequently, 10nM DTAF-NDP-MSH was added and cells were incubated 37 C, 5% CO2 for different time points and fixed after different

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60 timepoints (0 min, 1 min, 2 min, 5 min, 8 min, 12 min) with 1ml of 4% Formaldehyde/tube (end concentration in tube 2%) and held 15 min at RT. Cells were transferred into FACS tubes ( 0.5ml/tube) and centrifuged for 5 min at 600 x g. One ml FACS Buffer was added for washing and centrifuge 5 mi n at 600 x g. This step was repeated for 3 times. Cells treated with Isotype control (BD Biosciences) were used to set the background fluorescence staining fo r these analyses. BD Biosci ences FACS Calibur flow cytometers were used to collect stained cell percentages and mean fluorescence data were measured from a minimum of 10,000 collected ev ents per sample and each experiment was independently repeated two times. Deconvolution microscopy Following flow cytometry the cell suspensions we re transferred to cytospin cuvettes and 100 L DAPI stain (Sigma) was added. Cassettes were cytospinned onto microscope slides washed and mounted with Biomeda Gel Mount (Fishe r). The slides were st ored in dark at 4C until analyzed on the deconvolution microscope (Delta Vision/Olympus System, Applied Presicion). Images were reconstr ucted using 20-30 optical slices of 0.1 to 0.2 micron thickness to generate 3-dimensional (3D) projecti ons of cells for further analysis.

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61 Figure 2-1. General strategy for Fmoc solid phase synthesis of peptides.

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62 Figure 2-2. Chemical structure of the Wang and Rink amide resins. X1 = CH2 or COCH2. Figure 2-3. Fmoc deprotection reaction.

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63 Figure 2-4. Generation of an OBt ester using HBTU.

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64 Figure 2-5. Ninhydrin test.

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65 Figure 2-6. Schematic representation of the PC R strategy used for epotipe tagging of the mouse MC3R with Flag.

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66 Figure 2-7. Verification of Flag-mMC3R cell ex pression. (left) Cell surface and total receptor expression of the Flag-mMC3R, stably e xpressed in HEK293 cells, determined by flow cytometry. Data Points were derived from the mean fluorescence intensity of 10000 cells. (right) Deconvolution microscope picture of the Flag-mMC3R expressed on the cell surface of HEK293 cells Blue=Nucleus, Red=FlagmMC3R -12 -11 -10 -9 -8 -7 -6 -5 0.00 0.25 0.50 0.75 1.00 1.25 NDP-MSH EC50(nM) 0.13 + 0.052 Log Peptide Conc (M)-Galactosidase Activity normalized to Protein & Forskolin Figure 2-8. NDP-MSH agonist cu rve at the FlagmMC3R stably expressed in HEK293cells. EC50 (nM) + S.D. was determined in two indepe ndent experiments done in duplicate. Surface Total 0 10 20 30 40 50 60 70 80 90 100Receptor Surface Expression Receptor Total Expression Wt FlagmMC3R% Cell Receptor Expression

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67 Figure 2-9. Potential pharmaco logical effects caused by intr oducing mutations into a GPCR. Figure 2-10. Representation of the -MSH primer design. Table 2-1. Properties of the flourescent dyes FITC and APC. Dye Laser Excitation Line (nm) Maximal Absorbance (nm) Maximal Emission (nm) FITC 488495520 APC 630650660FITC: Fluorescein isothiocyantat e; APC: Allophycocyanin. The va lues are taken from Ref ( 99 ).

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68 Figure 2-11. Schematic representation of the -Galactosidase/CRE reporter gene assay. Figure 2-12. Schematic representa tion of possible pharmacology curves.

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69 Table 2-2. Analytical data for pe ptides synthesized in this study. Peptide Structure HPLC k (system 1) HPLC k (system 2) M+1 (calculated) Mass spectral analysis (M+1) Purity (%) BP6-120 Ac-His-DPhe-Arg-Trp-NH2 4.16.7686.8686.5>96 BP6-121 Ac-His-DTyr-Arg-Trp-NH2Tyr 3.65.4702.8702.3>97 BP7-29 Ac-His-(pNH2)DPhe-Arg-Trp-NH2 2.15.0701.8701.5>97 BP6-124 Ac-His-(pCH3)DPhe-Arg-Trp-NH2 4.68.0700.8700.5>97 BP6-126 Ac-His-(pt-Bu)DPhe-Arg-Trp-NH2 6.110.4742.9742.5>96 BP-129 Ac-His-(pBz)DPhe-Arg-Trp-NH2 4.59.5776.9767.1>97 BP6-128 Ac-His-(pCN)DPhe-Arg-Trp-NH2 4.26.9711.8711.3>97 BP7-23 Ac-His-(pNO2)DPhe-Arg-Trp-NH2 4.87.3731.8731.5>98 BP7-25 Ac-His-(pF)DPhe-Arg-Trp-NH2 5.57.3704.8704.6>98 BP7-24 Ac-His-(pCl)DPhe-Arg-Trp-NH2 4.48.2721.2720.4>97 BP7-30 Ac-His-(pBr)DPhe-Arg-Trp-NH2 4.98.5765.7766.4>99 BP7-27 Ac-His-(pI)DPhe-Arg-Trp-NH2 5.18.9812.7812.5>98 BP7-26 Ac-His-(pCF3)DPhe-Arg-Trp-NH2 5.09.1754.3754.7>98 BP7-147 Ac-His-(3,4-diCl)DPhe-Arg-Trp-NH25.29.2755.6754.3>99 BP7-148 Ac-His-(m-Cl)DPhe-Arg-Trp-NH2 4.58.3721.2720.3>99 The HPLC k value equals [(peptide retention time solvent re tention time)/ solvent retention time]. Two different solvent systems were used. Solvent system 1 equals ( 10% acetonitrile in 0.1% trifluoroacetic acid/H2O and a gradient to 90% acetonitrile over 35 min). Solvent system 2 equals (1 0% methanol in 0.1% trifluoroacetic acid/ H2O and a gradient to 90% methanol over 35 min). The analytical Vydac C18 column (Vyd ac 218TP104) in combination with a flow rate of 1.5 ml/min was used for analytical characterizati on. The peptide purity was determined by HPLC at a wavelength of 214 nm.

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70 Table 2-3. Sequences of primers used for the generation of FlagmMC3 receptor mutants. Mutation Primer Sequences S52A Forward: 5' GGCTCTGGGCATCGTC GC TCTGATGGAAAACATC 3' Reverse: 5' GATGTTTTCCATCAGA GC GACGATGCCCAGAGCC 3' N56A Forward: 5' CGTCAGTCTGATGGAA GC CATCCTGGTGATCCTG 3' Reverse: 5' CAGGATCACCAGGATG GC TTCCATCAGACTGACG 3' N56S Forward: 5' GTCAGTCTGATGGAAA G CATCCTGGTGATCCTG 3' Reverse: 5' CAGGATCACCAGGATG C TTTCCATCAGACTGAC 3' N91A Forward: 5' CTGGTGAGCCTGTCC GC CTCCCTGGAGACCATC 3' Reverse: 5' GATGGTCTCCAGGGA GG CGGACAGGCTCACCAG 3 N91S Forward: 5' CTGGTGAGCCTGTCCA G CTCCCTGGAGACCATC 3' Reverse: 5' GATGGTCTCCAGGGAG C TGGACAGGCTCACCAG 3' E94A Forward: 5' GTCCAACTCCCTGG C GACCATCATGATCGC 3' Reverse: 5' GCGATCATGATGGTC G CCAGGGAGTTGGAC 3' E94S Forward: 5' CTGTCCAACTCCCTG TC GACCATCATGATCGCC 3' Reverse: 5' GGCGATCATGATGGTC GA CAGGGAGTTGGACAG 3' I96A Forward: 5' CAACTCCCTGGAGACC GC CATGATCGCCGTGATC 3' Reverse: 5' GATCACGGCGATCATG GC GGTCTCCAGGGAGTTG 3' I96S Forward: 5' CAACTCCCTGGAGACCA G CATGATCGCCGTGATC 3' Reverse: 5' GATCACGGCGATCATG C TGGTCTCCAGGGAGTTG 3' D117A Forward: 5' CAGTTTATCCAGCACATGG C TAATATCTTCGACTCTATG 3' Reverse: 5' CATAGAGTCGAAGATATTA G CCATGTGCTGGATAAACTG 3' D117S Forward: 5' CAGTTTATCCAGCACATG TC TAATATCTTCGACTCTATG 3' Reverse: 5' CATAGAGTCGAAGATATTA GA CATGTGCTGGATAAACTG 3' F120A Forward: 5' GCACATGGATAATATC GC CGACTCTATGATTTGC 3' Reverse: 5' GCAAATCATAGAGTCG GC GATATTATCCATGTGC 3' F120S Forward: 5' GCACATGGATAATATCT C CGACTCTATGATTTGC 3' Reverse: 5' GCAAATCATAGAGTCG G AGATATTATCCATGTGC 3' D121A Forward: 5' CATGGATAATATCTTCGCC T CTATGATTTGCATC 3' Reverse: 5' GATGCAAATCATAGAGGCG A AGATATTATCCATG 3' D121S Forward: 5' CATGGATAATATCTTCTCC TC TATGATTTGCATC 3' Reverse: 5' GATGCAAATCATAGAGGAG AA GATATTATCCATG 3' S122A Forward: 5' GGATAATATCTTCGACGCT A TGATTTGCATCTCC 3' Reverse: 5' GGAGATGCAAATCATAGC G TCGAAGATATTATCC 3' L128A Forward: 5' CTATGATTTGCATCTCC GC GGTGGCCTCCATCTGC 3' Reverse: 5' GCAGATGGAGGCCACC GC GGAGATGCAAATCATAG 3' L128S Forward: 5' CTATGATTTGCATCTCC TC GGTGGCCTCCATCTGC 3' Reverse: 5' GCAGATGGAGGCCACC GA GGAGATGCAAATCATAG 3' I132A Forward: 5' CTCCCTGGTGGCCTCCGCC TG CAACCTCCTGGCC 3' Reverse: 5' GGCCAGGAGGTTGCAGGCG GA GGCCACCAGGGAG 3' I132S Forward: 5' CTCCCTGGTGGCC T CCAGCTGCAACCTCCTGGCC 3' Reverse: 5' GGCCAGGAGGTTG C AGCTGGAGGCCACCAGGGAG 3' T145A Forward: 5' CATCGACAGGTACGTC G CCATCTTCTATGCCC 3' Reverse: 5' GGGCATAGAAGATGG C GACGTACCTGTCGATG 3' T145S Forward: 5' CATCGACAGGTACGTCA G CATCTTCTATGCCCTTC 3' Reverse: 5' GAAGGGCATAGAAGATG C TGACGTACCTGTCGATG 3'

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71 Table 2-3. continued Mutation Primer Sequences Y152A Forward: 5' CTTCTATGCCCTTCGG GC CCACAGCATCATGAC 3' Reverse: 5' GTCATGATGCTGTGG GC CCGAAGGGCATAGAAG 3' Y152S Forward: 5' CTATGCCCTTCGGT C CCACAGCATCATGAC 3' Reverse: 5' GTCATGATGCTGTGG G ACCGAAGGGCATAG 3' K160A Forward: 5' CATCATGACAGTGAGG GC AGCCCTCACCTTGATC 3' Reverse: 5' GATCAAGGTGAGGGCT GC CCTCACTGTCATGATG 3' K160S Forward: 5' CATCATGACAGTGAGGA GC GCCCTCACCTTGATCG 3' Reverse: 5' CGATCAAGGTGAGGGCG CT CCTCACTGTCATGATG 3' I165A Forward: 5' GAAAGCCCTCACCTTG GC CGGGGTCATCTGGGTC 3' Reverse: 5' GACCCAGATGACCCCG GC CAAGGTGAGGGCTTTC 3' I165S Forward: 5' GAAAGCCCTCACC T TGAGCGGGGTCATCTGGGTC 3' Reverse: 5' GACCCAGATGACCC C GCTCAAGGTGAGGGCTTTC 3' C175A Forward: 5' GTCTGCTGCGGCATC GC CGGCGTGATGTTCATC 3' Reverse: 5' GATGAACATCACGCCG GC GATGCCGCAGCAGAC 3' C175S Forward: 5' GTCTGCTGCGGCATC AG CGGCGTGATGTTCATC 3' Reverse: 5' GATGAACATCACGCCG CT GATGCCGCAGCAGAC 3' F179A Forward: 5' CATCTGCGGCGTGATG GC CATCATCTACTCCGAG 3' Reverse: 5' CTCGGAGTAGATGATG GC CATCACGCCGCAGATG 3' F179S Forward: 5' CATCTGCGGCGTGATGT C CATCATCTACTCCGAG 3' Reverse: 5' CTCGGAGTAGATGATG G ACATCACGCCGCAGATG 3' Y182A Forward: 5' CGTGATGTTCATCATC GC CTCCGAGAGCAAGATGG 3' Reverse: 5' CCATCTTGCTCTCGGAG GC GATGATGAACATCACG 3' Y182S Forward: 5' CGTGATGTTCATCATCT C CTCCGAGAGCAAGATG 3' Reverse: 5' CATCTTGCTCTCGGAG G AGATGATGAACATCACG 3' E184A Forward: 5' GTTCATCATCTACTCCG C GAGCAAGATGGTCATC 3' Reverse: 5' GATGACCATCTTGCTC G CGGAGTAGATGATGAAC 3' E184S Forward: 5' GTTCATCATCTACTCC TC GAGCAAGATGGTCATCG 3' Reverse: 5' CGATGACCATCTTGCTC GA GGAGTAGATGATGAAC 3' M195A Forward: 5' GTGTGTCTCATCACC GC GTTCTTCGCCATGGTG 3' Reverse: 5' CACCATGGCGAAGAAC GC GGTGATGAGACACAC 3' M195S Forward: 5' GTGTGTCTCATCACCA GC TTCTTCGCCATGGTG 3' Reverse: 5' CACCATGGCGAAGAAG CT GGTGATGAGACACAC 3' F196A Forward: 5' GTGTCTCATCACCATG GC CTTCGCCATGGTGCTC 3' Reverse: 5' GAGCACCATGGCGAAG GC CATGGTGATGAGACAC 3' F196S Forward: 5' GTCTCATCACCATGT C CTTCGCCATGGTGCTC 3' Reverse: 5' GAGCACCATGGCGAAG G ACATGGTGATGAGAC 3' F197A Forward: 5' GTCTCATCACCATGTTC GC CGCCATGGTGCTCCTC 3' Reverse: 5' GAGGAGCACCATGGCG GC GAACATGGTGATGAGAC 3' F197S Forward: 5' CTCATCACCATGTTCT C CGCCATGGTGCTCCTC 3' Reverse: 5' GAGGAGCACCATGGCG G AGAACATGGTGATGAG 3' M199A Forward: 5' CCATGTTCTTCGCC GC GGTGCTCCTCATGG 3' Reverse: 5' CCATGAGGAGCACC GC GGCGAAGAACATGG 3' M199S Forward: 5' CATGTTCTTCGCCA GC GTGCTCCTCATGGGC 3' Reverse: 5' GCCCATGAGGAGCAC GC TGGCGAAGAACATG 3'

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72 Table 2-3. continued Mutation Primer Sequences L247A Forward: 5' CTGTCACCATCACTATC GC GCTGGGTGTTTTCATC 3' Reverse: 5' GATGAAAACACCCAGC GC GATAGTGATGGTGACAG 3' L247S Forward: 5' CTGTCACCATCACTATC AGC CTGGGTGTTTTCATC 3' Reverse: 5' GATGAAAACACCCAGGCT GAT AGTGATGGTGACAG 3' W255A Forward: 5' GTGTTTTCATCTTCTGC GC GGCGCCTTTCTTCCTC 3' Reverse: 5' GAGGAAGAAAGGCGCC GC GCAGAAGATGAAAACAC 3' W255S Forward: 5' GTTTTCATCTTCTGCT C GGCGCCTTTCTTCCTC 3' Reverse: 5' GAGGAAGAAAGGCGCC G AGCAGAAGATGAAAAC 3' P257A Forward: 5' CATCTTCTGCTGGGCG G CTTTCTTCCTCCACC 3' Reverse: 5' GGTGGAGGAAGAAAG C CGCCCAGCAGAAGATG 3' P257S Forward: 5' CATCTTCTGCTGGGCG T CTTTCTTCCTCCACC 3' Reverse: 5' GGTGGAGGAAGAAAG A CGCCCAGCAGAAGATG 3' F258A Forward: 5' CTTCTGCTGGGCGCCT GC CTTCCTCCACCTGGTC 3' Reverse: 5' GACCAGGTGGAGGAAG GC AGGCGCCCAGCAGAAG 3' F258S Forward: 5' CTTCTGCTGGGCGCCTT CT TTCCTCCACCTGGTCC 3' Reverse: 5' GGACCAGGTGGAGGAA AG AAGGCGCCCAGCAGAAG 3' F259A Forward: 5' GCTGGGCGCCTTTC GCC CTCCACCTGGTCCTC 3' Reverse: 5' GAGGACCAGGTGGAG GGC GAAAGGCGCCCAGC 3' F259S Forward: 5' GCTGGGCGCCTTTCT CT CTCCACCTGGTCCTC 3' Reverse: 5' GAGGACCAGGTGGAG AG AGAAAGGCGCCCAGC 3' H261A Forward: 5' GCGCCTTTCTTCCTC GC CCTGGTCCTCATCATC 3' Reverse: 5' GATGATGAGGACCAGG GC GAGGAAGAAAGGCGC 3' H261S Forward: 5' GCGCCTTTCTTCCTC AG CCTGGTCCTCATCATC 3' Reverse: 5' GATGATGAGGACCAGG CT GAGGAAGAAAGGCGC 3' L264A Forward: 5' CTTCCTCCACCTGGTC GC CATCATCACCTGCCC 3' Reverse: 5' GGGCAGGTGATGATG GC GACCAGGTGGAGGAAG 3' L264S Forward: 5' CTTCCTCCACCTGGTC AG CATCATCACCTGCCC 3' Reverse: 5' GGGCAGGTGATGATG CT GACCAGGTGGAGGAAG 3' I265A Forward: 5' CTCCACCTGGTCCTC GC CATCACCTGCCCCAC 3' Reverse: 5' GTGGGGCAGGTGATG GC GAGGACCAGGTGGAG 3' I265S Forward: 5' CCACCTGGTCCTCA G CATCACCTGCCCCAC 3' Reverse: 5' GTGGGGCAGGTGATG C TGAGGACCAGGTGG 3' T267A Forward: 5' CTGGTCCTCATCATC G CCTGCCCCACCAATC 3' Reverse: 5' GATTGGTGGGGCAGG C GATGATGAGGACCAG 3' T267S Forward: 5' GGTCCTCATCATCA G CTGCCCCACCAATC 3' Reverse: 5' GATTGGTGGGGCAG C TGATGATGAGGACC 3' C268A Forward: 5' GTCCTCATCATCACC GC CCCCACCAATCCCTAC 3' Reverse: 5' GTAGGGATTGGTGGG GG CGGTGATGATGAGGAC 3' C268S Forward: 5' GTCCTCATCATCACC A GCCCCACCAATCCC 3' Reverse: 5' GGGATTGGTGGGGC T GGTGATGATGAGGAC 3'

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73 Table 2-3. continued Mutation Primer Sequences T270A Forward: 5' CATCATCACCTGCCCC G CCAATCCCTACTGCATC 3' Reverse: 5' GATGCAGTAGGGATTGG C GGGGCAGGTGATGATG 3' T270S Forward: 5' CATCATCACCTGCCCC T CCAATCCCTACTGCATC 3' Reverse: 5' GATGCAGTAGGGATTG G AGGGGCAGGTGATGATG 3' T278A Forward: 5' CTACTGCATCTGCTAC G CGGCCCATTTCAACAC 3' Reverse: 5' GTGTTGAAATGGGCCG C GTAGCAGATGCAGTAG 3' T278S Forward: 5' CTACTGCATCTGCTAC T CGGCCCATTTCAACAC 3' Reverse: 5' GTGTTGAAATGGGCCG A GTAGCAGATGCAGTAG 3' F281A Forward: 5' CTGCTACACGGCCCAT GC CAACACCTACCTGGTTC 3' Reverse: 5' GAACCAGGTAGGTGTTG GC ATGGGCCGTGTAGCAG 3' F281S Forward: 5' CTGCTACACGGCCCAT AG CAACACCTACCTGGTTC 3' Reverse: 5' GAACCAGGTAGGTGTTG CT ATGGGCCGTGTAGCAG 3' N282A Forward: 5' CTACACGGCCCATTTC GC CACCTACCTGGTTCTC 3' Reverse: 5' GAGAACCAGGTAGGTG GC GAAATGGGCCGTGTAG 3' N282S Forward: 5' CACGGCCCATTTCA G CACCTACCTGGTTC 3' Reverse: 5' GAACCAGGTAGGTG C TGAAATGGGCCGTG 3' D295A Forward: 5' CAACTCCGTCATCG C CCCCCTCATCTAC 3' Reverse: 5' GTAGATGAGGGGG G CGATGACGGAGTTG 3' D295S Forward: 5' GCAACTCCGTCATC AG CCCCCTCATCTACGC 3' Reverse: 5' GCGTAGATGAGGGGG CT GATGACGGAGTTGC 3' R302A Forward: 5' CTCATCTACGCCTTC GC CAGCCTGGAGCTGC 3' Reverse: 5' GCAGCTCCAGGCTG GC GAAGGCGTAGATGAG 3' R302S Forward: 5' CTCATCTACGCCTTC A GCAGCCTGGAGCTG 3' Reverse: 5' CAGCTCCAGGCTGC T GAAGGCGTAGATGAG 3' The nucleotides differing from the wild-type r eceptor sequence are highlighted in red. Table 2-4. Sequences of primers used for the generation of the FlaghMC4 receptor mutants. Mutation Primer Sequences L250A Forward: 5' CGATTACCTTGACCATC GC GATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAATC GC GATGGTCAAGGTAATCG 3' L250E F Forward: 5' CGATTACCTTGACCATC GA GATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAATC TC GATGGTCAAGGTAATCG 3' L250F Forward: 5' CGATTACCTTGACCATC T T T ATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAAT A A A GATGGTCAAGGTAATCG 3' L250K Forward: 5' CGATTACCTTGACCATC AA GATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAATC TT GATGGTCAAGGTAATCG 3' L250N Forward: 5' CGATTACCTTGACCATC AAC ATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAAT GTT GATGGTCAAGGTAATCG 3' L250Q Forward: 5' CGATTACCTTGACCATCC A GATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAATC T GGATGGTCAAGGTAATCG 3' L250R Forward: 5' CGATTACCTTGACCATCC G GATTGGCGTCTTTGTTG 3' Reverse: 5' CAACAAAGACGCCAATC C GGATGGTCAAGGTAATCG 3' The nucleotides differing from the wild-type r eceptor sequence are highlighted in red.

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74 Table 2-5. Concentration of DNA used in the transient transfection assay. concentration plotted (DNA transfected) 0 ng5 ng25 ng50 ng 75 ng 100 ng mutant/wild-type 0 ng5 ng25 ng50 ng 75 ng 100 ng pCDNA3 plasmid 100 ng95 ng75 ng50 ng 25 ng 0 ng CRE/ -gal 4 g4 g4 g4 g4 g 4 g Flag-mMC3R and Flag-hMC4R were used as w ild-type DNA in the respective experiments. Table 2-6. Analytical data for pe ptides synthesized in this study. Peptide Structure HPLC k (system 1) HPLC k (system 2) M+1 (calculated) Mass spectral analysis (M+1) Purity (%) BP2-38 MSYSMEHFRWGKPV-NH24.79.517541754>98 BP2-67 MSYSMEHFRWGKPV-OH 5.39.117551756>98 The HPLC k value equals [(peptide retention time solvent retention time)/ solvent retention time]. Two different solvent systems were used. So lvent system 1 equals (10% acetonitrile in 0.1% trifluoroacetic acid/H2O and a gradient to 90% acetonitrile over 35 min). Solvent system 2 equals (10% methanol in 0.1% trifluoroacetic acid/ H2O and a gradient to 90% methanol over 35 min). The analytical Vydac C18 column (Vydac 218TP104) in combination with a flow rate of 1.5 ml/min was used for analytical characterization. The peptide purity was determined by HPLC at a wavelength of 214 nm.

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75 CHAPTER 3 STRUCTURE-ACTIVITY RELATIONSHIP ST UDIES OF TETRAPEPTIDE LIGANDS AT THE MOUSE MELANOCORTIN RECEPTORS All peptides were designed, synthesized, purif ied and analytically characterized by Bettina Proneth under the supervision of Dr. Carrie Haskel l-Luevano. The functional assays at the mouse melanocortin receptors were carried out by Federico Portillo, technician of the Haskell-Luevano laboratory. Data analysis was perfor med by Dr. Carrie Haskell-Luevano. Introduction The centrally located Melanocortin-3 and -4 receptors (MC3R and MC4R) ( 4-7 ) have been identified in knockout mice to be involved in energy homeostasis, obesity and metabolism ( 25, 26, 31 ) and are potential drug target s for the treatment of obesity and related diseases. While MC4R knockout mice are extremely obese and hyperphagic, MC3R knockout mice possess normal body weight with increased fa t mass and decreased lean body mass ( 25, 26, 31 ). Extensive studies have been car ried out to understand the physio logical role of the MC4R but little is known about the MC3R. To date, only a few ligands have been identified that are 100to 200-fold selective for the MC 3R over the MC4R subtype ( 115-117 ). The identification of ligands that are selective for th e MC3R, we hereby define selectivity as > 500 fold difference in potency between the receptor subtypes, can be used as in vivo tools to explore the vast array of physiological functions of this receptor subtype and ultimately aid in the discovery of new antiobesity agents. The design of ligands with a specific activity and the desired pharmacological profile is a complex task. One approach to accomplish this obj ective is the use of structure-activity studies (SAR) that utilize systematic m odifications of a lead peptide a nd are designed to provide insight into potential interactions involved in the form ation of ligand-receptor complexes. Once a lead peptide or pharmacophore is identified the next st ep is to explore ligand side chain requirements.

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76 The study presented herein investigates the ro le of side-chain functional groups in ligandreceptor interactions. Replacement of hydrogen atom s by substituents can modify the solubility, bioavailability, pharmacokinetics electronic density, conformation and the degree of direct interactions between the subs tituted ligand and the receptor of interest (Figure 3-1) ( 118 ). SAR studies which utilize substituent effects are commonly used in medicinal chemistry and aromatic rings are popular targets for substitution ( 118 ). Table 3-1 illustrates the theoretical descriptors for substituents used in this study and Figure 3-2 represents these properties graphically ( 119 ). The Hanschs constant is a measure of the cont ribution of lipophilicity, the Hammets constant accounts for the electronic effects and the molecular refracti on (MR) reflects the volume of the substituent ( 118 ). In the series of para-substituen ts (Figure 3-3) the methyl-, t-butyl and benzyl groups are chosen as electron donating groups and hydrophobic groups. Alkyl groups are the only substituents which are solely ex erting inductive electron-donating effects. Both, the t-butyl and the aromatic benzyl-group are very voluminous and may cause steric hindrance in ligandreceptor interactions, when used as substituent. The hydrophilic hydroxyland amino groups are electron donating groups in neutral media a nd may serve as hydrogen bond donors in ligandreceptor complexes. The polar cyanoand nitrogroups are electron withdrawing when used as para substituents. The electronic effects of halogens are due to their inductive electron withdrawing properties. These are maximal for chlorine and br omine, less for iodine and weak for fluorine. Both, lipophilicity and size of th e substituents increase in the se ries of halogens, when going from fluorine, chlorine, bromine to iodine. Halo gens are also known for their ability to exert charge-transfer interactions, which are almost non existent for fluorine and increase in the order

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77 Chlorine
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78 distinct potency and/or functional activity at the receptor subtypes. The MC2R was not included since it can be only stimulated by ACTH ( 3 ). Ac-His-DPhe-Arg-Trp-NH2 (BP6-120), Ac-HisDTyr-Arg-Trp-NH2 (BP6-121) and Ac-His -(pI)DPhe-Arg-Trp-NH2 (BP7-27), have been previously reported and have been included in this study as control compounds to complement SAR trends ( 108, 121 ). Ac-His-DNal2-Arg-Trp-NH2 (JRH887-12) has been previously reported ( 108 ) and was integrated herein due to the put ative involvement of the bulky DNal2 group of melanocortin based peptides into antagonist act ivity at the brain MC3 and MC4 receptors ( 52 ). BP-147 was designed to mimic the para and meta DPhe substitution of the DNal2 group and BP7-148 was included to investigate single su bstitution at the meta position. The unsubstituted tetrapeptide BP6-120 possessed 112, 126, 2.28 and 2.26 nM agonist activity at the mMC1R and mMC3-5R, respectively. Substitution of the DPhe phenyl ring in para position with the hydr ophilic OH (BP6-121) led to decreased agonist potencies of 23-fold at the mMC1R and 99-, 171-, and 323-fold at the MC3-5 receptors, respectively, as co mpared to BP6-120. Similarly, the (pNH2-DPhe) containing tetrapeptide BP7-29 possessed de creased agonist potencies of 21-fold at the mMC1R and 130-, 96-, and 100-fold at the MC3-5 receptors, respectively, compared to the unsubstituted tetrapeptide BP6-120. The electron donati ng and lipophilic (pCH3-DPhe) containing peptide BP6-124 maintained equipotency at the mouse melanocortin receptors, within the experimental error. Tetrapeptide BP6-126 containing the bulky tBugroup at the DPhe phenyl para position possessed equipotent EC50 values at the mMC1R, 1912and 150fold reduced potency at the mMC4 and mMC5 receptors, respectively, compared to BP6-120, but was completely devoid of stimulatory or antagonist activity at up to 100M concentration at the mMC3R. In contrast, BP6-129 (pBz-

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79 DPhe) which might be similar in bulk but cause less steric hindrance than BP6-126 due to free rotation around the methylene group was equi potent to BP6-120 at the mMC1R, almost equipotent at the mMC3R and 28and 23-fold le ss potent at the MC4R a nd MC5R, respectively, compared to the DPhe containing peptide BP6-120. The polar (pCN-DPhe) contai ning tetrapeptide (BP6-128) possessed equipotency at the mMC1R, mMC3R, mMC4R and mMC5R within the inherent 3-fold experimental error, as compared to BP6-120. BP7-23, which contains a n itro group at the DPhe para position, resulted in slight, but nearly equal 5-9 fold decreased potency as compared with BP6-120 at the mMC1-5 receptors. Substitution with the halogens -Cl and -F at the DPhe para position led to equipotency at all melanocortin receptors, compared to BP6-120 and within experimental errors. The (pBrDPhe) and the previously reported (pI-DPhe) cont aining tetrapeptides retained almost equipotent agonist activity at the mMC1, mMC4 and mMC5 receptors, within the experimental error compared with BP6-120. Interestingly, at the mMC3R these tetrapeptides were converted to potent antagonists (pA2=7.54 and 7.41, respectively) with part ial agonist activ ity (Table 3-2, Figure 3-5). BP7-26, containing the -CF3 group at the DPhe para posi tion resulted in a similar pharmacological profile with almost equipot ency at the MC1, MC4 and MC5 receptors compared to BP-120 and partial agonist/antagonis t activity (pA2=6.79) at the brain mMC3R (Table 3-2, Figure 3-6). In JRH887-12, DPhe, is replaced by the bul ky and lipophilic DNal2 group. This peptide resulted in full agonist activity at the periphera l MC1 and MC5 receptors with equipotency at the MC1R and 10-fold decreased potency at th e MC5R, compared to the DPhe containing tetrapeptide BP6-120, but resulted in partial agonist/antagonist activity at the MC3R (pA2=6.70)

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80 and antagonist activity at the MC4R (pA2=8.28) (Table 3-2, Figure 3-7). Interestingly, substitution of the DPhe phenyl ring with Cl in position 3 and 4 at the same time led to equipotent agonist acitivity at the MC1, MC4 and MC5 receptors, compared to the unsubstituted BP6-120, and partial agonist/antagon ist activity at the MC3R (pA2=7.41) (Table 3-2, Figure 37). The (mCl-DPhe) containing tetrapeptide poss essed full agonist activity at all melanocortin receptors, and was equipotent at the MC1 and MC 4 receptors and 11to 13fold less potent at the MC3 and MC5 receptors, respectively, as co mpared to the DPhe containing tetrapeptide BP6-120 (Table 3-2, Figure 3-7). Discussion The study reported herein extends previous repo rted structure-activit y relationship studies at the tetrapeptide template Ac-His-DPhe-Arg-Trp-NH2, where DPhe was substituted by a series of natural and unnatural aromatic amino acids. These studies id entified the compound Ac-His(pIDPhe)-Arg-Trp-NH2 which resulted in mixed pharmacology and possessed partial agonist/antagonist activity at the brain MC3R while retaining full nM agonist activity at the brain MC4R as well as the peripherally expressed MC1 and MC5 receptors ( 108 ). Both centrally expressed MC3 and MC4 receptors have been iden tified to participate in energy homeostasis and obesity. While the physiological functions of the MC4R are more obvious and extensively studied, little is known about the MC3R. To our knowledge, there is currently no compound available that is selective (> 500 fold) for the MC 3R subtype. The fact that para substitution with iodine at the DPhe phenyl ring l eads to antagonist activity at the MC3R while agonist activity at the MC4R is maintained, clearly indicates that this position can be exploited to examine ligand side-chain determinants which are important fo r receptor selectivity of MC3 versus the MC4R and the differentiation of agonist vers us antagonist activit y at the MC3R.

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81 For this purpose a series of functional groups was chosen for the substitution of the DPhe phenyl ring in para/meta position and the resu lting tetrapeptides were pharmacologically characterized at the cloned mouse melanocortin receptors, with the exception of MC2R which is only stimulated by ACTH. Substitution with Hydrophilic Groups Representatives for polar substituents are OH, NH2, CN and NO2. However, apart from their hydrophilic nature, these groups have little in common (Table 3-1 and Figure 3-2). The OH and -NH2 groups donate electrons to the phenyl ri ng in neutral medium and can serve as H-bond donors while CN and NO2 are electron attractors and have the potential of serving as H-bond acceptors during formation of the liga nd-receptor complexes. These differences are clearly reflected in the distinct pharmacological profiles of these tetrapeptide analogs at the melanocortin receptors (Table 3-2). (pOH-DPhe) BP6-121 and (pNH2-DPhe) BP7-29 containing analogs both lead to large potency drops at the mMC1 and MC3-5 receptors. Interestingly, the loss in potency at the mMC1R is less pronounced. Th is is in agreement with previously reported results where the tetrapeptide Ac-His-DPhe-Arg-Trp-NH2 was systematically modified with various natural and unnatural aromatic amino acids at the DPhe position ( 108 ). All of the tetrapeptide analogs tested were well tolera ted by the mMC1R and resulted in full agonist activity, unlike at the other melanocortin receptors ( 108 ). By using the pentapeptide template BuHis-DPhe-Arg-Trp-NH2 it was shown that the loss of potency of the DTyr containing molecule could be completely reversed by capping the OH group with CH3 or C2H5 ( 122 ). Receptor mutagenesis of the MC1, MC3 and MC4 recepto rs and GPCR homology modeling of the MC1 and MC4 receptors identified a putative hydrophobic receptor pocke t consisting of aromatic and hydrophobic amino acid residues that are proposed to interact with the Phe residue of melanocortin peptide ligands ( 85, 114, 123-130 ). The decrease in agoni st potency observed with

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82 the DTyr (BP6-121) and pNH2-DPhe (BP6-120) containing tetrapep tides, as well as the recovery of potency by using methoxy and ethoxy substituents is consistent with the receptor mutagenesis and modeling studies, suggesting that the ligand DPhe residue is inter acting with a hydrophobic network of receptor amino acids. It is therefor e interesting to see that substitution with the hydrophilic -CN (BP6-128) and -NO2 (BP7-23) groups in the tetrap eptide template resulted in equipotency and only slight decreas es in agonist potencies (5-9 fo ld), respectively, at the mMC1 and mMC3-5 receptors. One reason c ould be that both, the OH and NH2 groups are H-bond donors and may be disrupting the receptor hydroge n bond network necessary for its structural integrity and the conformational changes accomp anying receptor activat ion, whereas the CN and NO2 do not contain hydrogens. Another possible reason might be that electron rich aromatic rings of OH and NH2 are less favorable for arom atic-aromatic or hydrophobic interactions with the receptor residues, whereas the electron poor aromatic rings of the CN and NO2 substituted compounds might be more favorable during formation of ligand-receptor complexes. These two mechanisms remain speculati ve and the extent by wh ich they contribute to the observed pharmacological profile remains to be identified. Substitution with Hydrophobic A liphatic and Aromatic Groups In traditional SAR studies a methyl group is always included as representative of an electron donating group substituent, however this effect is less pronounced then with OH and NH2 groups ( 118 ). The hydrophobic (pCH3-DPhe) containing peptide BP6-124, retained equipotency and full agonist activ ity at all melanocortin receptors, compared to BP6-120 (Table 3-2). This observation is consistent with the existence of a putative hydrophobic receptor binding pocket, as described above. The tBu-DPhe containing peptide BP6-126 wa s included, mainly because of the bulkiness of this substituent. Surprisingl y, this tetrapeptide retained equipotency at the mMC1R, as

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83 compared to BP6-120, while it resulted in 1912-fold and 150-fold reduced potency at the mMC4 and MC5 receptors and it was unable to stimul ate the MC3R. This data strongly suggests high conformational freedom at the DPhe para posi tion of the mMC1R. Whereas the mMC3R DPhe binding pocket might be more constrained, th erefore tolerating less bulk than the other melanocortin receptors. BP6-129 which contains the -pBz group resulted in equipotency at the mMC1R, slightly reduced potency at the mMC3R an d a 28and 23-fold loss in pot ency at the mMC4 and mMC5 receptors. The smaller changes in agonist activity compared to BP6-126 are probably due to the methylene linker which allows more rotational fr eedom of the benzyl ring and renders it less rigid as compared to tBu substitution. JRH887-12 was published previously and cont ains the bulky and lipophilic DNal2 group in place of DPhe ( 108 ). Consistent with previous reports this peptide resulted in a partial agonist/antagonist at the MC3R, an antagonist at the MC4R and retained full agonist activity at the MC1 and 5 receptors. Earlier DNal(2) substitu tion in the MTII cyclic peptide template (AcNle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH2) resulted in the MC3R partial agonist/antagonist and MC4R antagonists SHU9119 ( 52 ). These original studies led to the hypothesis that bulky aromatic amino acid substitutions at the DPhe se ven position are responsible for differentiating agonist versus antagonist activi ties of melanocortin ligands at the brain MC3 and MC4 receptors ( 52 ). Substitution with Halogens Substitution of DPhe with (pI)DPhe in the MT II template resulted in the potent hMC3R and hMC4R partial agonist/an tagonist SHU8914 (Ac-Nle-c[AspHis-(pI)DPhe-Arg-Trp-Lys]NH2) ( 52 ). Interestingly, SHU8914 possessed full a gonist activity at the hMC1R and partial agonist activity at the MC5R ( 52 ). Modification of the phenyl ri ng at the para position with

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84 either fluorine or chlorine retained agonist ac tivity at all cloned human melanocortin receptors ( 52 ). Further (pI)DPhe substitution of the DPhe7 amino acid in the linear peptide NDP-MSH resulted in SHU9005 (Ac-Ser-T yr-Ser-Nle-Glu-His-(pI)DPhe-A rg-Trp-Gly-Lys-Pro-Val-NH2) with partial agonist and antagonist activity at the mMC3R and mMC4R ( 114 ). Substitution of the DPhe residue in the linear pentapeptide template Bu-His-DPhe-Arg-Trp-Gly-NH2 with (4Cl)DPhe and (4-Br)DPhe possessed similar poten cies at the hMC1 and hMC4 receptors compared to the unsubstituted lead pentapeptide ( 122 ). The substituted tetrapeptide Ac-His(pI)DPhe-Arg-Trp-NH2 BP7-27 resulted in a new pharmacol ogical profile and possessed partial agonist/antagonist activity only at the brain MC3R while it retained full nM agonist activity at the brain MC4R as well as the peripher ally expressed MC1 and MC5 receptors ( 108 ). The difference of the tetrapeptide in pharmaco logy compared to SHU8914 and SHU9005 was attributed to the additional amino acids at th e N and C terminus that may be modifying the secondary structure maybe in combination wi th the topography of how these ligands might interact with the MC4 recep tor putative binding pocket ( 108 ). To investigate the molecular mechanism by which substitution of DPhe with iodine in para position results into antagonist act ivity at the MC3R a series of peptides was synthesized containing F (BP7-25), -Cl (BP7-24), -Br (BP7-30) and -I (BP7-27). In th e series of halogens, substitution with either fluorine or chlorine resulted in full and equipotent agonist activity at the MC1, MC4 and MC5 receptors, compared to the unsubstituted BP6-120. Interestingly, upon substitution with Bromine and Iodine, the tetr apeptide analogs were converted into partial agonists/antagonists only at the mMC3R, while re taining similar agonist potencies as compared to BP6-120 at the other melanocortin recepto r subtypes (Figure 3-5). Both, the Bromine containing peptide BP7-30 and the Iodine cont aining peptide BP7-27 show partial agonist,

PAGE 85

85 activity besides competitive antagonism. When co mparing the efficacy of the partial agonists, BP7-30 reaches 77% maximal response, whereas BP7-27 only reaches only 55% maximal response (Figure 3-8). By comparing the para subs tituent effects of chlorine, bromine and iodine, all three have similar electronic properties (T able 3-1 and Figure 32). However, size and lipophilicity increase in the series of chlorine, bromine and iodine, and these parameters might be important considering the fact th at the DPhe phenyl ring putatively interacts with a network of aromatic and hydrophobic receptor amino acids in the melanocortin receptor binding pocket. Aromatic-aromatic interactions in ligand-receptor complexes have been suggested to occur between the side chains of aromatic amino acids of the receptor and the aromatic moieties like DPhe of the ligand ( 131 ). Aromatic interactions may consist of Van der Waals, hydrophobic and electrostatic forces, although the relative contribution of each of those components is still under investigation ( 132 ). Hereby, several geometries are possi ble, edge to face, which can be considered as CHinteraction and stacking, as illustrated in Figure 3-9 ( 131 ). Substituents could potentially interfere with both types of aromatic-aromatic in teractions. Invest igation of the effect of halogenation on edge-face aromatic in teractions of two phenylalanine residues in a model beta hairpin peptide, where one phenyl ring was substituted showed that the stability of the fraction folded of the substitu ted beta hairpin peptides increas ed from Fl, -Cl, -Br to I, which was attributed to the in crease in size and lipophilicity ( 133 ). Another report investigating aromatic stacking, suggested that electrostati c interactions between substituents on one ring and the -face of the other ring might cau se additional contributions ( 134 ). It is well known that halogens may also form charge transfer comple xes where they serve as acceptors and interact with a donor by transferring electronic charge ( 120 ). These interactions can occur in biological systems between halogens and the -electron clouds of benzen e rings as well as between

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86 halogens and the lone pair of oxygen, n itrogen and sulfur and the delocalized -electrons of peptide bonds or carboxy and amide groups ( 120 ). The magnitude of this interaction increases from Cl < Br < I, whereas fl uorine is usually not involved ( 120 ). The trifluoromethyl group CF3 (BP7-26) is comparable in size with chlorine and comparable in lipophilicity with bromine. Th is group is commonly used in SAR studies to replace chlorine ( 118 ). The (pCF3-DPhe) containing tetrapepti de showed a pharmacological profile comparable to -Br and I and resulted in a partial agonist/antagonist activity at the mMC3R (Figure 3-6), while retaining full agoni st activities at the remaining melanocortin receptor subtypes. It is quite astonishing, that lit tle differences, which might be attributed to the increase in lipophilicity and el ectron attracting effects of CF3 compared to Cl lead to a complete conversion of agonist to antagonist activity at one receptor subtype. Since CF3 is even smaller in size then Cl, steric effects do not seem to be important for these two compounds. Fluorine might not take part in charge transfer complexes ( 120 ) and the antagonistic effect of the (pCF3-DPhe) containing tetrapeptide mi ght be attributed to the low electron density of the phenyl ring/and or the high lipophilicity of -CF3. The CF3 group is also similar in size but more lipophilic then the CH3 group (BP6-124) which resulted in full agonist activity. Fluorin ated compounds are employed in drug design to increase drug activity which may be due to th e enhanced hydrophobic char acter of fluorinated substituents ( 135 ). Because of the fact that hydrophobic fo rces play important roles in protein folding and protein-protein rec ognition, fluorinated amino acids ar e being utilized to prepare peptides and proteins with e nhanced structural stability ( 135, 136 ). Natural peptides and proteins are generally considered as poor drug candida tes because they usually do not easily cross membranes and susceptible to degradation by proteases ( 137 ). Fluorination was suggested as

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87 way to increases the bioavailability of drugs, mainly through increased lipophilicity that may help in crossing cell membrane ( 137 ). Fluorinated peptides may al so be resistant to proteases rendering them more amenable for in vivo application ( 137 ). If the antagonist activity of BP7-26 is based on its high lipophilicit y, the electron poor phenyl ring or a combination of both remain to be identified. A slightly different molecula r mechanism for mMC3R antagonism compared to the Bromine and Iodine containing tetrapeptides may be indicated by the lower antagonist potency of BP7-26 (Table 3-2). In BP7-147 the DPhe phenyl ring was substitute d simultaneously at meta and para position with -Cl. This compound was designed to investig ate (i) if a second chlori ne substituent, which might enhance also the lipophilicity of the mol ecule and/or decrease electron density of the phenyl ring, is needed to confer antagonist activity at the mMC3R and (ii) to mimic the (DNal2) substitution of JRH887-12, which is an anta gonist at both MC3 and MC4 receptors ( 108 ). Surprisingly, BP7-147 resulted in partial agonist /antagonist activity only at the MC3R subtype, while retaining full and equipotent agonist activ ity at all other melanocortin receptor subtypes (Table 3-2 and Figure 3-7). To examine if this antagonist activity at the MC3R is due to substitution or a steric effect at the phenyl meta position, we synthesized the (mCl-DPhe) containing tetrapeptide BP7-148. This peptide ho wever, resulted in a full agonist at all melanocortin receptors, with slight decreased pot ency at the MC3 and 5 receptors and almost equipotency at the MC1 and MC 4 receptors. It can be postu lated therefore that double substitution of the phenyl ring is necessary for an tagonist activity at th e MC3R of a chlorine containing tetrapeptide. A previous study reported dichloro substitution of the DPhe phenyl ring, using the pentapeptide template Bu-His-DPhe-Arg-Trp-NH2 ( 122 ). This compound showed enhanced

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88 binding affinity at the hMC4R, but was devoid of any stimulatory activity at up to 50 M concentration. A functional antagonist assay was not carried out, but give n its proposed mimicry of DNal2 one can speculate that it w ould be an antagonist at the hMC4R ( 122 ). The difference of the dichloro containing tetrapeptide in pha rmacology compared to the pentapeptide could be attributed to the additional butanoyl group at the N-terminus that may be modifying the secondary structure maybe in combination wi th the topography, similar to SHU9005, of how these ligands interact with the MC4 receptor ( 108, 114 ). Additionally, capping of Ac-His(pIDPhe)-Arg-Trp-NH2 with an N-terminal octan oyl group (compound AT395-27, unpublished material of the Haskell-Luevano laboratory) re sulted in antagonist activ ity at both mouse MC3 and MC4 receptors, providing further experimental evidence of the importance of N-terminal extension for MC4R antagonist act ivity. In regards to the molecu lar mechanism of how the BP7147 ligand interacts with the MC3R it can be hy pothesized that halogen charge-transfer and aromatic-aromatic interactions with MC3R recep tor residues might be the main reason. Steric effects of dichloro substitution which might mimic DNal2 did not result into MC4R antagonist activity, however might contribute to antagonist activity at the MC3R, since meta substitution led to 10-fold decrea sed agonist potency. Results from these studies are quite intrigui ng in that a subtle difference in the DPhe phenyl ring para position confer s antagonism only at one recept or subtype while exerting full agonism at the other receptor subtypes. The current hypothesis states that large steric modifications and bulky aromatic amino acid su bstitutions, like in the case of SHU9119 and SHU9005 in the His-Phe-Arg-Trp message sequen ce are necessary for antagonism at the MC3 and MC4 receptors ( 52 ). However the requirements for antag onism at the MC3R as shown in the study presented herein are more subtle, and co mplex to explain. Three hypotheses have been

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89 reported that address the differential pharmacology of the DPhe containing agonist MTII and the DNal2 containing antagonist SHU9119. In one hypothesis steric hindrance caused by the DNal2 group was used as rationale. It was suggested from molecular modeling and mu tagenesis studies of the hMC4R that DNal2 might hinder side chain rotati ons of W258 (TM6) and L133 (TM3) (hMC4R numbering) that occur during receptor activation ( 128, 138 ), keeping the receptor in its inactive state ( 128, 138 ). The other hypothesis, is based on th e strength of aromaticaromatic interactions of the DNal2 group with Phe receptor residues ( 114 ). This was derived from in vitro mutagenesis studies of the mMC4R suggesti ng that receptor residues F254 and F259 in TM6 of the mMC4R (mouse MC4R numbering) may be responsible for differentiating agonist versus antagonist activity, since mutation of either of these residues to Ser resulte d in stimulation of the mMC4R mutants by the antagonists SHU9119 and SHU9005 ( 114 ). It was postulated that the mMC4R F254 and F259 residues may be engaged in hydrophobic interactions with the DNal2 (SHU9119), preventing rotation of TM6, theref ore hindering the receptor to undergo the conformational changes which ar e necessary to generate full agonist responses. A homologous F259 residue is absent in the MC1R and MC3R, which contain Leu at this position. This was used to explain the partial agonist activity in ad dition to antagonist activ ity of SHU9119 at the MC3R ( 114 ). In a third hypothesis it was pr oposed that the ligand residue Arg would interact with DNal2 through strong cationinteractions, which might alter positioning of the guanidine moiety and influence interaction of Arg with the hydrophilic receptor binding pocket of the MC4R ( 114 ). In the absence of crystal structures of GPCRs these hypotheses remain speculative and the extent by which each of those mechanis ms contributes to DNal2 antagonism requires

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90 further experimental verification. Additionally, these hypotheses may be sufficient to explain DNal2 antagonism at the MC4R but requirements for antagonism at MC3R seem to be more finely tuned. It also remains to be questi onable if the DNal2 containing compounds confer antagonism by a mechanism which is similar or di stinct to the halogen containing antagonists. Based upon the results of this study it might be envisioned that Iodine and Bromine containing compounds might interact with elec tron rich aromatic receptor residues through a combination of charge-transfer and interactions, thus prev enting the binding pocket to undergo conformational changes necessary fo r receptor activation and agonist binding. Introduction of second chlorine in BP7-147 might confer the necessary low -electron density of the DPhe phenyl ring, enable stronger charge tr ansfer interactions th en single chlorine substitution and increase li pophilicity as well. The CF3 group is probably not able to engage in charge-transfer interactions, however might possess high enough lipophilicity and due to its electron poor DPhe phenyl ring inte ractions with electron rich r eceptor residues might still be possible. These mechanisms might also explai n the lower antagonist potencies of the CF3 containing peptide, compared to Br, I and 3,4d iCl containing peptides. Based on results in this study it is hypothesized that th e electron rich and bulky arom atic DNal2 ring might cause antagonism by a mechanism distinct from the ha logen containing compo unds at the MC3R. In contrast, the halogen containi ng peptides might cause antagonism at the MC3R through a combination of hydrophobic, aromatic and charge-transfer interactions. Receptor Selectivity The study presented herein led to a better unde rstanding of the determinants at the ligand DPhe position that are important for antagonist activity at the mMC3R. Compounds have been identified that possess a unique pharmacological profile and are antagonist s at the MC3R while retaining full agonist activity at the remaining mouse melanocortin receptors. Substitution at the

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91 ligand DPhe phenyl ring is a major contributor in switching receptor func tion of the MC3R, and also resulted in slight changes in subtype sp ecificities. The unsubsti tuted tetrapeptide BP6-120 was 50-fold selective for the mMC4 and mMC5 receptors over the mMC1 and mMC3 receptors (Table 3-2). The same pattern of subtype selectivity was mainta ined for most of the ligands characterized in this study (Table 3-2). Substitution with the hydrophilic el ectron donating groups -OH and -NH2 did not result in substantial selectivity changes between the receptor subtypes, compared to BP6-120. This observation may be consistent w ith the hypothesis that melanocortin receptors possess a common hydrophobic melanocortin receptor binding pocket that is putatively interacting with the DPhe ligand residue ( 85, 114, 123-130 ). Interestingly, substitution with the hydrophilic electron withdrawing groups NO2 and CN resulted into increase d selectivity of the MC4 and MC5 receptors over the skin MC1R of 100and 80-fo ld respectively. In addition, substitution of the DPhe phenyl ring in para position with the hydr ophobic electron withdrawing halogens F and Cl resulted in compounds which are 100to 200 -fold selective for the MC4 and MC5 receptors over the MC1 and MC3 receptors. Conversely, subs titution of the DPhe phenyl ring with -Cl in meta position resulted in 140-fold selectivity of the MC4 over the MC3 receptor subtype but only 40-fold selectivity of the MC4 over the MC1 receptor. When comparing the antagonist potency of the halogen containing antagonist ligands BP7-30 (Ki=35 nM), BP7-27 (Ki=39 nM), BP7-147 (Ki=38 nM) and BP7-26 (Ki=162 nM) at the MC3R with the corresponding agonist potency values at the MC4R, MC3R/MC4R subtype selectivity is decreased, as compared to the control peptide BP6-120. In summary, substitutions at the DPhe phenyl ring led to slight changes in receptor subtype specifi cities and this knowledge is valuable for further SAR st udies of melanocortin ligands.

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92 Conclusion In the study presented herein, we have extens ively investigated the requirements at the tetrapeptide DPhe position for antagonist activit y at the MC3R. We have generated a hypothesis by which a combination of aromatic halogen charge-transfer interactions and lipophilicity might cause antagonism at the MC3R maybe di stinct from the DNal2 containing peptides. These results highlight the differential recepto r activation and inactivation mechanism of the MC3R versus the MC4R and are highly valuable for future drug design of melanocortin selective ligands. In addition, new compounds with mixed pharmacology have been identified (BP7-30, BP7-147 and BP7-26) which are stably and therefore more useful for in vivo studies then a pI containing tetrapeptide.

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93 Table 3-1. Common substituant constants for aromatic sustituents ( 118 ). Hansch lipophilic effect Hammet electronic effect MR size p-CN -0.57 0.66 0.633 p-NO2 -0.28 0.78 0.736 p-F 0.14 0.06 0.092 p-Cl 0.71 0.23 0.603 p-Br 0.86 0.23 0.888 p-I 1.12 0.18 1.394 p-CF3 0.88 0.54 0.502 p-OH -0.67 -0.37 0.285 p-NH2 -1.23 -0.66 0.542 p-CH3 0.56 -0.17 0.565 p-tBu 1.98 -0.20 1.962 p-Bz 2.01 -0.09 3.001 m-Cl 0.71 0.37 0.603 Figure 3-1. Schematic representation of parameters that can be affected by substituents in a drug molecule (modified from ( 118 ))

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94 Figure 3-2. Graphic representation of the sustituent constants for para phenyl substituents used in this study. Figure 3-3. Schematic representation of the substituents in DPhe para position and their properties used in this study. EDG d onated electron donating group. EWG donates electron withdrawing group.

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95 Figure 3-4. Structures of amino acids used to replace DPhe in the peptide template Ac-DPheXaa-Arg-Trp-NH2.

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96 Table 3-2. Functional activiti es of the DPhe modified tetrapeptides at the mouse melanocortin receptors. mMC1R mMC3R mMC4R mMC5R Peptide Structure EC50 (nM) fold diff EC50 (nM) fold diff EC50 (nM) fold diff EC50 (nM) fold diff NDP-MSH Ac-Ser-Tyr-Ser-Nle-Glu-HisDPhe Arg-Trp-Gly-Lys-Pro-Val-NH2 0.0880.0310.330.080.120.010.260.05 BP6-120 Ac-HisDPhe -Arg-Trp-NH2 1122811267.812.28.5212.26.601 BP6-121 Ac-HisDTyr -Arg-Trp-NH2Tyr 2600023125007009939048171730210323 BP7-29 Ac-His(pNH2) DPhe-Arg-Trp-NH2 23000211640048013022015.09622610.2100 BP6-124 Ac-His(pCH3) DPhe-Arg-Trp-NH2 170201.552513045.65.8825.20.712 BP6-126 Ac-His(pt-Bu) DPhe-Arg-Trp-NH2 100131> 100 000436000191234073150 BP-129 Ac-His(pBz) DPhe-Arg-Trp-NH2 180722770108663.82.52851.1.2223 BP6-128 Ac-His(pCN) DPhe-Arg-Trp-NH2 280272.52404922.84.2612.85.271 BP7-23 Ac-His(pNO2) DPhe-Arg-Trp-NH2 10305961544513.0.92617.6.18 BP7-25 Ac-His(pF) DPhe-Arg-Trp-NH2 53.78.62-253.75.30-20.450.10-52.040.271 BP7-24 Ac-His(pCl) DPhe-Arg-Trp-NH2 38.23.3-384.821.0-1.50.400.12-61.470.441.5 BP7-30 Ac-His(pBr) DPhe-Arg-Trp-NH2 40.810.4-3 pA pA2=7.540.12 ANT 1.070.46-22.601.521 BP7-27 Ac-His(pI) DPhe-Arg-Trp-NH2 40.72.733 pA pA2=7.410.08 ANT 3.941.3227.410.083 BP7-26 Ac-His(pCF3) DPhe-Arg-Trp-NH2 5401205 pA pA2=6.790.15 ANT 12.81.9468.031.333 JRH887-12 Ac-His( DNal2) DPhe-Arg-Trp-NH2 92.842.71 pA pA2=6.700.12 ANT pA2=8.280.13 ANT 22.45.010 BP7-147 Ac-His(3,4-diCl) DPhe-Arg-Trp-NH2140301 pA pA2=7.420.26 ANT 3.400.6617.420.263 BP7-148 Ac-His(m-Cl) DPhe-Arg-Trp-NH2 32057.331240270108.641.73430.013.613The indicated errors represent the sta ndard error of the mean from at least three independent experiments. > 100000 EC50 values denotes that compounds were not found to possess any agonist or an tagonist activity at up to 100 M ligand concentrations. The compounds possessing partial agonism were assayed for competitive antagonism using Schild pA2 analysis using MTII as agonist. Ki=-log pA2. ANT=Antagonist; pA=partial agonist.

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97 Figure 3-5. Pharmacology curves of the tetrapeptides substituted w ith halogens at the DPhe para position at the mMC3R, showi ng full agonist activity for H, -F, -Cl and partial agonist/antagonist activity of Br and I substituents. Figure 3-6. Antagonist pharmacology cu rves of BP7-26 at the mMC3R.

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98 Figure 3-7. Pharmacology curves of the tetrapep tides substituted with chlorines and DNal2 at the mouse MC3R. Figure 3-8. Pharmacology curves of the tetrapeptides substituted w ith halogens at the DPhe para position at the mMC3R, showi ng full agonist activity of the H, -F, Cl and partial agonist activity of Br and I.

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99 Figure 3-9. Illustration of po ssible geometries of aromatic edge-to-face (left) and stacking interactions (right) using BP 7-27 as example. The substituted phenyl ring is electron deficient as depicted by +. Iodine substitution may potentiate the aromatic interactions through formation of charge-transfer complexes. A phenyl ring is used as example for an aromatic receptor residue.

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100 CHAPTER 4 SITE-DIRECTED MUTAGENESIS STUDIES AT THE MOUSE MELANOCORTIN-3 RECEPTOR Residue selection for mutagenesis and prim er design was done by Bettina Proneth and Dr. Carrie Haskell-Luevano. The wild-type and mu tant Flag-mMC3 receptors were generated by Bettina Proneth. Flow Cytometry for detection of receptor cell surface and total cell expression was performed by Bettina Proneth in collaboration with Dr. Sally Litherland, Department of Pathology, Immunology and Laborat ory Medicine. Functional assays and competitive binding assays were performed by Bettina Proneth. Iodination of NDP-MSH was done by Bettina Proneth in collaboration with Dr. William Millard, Department of Pharmacodynamics. Functional assay data analysis was done by Bettina Proneth and binding data analysis was done by Dr. Carrie Haskell-Luevano. Introduction The first step in the rational drug design pro cess is the identification of specific ligand residues or a core pharmacophore sequence importa nt for the molecular recognition of ligandreceptor complexes and ligand induced receptor stimulation and/or inhibition, as shown in Chapter 3. However, it might be envisioned that residues of both, the ligand and the receptor are critical for molecular recognition, receptor se lectivity and differen tiating agonist versus antagonist activity. Site-directed mutagenesis of G-Protein coupled receptors (GPCRs) is therefore an important tool to identify specific residues that are involved in generating these pharmacological effects. Considering the fact that to date there is only limited availability of selective ligands ( 115-117 ) (> 500-fold selective) for the Me lanocortin-3 Receptor (MC3R) it is important to gain insight into specific ligand-r eceptor interactions and the mechanism of receptor activation and inactivati on of the MC3R. This study was undertak en to identify receptor residues involved in molecular recognition of structurally different mela nocortin agonists and antagonists

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101 and to identify receptor residues important for the differentiation of agonist versus antagonist activity as well as for the differentiation of MC 3 and MC4 receptor subtypes. This mutagenesis data will provide significant insight into the pu tative ligand-receptor in teractions and receptor activation and inhibition m echanisms and can then be used together with ligand SAR studies as guideline for creating 3D receptor homology mode ls for the docking of agonists and antagonist ligands. Results Figure 4-1 summarizes the mouse MC3R mutati ons generated in this study. A Flag-tag was inserted at the N-terminus of the MC3R dow nstream of the Methionine start codon to allow for immunocytochemical detection of the rela tive receptor cell surface and total expression. Receptor residues for mutation where systematica lly selected based upon previous mutagenesis studies of the human and mouse MC1 and MC4 recep tors that were identified as important for ligand receptor interaction a nd/or receptor activation ( 85, 114, 129, 130, 138, 139 ). These studies identified a putative binding pocket for the DPheArg-Trp ligand core se quence that exists of one region formed by acidic amino acid residue s and one region forming a hydrophobic network of aromatic amino acids ( 85, 114, 129, 130, 138, 139 ) (Figure 4-2). Site-directed mutagenesis of GPCRs as well as most other proteins is commonly performed through (Alanine) Ala scan mutagenesis and was included in this study to be able to compare our results with those performed by other laboratories at the melanoc ortin receptors. In this procedure, the side chain at a particular position is basically "removed" which allows for th e investigation of the role a specific side chain may play in the formation of ligand-receptor complexes and/or intramolecular interactions within the receptor. This method is generally safe in terms of minimally disturbing the overall structure of the receptor. Howe ver, Ala substitutions may not be very effective at locating contact amino acids in receptor bindi ng sites, especially with other hydrophobic ligand

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102 residues. For example, the ligand DPhe7 and Trp9 residues are putatively interacting with the melanocortin receptor hydrophobic binding pocket cons isting of several aromatic amino acids. Since Ala possesses a hydrophobic met hyl side chain it is possible that when the Phenylalanine (Phe) to Ala substituted residue interacts with the ligand aromatic resi dues then both ligand and receptor might still occupy the same binding poc ket space. However, if a hydrophilic residue, such as Ser, replaces the hydrophobic aromatic receptor Phe residues, significantly larger changes in ligand binding and/or potency may be observed that result from electrostatic, van-derWaals forces or repulsive ligand-receptor interac tions that might ultimately result in larger differences in pharmacological binding of func tional values. This method was previously successfully applied to mutage nesis studies of the mMC4R ( 114 ) and was shown to work well for other types of side chain moieties like Aspa rtic Acid (Asp), Glutamic Acid (Glu) and Cysteine (Cys) as well ( 85 ). Functional Characterization of Wild -type and Mutant Flag-mMC3 Receptors Each mutant Flag-mMC3R was pharmacological ly characterized by several agonists and antagonists and their structur es are shown in Table 41. The functional activity EC50 values (nM) of the agonists at the Flag-mMC 3 receptors are summarized in Table 4-2 and the antagonist potency pA2 values of SHU9119 and AGRP are presented in Table 4-3. In order to compare the fold differences of the antagonist potency values to the potencies observed at the wild-type FlagmMC3R, the pA2 values were converted into their corresponding Ki values (nM), where pA2 = logKi ( 103 ). The agonist and antagonist pharmacology cu rves of the wild-type Flag-mMC3R are illustrated in Figure 4-3. Due to space issues only selected pharmacology curves are shown throughout this chapter. The remaining pharmacol ogy curves are summarized in Appendix B in the end of this dissertation. Because of the inhere nt experimental errors of the functional reporter gene assay used in this study potency changes of 1-3 fold were considered as essentially

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103 equipotent, changes of 4-9 fold were defined as slightly decreased potency and changes of >10 fold were defined as significantly reduced potency. Synthetic Melanocortin Agonists NDP-MSH is a 13 amino acid peptide that resembles the -MSH structure in that the MSH residues Met4 and LPhe7 are substituted with Nle4 and DPhe7 and results into increased potency compared to -MSH ( 140, 141 ). NDP-MSH possessed equipotency or decreased potency of up to 3-fold at the majority of th e mutant Flag-mMC3 receptors, consistent with a previous report of 19 mutations of the human MC3R ( 123 ). Potency decreases of 4-9 folds were observed at the E94S, D117A, D117S, D 295A, R302A, R302S Flag-mMC3 receptors. Interestingly, at the M199S and T270S NDP-MSH possessed increased potencies in the 4-9 fold range. Changes in potencies of >10 fold were observed at the D121A, D121S and D295A FlagmMC3 receptors. MTII is a very potent cyclic seven amino acid peptide agonist where cyclization is achieved through the use of a lactam bridge around the core His-DPhe-Arg-Trp sequence ( 142 ). Cyclic peptides are generally more constrai ned and are believed to mimic the bioactive conformation of a ligand. Similar to NDP-MSH, MT II was essentially equipotent in the 1-3 fold range at the majority of mutant Flag-mMC3 recep tors. Potency decreases of 4-9 folds could be observed at the E94S, F120S, I132A, I132S, T145A, Y182A, Y182S, F1 96S, W255A, W255S, F259S, D295A, D295S, R302A and R302S Flag -mMC3 receptors. At the D117A, D117S and D295A Flag-mMC3 receptors, the cyclic MTII peptide possessed considerable reduced potency of more then 10 fold, as compared to the wild-type receptor. AMW3-130 is a cyclic eleven amino acid peptid e which contains the putative agonist core recognition sequence His6-DPhe7-Arg8-Trp9 incorporated into the antagonist AGRP (108-119) template instead of the antagonist recognition sequence Arg111-Phe112-Phe113 ( 143 ). This

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104 chimeric ligand resulted in converting the endogenous AGRP an tagonist template into an agonist. It might be envisioned th at this compound, containing an tagonist and agonist amino acid residues may interact with the r eceptor distinct from the other ligands and was incorporated in this study to investigate this possibility. AM W3-130 possessed almost equipotency (1-3 fold range) at the majority of mutant receptors te sted. Potency decreases of 4-9 fold could be observed at the Y182S, W255S, F281A, F281S R302A and R302S Flag-mMC3 receptors. Larger decreases in potency of more then 10-fold occurred at the E94S, D117A, D117S and D295A Flag-mMC3 receptors. The linear synthetic tetrapep tide JRH887-9 contains the me lanocortin core sequence His6DPhe7-Arg8-Trp9 putatively involved in melanocortin receptor molecular recognition and stimulation ( 108 ). It was included in the study presented herein to investigate if ligand-receptor interactions are mainly base d upon interaction of the melanoc ortin core sequence or if contribution of additional N-and C-terminal amino acids are required. JRH887-9 resulted in potency changes of 4-9 fold at the I96S F120A, Y152S, E184A, E184S, M195A, F197A, H261S, I265A, and N282S Flag-mMC3 receptors and into potency changes of >10 fold at the Y182A, F196A, F196S, W255A, D2 95S Flag-mMC3 receptors. JRH887-9 was unable to fully stimulate the E94S, D117A, D117S, Y182S, M195S and W255S and no stimulatory activity at up to 10M concentration was observed at th e D121A, D121S, L128A and F285A Flag-mMC3 receptors. At the N282A and D295A Flag-mMC3 recep tors JRH887-9 resulted in slight or partial agonist activity, however an EC50 value could not be determined. Interestingly, JRH887-9 resulted in increased potencies compared to wi ld-type at several Flag-mMC3 mutant receptors. Increases in potency in the 4-9 fold range we re observed at the T145S, T270A, T270S, F281A

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105 and F281S and remarkable potency increases of more then 10-fold at the M199A and M199S Flag-mMC3 receptors. Endogenous Agonists All endogenous agonist liga nds possess the natural LPhe7 amino acid which is replaced by DPhe7 in the synthetic agonists, tested in th is study. The endogenous agonists are derived through posttranslational processing from the common precursor hormone proopiomelanocortin (POMC) in vivo (9, 10) The -MSH endogenous peptide ligand is a linear 13 amino acid peptide that is N-acetylated at the N-terminus a nd C-amidated at the C-terminus. Betaand -MSH do not possess these posttranslational modificati ons and are 22 amino acid and 12 amino acid peptides respectively. The -MSH peptide possessed potency changes of 4-9 fold at the S122A, T145A, C175S, M199A and T270A mutant Flag-mMC3 receptors For the functional assay, the endogenous ligands were tested at co ncentrations ranging from 10-6 to 10-12M at the wild-type and mutant Flag-mMC3 receptors. At several mutant Flag-mMC3 receptors, the -MSH ligand was not able to generate a full response at 1 M concentration; therefore an EC50 value could not be determined at these mutant receptors. This was observed at the D117A, D117S, F120S, I132S, Y182S, M195S, F196S, W255S, F259S, I265A, N282S and F281S Flag-mMC3 receptors. Mutant receptors, at which an EC50 value was still generated with NDP-MSH, but -MSH resulted in either partial, s light agonist or no functional resp onse, are the E94S, D121A, D121S, H261A, H261S and N282A Flag-mMC3 receptors. The -MSH endogenous agonist differed from the pharmacological profile of and MSH in that it resembled the functional activities of the synthetic agonist s. One explanation for this could be that the longer N-terminus a nd different amino acid composition around the HisPhe-Arg-Trp region may be modifying the co nformation of the melanocortin core sequence

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106 more favorable for interaction with the mMC3R. Decreased potency ranges of 4-9 fold were observed at the N91A, F120S, I132A, I132S, T145A, Y152S, F179A, Y182S, E184S, F196S, W255A, W255S, F259S, H261A, H261S, N282S, F281S, R302A and R302S Flag-mMC3 receptors and interestingly at the M199S Flag-mMC3R, -MSH possessed increased potency of 4-fold. At the Y182S, I265A, D295 A and D295S Flag-mMC3 receptors -MSH resulted in decreased potency of more then 10-fold a nd at the E94S, D117A and D117S Flag-mMC3 receptors -MSH resulted in only 50-70% stimulator y activity at 1M co ncentration. No EC50 value could be generated at the D121A, D121S and N282A Flag-mMC3 receptors. The -MSH endogenous agonist possessed reduced potencies of 4-9 fold at the N91A, S122A, T145A, Y152S, E184S, Y184A, M195A, W255A and F281A Flag-mMC3 receptors and increased potencies of 4-9 fold at M199A and M199S Flag-mMC3 receptors. Flag-mMC3 receptors which could be only partially activat ed at up to 1M concentration or where no EC50 value could be determined are E94S, D 117A, D117S, F120A, F120S, I132S, Y182S, M195S, F196S, W255S, F259S, H261A, H261S, I265A, N282A, N282S, F281S, D295A, D295S, R302A and R302S. Endogenous and Synthetic Antagonists The endogenous antagonist AGRP and the synthe tic antagonist SHU9119 were included in this study in attempts to identify Flag-mMC3 recep tor residues, that differe ntiate agonist versus antagonist activity and to inve stigate if the structurally different AGRP and SHU9119 antagonists would share similar mechanisms of ligand-receptor interactions. The antagonist pharmacology curves at the wild-type Flag-mMC3R are illustrated in Figure 4-3. AGRP is a 131 amino acid peptide that contains ten cysteine residues capable of forming five potential disulfide bridges ( 14, 44, 53, 144 ). A loop of 8 residues which is fla nked by two cysteine residues contains the Arg111-Phe112-Phe113 region which has been demonstrated to be critical for antagonism at the

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107 melanocortin receptors ( 14, 43, 145 ). In addition to competitive antagonism, AGRP possesses inverse agonist activity in vitro at the MC4R which was demons trated using overexpressed MC4 receptors in cells and a constitutively active mutant mouse MC4R ( 15-17 ). Due to experimental conditions and the sensitivity of th e assay used it is often not po ssible to observe inverse agonist activity which does not mean it is absent ( 104, 146 ). In the study presented herein, AGRP inverse agonism was only observed at the N91A Flag-mMC3 receptor. To compare the antagonist potencies, pA2 values were converted into their corresponding Ki values. AGRP possessed decreased potencies in the 4-9 fold range at the M195A, L264A, T270A and D295A Flag-mMC3 receptors and decreased potencies of 10-fold or more at the M199A, F281A and F281S Flag-mMC3 receptors. Increased potencies were observed at F197A, I265A (4-fold) and F120S (10-fold) and H261A (100-fold) Flag-mMC3 receptors. SHU9119 is a cyclic seven amino acid peptid e antagonist which differs from the MTII agonist only by substitution of the DPhe7 ligand residue with the bulky aromatic amino acid (DNal2)7 ( 52 ). Interestingly, this compound resulted in a competitive antagonist at the MC4R, a competitive antagonist with partial agonist activity at the MC3R and full agonist at the peripheral MC1 and MC5 receptors, respectively ( 52 ). It was therefore hypothe sized that bulky aromatic amino acid substitutions at the ligand DPhe position confer antagonist activity at the brain MC3 and MC4 receptors ( 52 ). SHU9119 resulted in a competitiv e antagonist with partial agonist activity at the wild-type Flag -mMC3R. Unexpectedly, at th e T145A and N91A Flag-mMC3 receptors SHU9119 lost the part ial agonist activity observed at the wild-type receptor and resulted in a pure competitive antagonist. The I132S, M199A, H261A and T270S Flag-mMC3 receptors showed increased part ial agonist activity of SHU9119, while still retaining competitive antagonism. Flag-mMC3 mutant receptors where SHU9119 lost its antagonist activity, while

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108 possessing elevated partial agonist activity are the D117A, D117S and F179S Flag-mMC3 receptors and mutations where SHU9119 was convert ed into full agonist activity are the I132A, M199S, W255A, W255S, F259S, T270A, F281A and F281S Flag-mMC3 receptors. The L128A and L128S Flag-mMC3 receptors possessed partial agonist activity for all agonists tested. When SHU9119 was tested at these mutant receptors, no antagonist activity could be determined and this compound resulted in partial agonist activity with similar efficacy as observed with the agonist MTII. SHU9119 showed decreases in pote ncy in the range of 49 fold at the N91A, D117A, T145A, T145S and F196S Flag-mMC3 recepto rs and decreases in potency of 11-fold at the D117S Flag-mMC3R. Interestingly, several receptor mutations caused an increase in SHU9119 potencies, like S52A, I96A, I96S Y152S, C175S, E184A, M199A, T278S, N282A, D295A and D295S (4-9 fold range), Y 152A (16-fold) and R302S (13-fold). Flag-mMC3R Mutations resulting in a Loss of Functional Activity Loss of ligand induced receptor stimulation of all agonists te sted, resulted when several residues were mutated to Ala and/or Ser in a (T able 4-2). Receptor mutations that did not possess any stimulatory or inhibitory activity at ligand concentratio ns up to 1M are N56A, N56S, D84A, D84S, N91S, E94A, K160S, I165A, I165S, L247A, L247S, P257A, P257S, F258S, F259A, L264S, I265S, T267A, T267S, C268A and C 268S (Table 4-2). Ther e are various reasons for the loss of receptor functi onal activity and the molecular mechanisms underlying this phenotype need to be carefully investigated. Poss ible explanations can be that the receptor residue might be directly involved in molecular recognition of the ligand-re ceptor complex or the ligand might still bind to the mutant receptor but is unable to stimulate the receptor, which would be indicative for involvement in receptor activat ion processes. Another po ssibility might be the involvement of this particular receptor residue in maintaining the overall structural integrity of the receptor. Improperly folded GP CRs might be retained fully or partially in the endoplasmic

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109 reticulum (ER) and are not or only partially processed to the cell surface. These mechanisms will be investigated in this study using competitiv e ligand binding assays to investigate the ligand binding affinity and Flow Cytometry to inves tigate the receptor cell surface expression. Flag-mMC3R Mutations Causing Partial Agonism Flag-mMC3R residues that when mutated to Al a and Ser and resulted in partial agonist activity of all agonists tested are I96S, F 120A, S122A, L128A, L128S, Y152A, Y152S, C175A, C175S, Y182A, F258A, H261A, H261S, N282A, D 295A, D295S, R302A, R302A (Table 4-2). The molecular mechanism by which partial agonism is caused in GPCRs is unknown and still under investigation. However it may be speculated that receptor mutations th at are only able to generate a partial response when stimulated by agonists, but retain normal ligand binding affinities are involved in recep tor activation processes. Cell Surface Expression of Flag-mMC3 Receptors The wild-type and mutant Flag-mMC3 receptors contain an extracellular Flag-tag that was inserted between the Methionine start codon and th e first receptor amino acid at the N-terminus (Figure 4-1). Figure 4-4 and Ta ble 4-4 summarize the percentage of total cellular receptor expression and receptor cell surface expression of the mutant Flag-mMC3 receptors that were stably expressed in HEK293 cells, relati ve to the wild-typ e Flag-mMC3R, using immunocytochemical staining and Flow Cytometr y. Determining the cell surface expression is especially important for receptor mutations causing the loss of function phenotype or those possessing differences in ligand pharmacology, comp ared to the wild-type receptor. Specific amino acid residues are of crucial importance for r eceptor folding and structural integrity, correct insertion of the transmembrane helices into th e cell membrane or posttranslational processing from the endoplasmic reticulum (ER) to the cell surface. Mutations can affect either of these states and prevent the receptor to be fully expresse d at the cell surface to be able to interact with

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110 ligands. Using flow cytometry in combination w ith intraand extracel lular staining techniques allows for quantification as well as for localization of the receptor protein with in the cell. All mutant Flag-mMC3 receptors were generally ex pressed on the cell surfac e. Several Flag-mMC3 receptors were expressed less then 50% relati ve to the wild-type Flag-mMC3R on the cell surface. These include N56A, I96S, F120A, I132S, Y152A, Y152S, K160S, I165S, F258A, F259A, L264S, I265S, C268S, N282A a nd D295S Flag-mMC3 receptors. Competitive Displacement Binding Studies Table 4-5 summarizes the competitive binding affinity IC50 values of the NDP-MSH ligand at the wild-type and mutant Flag-mMC 3 receptors, compared to the functional EC50 values. The binding affinity dose-response cure of the wild-type Flag-mMC3R is illustrated in Figure 4-3. Nonlabeled NDP-MSH was us ed to displace the radio labeled I125 NDP-MSH in a competitive manner. NDP-MSH has become the lig and of choice for binding studies at the melanocortin receptors, and was used in this stu dy for the purpose of being able to compare our results with findings from the lit erature. Additionally, NDP-MSH is highly potent, biologically stable and amenable for iodinati on. Eight Flag-mMC3 receptors lost the ability to bind and to be stimulated by NDP-MSH (N56A, N56S, I165S, L247S, P257A, I265S, T267A, T267S and C268A). The N56, I165, L247 Flag-mMC3 receptor re sidues are generally not located within the putative ligand binding pocket and might be of crucial importance for the overall structural integrity of the Flag-mMC3R and/or important for receptor activation mechanisms. The relative contribution of the P257A, I265S, T267A, T 267S and C268A Flag-mMC3 receptors into molecular recognition of NDP-MSH and/or receptor activation still needs to be investigated and will be discussed below. Thirteen Flag-mMC3 recep tors were identified that might be important for receptor activation mechanisms since NDP-MSH was still able to bind to these receptors but unable to cause ligand induced receptor stimulation (D84A, D 84S, N91S, E94A, K160S, I165A,

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111 L247A, P257S, F258S, F259A, L264S). Due to th e inherent experimental errors of the competitive binding assay used in this study differences in IC50 values of up to 1-3 folds were considered as similar, differences of 4-9 folds as slightly decreased and differences of more then 10 fold were defined as significan tly decreased binding affinity, co mpared to the wild-type FlagmMC3R. Binding affinity ranges of 4-9 fold s were observed at the N91A, C175S, L247A, W255A, F258A and N282A mutant receptors The Y182A and the C268S Flag-mMC3R possessed decreased binding affinities of 16a nd 27-fold respectively. Interestingly, the D121S and F197A Flag-mMC3 receptors possessed increases in ligand binding affi nities of 9and 10fold, respectively, when compared to the wild-type Flag-mMC3R. Discussion The MC3R has been identified to be i nvolved in energy homeostastis and obesity. However, the exact physiological function of th is particular receptor subtype is yet unknown. This is partly due to the difficulty encountered in the design of MC3R selective agonists or antagonist ligands. The study presented herei n, was undertaken to understand the molecular mechanism by which some ligands activate the MC 3R while others do not, which is important for the development of agonist and antagonists as in vivo tools to investigate the physiological functions of the MC3R as potential drug targ et. An additional purpose of this study was to identify receptor residues that discriminate between the MC3 and MC4 receptor subtypes, which can be useful in the design of ligands, partic ularly targeting these residues. Thus, 41 FlagmMC3R residues have been mutated to Ala and 37 have been mutated to Ser. The wild-type and mutant Flag-mMC3receptors have been character ized for functional activity and ligand binding affinity using seven structurally diverse agonist and two antagoni st ligands to identify putative ligand-receptor interactions that differentiate between the MC3/MC4 receptor subtypes and agonist/antagonist activ ity at the MC3R.

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112 AgonistReceptor Interactions: The Hydrophilic Region Previous studies suggested that the ligand Arg8 residue ( -MSH numbering) is putatively interacting with a hydro philic binding pocket consisting of one or several of the conserved residues E94 (TM2), D117 (TM3) and D 121 (TM3) (mouse MC3R numbering) ( 114, 123-125, 129, 130, 139 ). This hydrophilic cluster is highly cons erved in the melanocortin receptor family. Figure 4-5 illustrates an alignment of bovine rhodopsin, and the human and mouse melanocortin receptors, where the transmembrane helices were aligned based upon a previous report ( 147 ). Several hypotheses have been proposed to explain the interaction of Arg8 with these residues. Genetic studies in mice showed that mutation of Gl u in TM2 to Lys resulted in a constitutively active MC1R ( 19 ). Interestingly, when the TM2 Glu was mutated to Lys, a constitutively active receptor resulted with a basal cAMP activity of 50 % of the maximal response, and when this Glu was mutated to an Arg, nearly 100% maximal cA MP activity was observe d in the absence of ligand ( 139 ). These MC1R mutagenesis studies indi cate that upon modification of the TM2 Glu to substitutions resembling the melanocortin ligand Arg8 amino acid, constitutively active receptors result, suggesting that these recepto r mutations may be mimicking the ligand Arg8 side chainreceptor interactions important for melanocortin receptor activation ( 139 ). Based on these findings it could be speculated that ionic interactions, obser ved with the Arg mutation, are preferred over the hydrogen bondi ng interactions, observed with the Lys mutation. When the TM2 Glu is substituted with Arg or Lys in the mouse MC4R, no constitutive activity was observed ( 114 ), which might indicate that the receptor subtypes interact differentially with the Arg8 residue. However, mutation to Lys retained reduced binding affinity and mutation to Arg and Ser abolished both binding and functional ac tivity of the NDP-MSH ligand, further implying that these residues are involved in mol ecular recognition of the NDP-MSH ligand ( 114 ). Several independent melanocortin receptor mutagenesis studies of the MC1R and MC4R support the

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113 concept of a hydrophilic receptor pocke t interacting with the ligand Arg8 side chain albeit the exact nature of these interacti ons is still unknown. Hypotheses have been proposed to explain the putative Arg8 interactions with the hydrophilic me lanocortin receptor binding pocket: (i) Arg8 might form ionic interactions with E94, D117 and D121 through its positive charge ( 85, 139 ) (ii) the guanidine moiety may be involved in hydrogen bonding ( 148 ) and/or (iii) the stereochemistry of the hydrophobic side chains may be more import ant since small molecule ligands lack a group mimicking Arg8 ( 149 ). In another hypothesis it was sugge sted that the DNal2 group of the antagonist SHU9119 at postion 7 may influence the orientation of how Arg8 interacts with the hydrophilic receptor binding pocket ( 114 ). This possibility will be disc ussed later in this chapter. In the study presented herein, the E94 resi due might be important, not only for ligand molecular recognition, but also ligand induced receptor stimulation. Mutation of E94 to Ala resulted in loss of ligand functional activity but retained binding affinity to NDP-MSH (Table 42, Table 4-5 and Figure 4-6). In mutagenesi s studies of the human MC3 receptor, the corresponding mutation resulted in partial cAMP response with reduced potency upon stimulation with NDP-MSH ( 123 ). An explanation for this might be the use of transient transfection, which allows for higher initial receptor expressi on levels as opposed to stable transfection used in this study ( 123 ). However, the observation of partial agonism of the human MC3R E94A mutant further indicat es this residue as important fo r MC3R activation. Mutation of E94 to Ser resulted in either reduced agonist potencies (for NDP-MSH, AMW3-130, -MSH and JRH887-9) or loss of functional activity ( -MSH and -MSH) at the ligand c oncentrations tested, whereas the antagonists SHU9119 a nd AGRP were not affected (Tab le 4-2, Table 4-3, Table 4-5 and Figure 4-6). Interestingly, when the corres ponding residue in the mouse MC4R (E92) is mutated to Ser, this mutant receptor resulted in loss of functional ac tivity as well as binding

PAGE 114

114 affinity for the agonists NDP-MSH and MTII, whereas decreased binding affinity to the antagonist SHU9119 was retained ( 114 ). Summarizing these observations, it seems that the E94 residue is important for molecula r recognition of the agonist Arg8 moiety and receptor stimulation of the mMC3R (Table 4-2, Table 4-5 and Figure 4-6). The D117 residue might be involved in molecu lar recognition and potencies of the agonist ligands since both functional activities and NDP-MSH binding affinities are decreased at the D117A and D117S mutant receptor s (Table 4-2, Table 4-5 and Fi gure 4-6). D121 is located one helical turn below D117 and might be involved in receptor stimulation as well as in molecular recognition of the Arg8 ligand residue. At the D121A muta nt receptor some stimulatory cAMP response could be observed, only upon NDP-MSH stimulation, however no IC50 binding affinity value could be determined (Table 4-2, Table 4-5 and Figure 4-6). A similar result has been reported in a recent study, pharmacologically ch aracterizing the corresponding hMC4R mutation D126N with NDP-MSH ( 150 ). The reason why some stimulat ory activity was observed but no IC50 value could be generated may be that the re ceptor competitive displ acement studies directly depend on receptor number, while functional stud ies are less dependent on the receptor number and potentially more sensitive ( 114 ). When comparing the to tal specific counts at 10-11M NDPMSH concentration, the counts pe r minute (cpm) observed with D 121A are fairly low, which could be a reason why no inhibito ry dose-response curve could be generated. The same trend was observed with the relative cell surface expression of the D 121A mutant (54% relative to wild-type) compared to the D121S mutant (75% relative to wild -type) (Figure 4-7). The D121S Flag-mMC3R was able to generate reduced st imulatory response with NDP-MSH, but possessed high binding affinity towards NDP-MSH, which i ndicates this residue as important for ligand recognition aspects as well as ligand induced r eceptor activation (Table 4-2, Table 4-5 and

PAGE 115

115 Figure 4-6). These results, in combination with previous mutagenesis studies of the MC1, MC4 and human MC3 receptors highlight the importan ce of the acidic region of E94, D117 and D121. The use of Ser substitution in addition to Ala was highly benefi cial since this study provided experimental evidence that the hydrophilic re ceptor residues in TM2 and TM3 are not only important for molecular recognition of the Arg8 ligand but additionally might be playing a key role in ligand induced re ceptor activation. Furtherm ore it can be speculated that a combination of Arg8 with DPhe7is important, since LPhe7 containing peptides resu lted in higher losses in potency and efficacy. The DPhe7 residue might be the reason that NDP-MSH was still able to bind to these mutant receptors with high affi nity, except D121A as explained above. This hypothesis is experimentally supported by Ala sc anning of NDP-MSH (unpublished results from the Haskell-Luevano laboratory) and the tetrapeptide JRH887-9 ( 108-111 ). Substitution of Arg8 in NDP-MSH (Ala8-NDP-MSH = RDP-3-21-3) and JRH887-9 (Ala8-JRH887-9 = JRH3107-29) resulted in less dramatic changes in potency comp ared to the parent pep tides as did substitution of DPhe7 with Ala (Ala7-NDP-MSH = RDP-25-4; Ala7-JRH887-9 = JRH3107-24), further implicating the high importance of DPhe7 for mMC3R binding and stimulation. The following residues might not be directly involved in MC3R ligand recognition but given the close proximity to E94, D117 and D121 th ese residues might affect either positioning of these hydrophilic side chains and/or positi oning of TM3. This may then provide further experimental evidence of the importance of th e three acidic residues in ligand binding and signaling. Mutation of F120 to Ala resulted in partial agonism for all the a gonists tested, whereas F120S maintained full agonist activity and almo st equipotency to NDP-MSH and MTII, however -MSH, -MSH and JRH887-9 were not able to ge nerate full response at 1 M and 10 M

PAGE 116

116 concentration, respectively (Table 4-2 and Tabl e 4-5). F120 corresponds to I125 in hMC4R, and this residue, when mutated to Ala resulte d in partial agonist activity as well ( 126 ). F120 might not be involved into dire ct contacts with the Arg8 ligand but rather direct positioning of acidic residues in TM2 and TM3 in the right orient ation for receptor activation or molecular recognition. Space generating substitution to Ala would therefore abolish this stabilizing interaction and indeed the F120A mutant resulted in partial agonist activity whereas F120S resulted in full agonist activity (Table 4-2 and Tabl e 4-5). Maybe Ser is still able to interact with the acidic residues from TM2 and TM3 forcing th em into an orientation suitable for receptor stimulation. Mutation of S122 to Ala resulted in partial a gonist activity of ligand potencies similar to the wild-type receptor, further implicating this region in receptor activation mechanisms (Table 4-2 and Table 4-5). The corresponding mutation S 127L in the hMC4R was identified in obese patients, and when characterized pharmacologically resulted in reduced potencies for the agonist ligands tested ( 95 ) which may be due to introduction of the bulky Leu group. Mutation of N91 to Ser resulted in loss of functional activity, wh ile retaining normal binding affinity to the NDP-MSH ligand, whereas mutation to Ala did not significantly interfere with agonist or antagonist potenci es (Table 4-2 and Table 4-5). N 91 is putatively located in TM2 in close proximity to E94. Mutation to Ser might lead to interaction with e ither one or multiple of the acidic residues E94, D117 and D121, dist urbing ionic interactions with the Arg8 ligand residue. S91 might hydrogen bond to these side chai ns, changing their orient ation and preventing them to interact with Arg8. Another possible explanation might be that S91 is interacting with the conserved acidic amino acid re sidues from TM2 or TM3 and pr event conformational changes that accompany agonist binding and receptor ac tivation. N91 does not seem to be directly

PAGE 117

117 involved in molecular recognition of the Arg8 ligand residue, and the exact mechanism by which this residue is involved into rece ptor activation remains speculative Another interest ing aspect is that N91 aligns with Ser in hMC5R, Ile in mM C1R and Lys in MC2R (Figure 4-5), which might implicate this residue in subtype specif ic melanocortin receptor activation. The N282 residue is located at the extra ce llular end of TM7 and in the model of the human MC4R is situated in close proximity to the acidic residues in TM2 (E94) and TM3 (D117 and D121) ( 128 ). Similar to the F120 Flag-mMC3R muta tion N282A resulted in partial agonist activity and N282S retained full agonist activity with decreases in potency for the endogenous agonists (Table 4-2 and Table 4-5). These findings provide e xperimental evidence that N282 may potentially be participating in the hydr ophilic region of the MC3R, probably through hydrogen bonding. In summary, the use of Ala in combination with Ser mutations was highl y beneficial in that the hydrophilic region of the MC3R was identifi ed as being involved in receptor activation processes in addition to the current hypothesis of ligand molecular recognition aspects of Arg8. The differential pharmacology of endogenous and s ynthetic ligands is probably due to the stereochemistry of DPhe7 residue. The potency losses of JR H887-9 at these re ceptor residues may be explained by the additional amino acids of NDP-MSH and MTII which may be modifying Arg8 ligand positioning as well. It can be ther efore hypothesized that a combination of Arg8 and DPhe7 is important for receptor binding and stimulation. The extent by which each of these residues is involved in the ligand rec ognition and/or ligand indu ced receptor activation, which the exception of D117 which seems to be involved in ligand recepto r interaction, remains to be identified.

PAGE 118

118 Agonist Receptor Interactions : The Hydrophobic Region The melanocortin ligand hydrophobic Phe7 and Trp9 residues are puta tively interacting with hydrophobic binding pocket consisting of residues from TM4, TM5, TM6 and TM7 ( 85, 114, 123, 124, 126-130 ) (Figure 4-2). NDP-MSH is a highly potent and stable peptide agonist. Recent studies using NDP-MSH for the charac terization of forty human polymorphic MC4 receptors side by side with the endogenous mela nocortin peptides showed that potency and binding affinity of this ligand at mutant hMC4 receptors mostly does not differ from wild-type whereas the endogenous ligands result ed in greater potency reduction ( 95 ). Therefore, FlagmMC3R mutations that s how differences of changes in poten cies between the linear agonists NDP-MSH and -MSH, -MSH and -MSH that primarily differ by the stereochemistry around the Phe7 residue were hypothesized herein as pa rticipating in the hydrophobic ligand binding pocket. These residues are Y182, M195, F196, W255, H261, I265, and F281 (Table 4-2). The same receptor residues show also differences in change of potencies between JRH887-9 which contains the NDP-MSH DPhe7 residue, however is missing the additional Nand C-terminal amino acids. These additional ligand amino acids might play a role in stabilizing the melanocortin core sequence and s econdary structure of the ligand, more suitable for interaction with the MC3R. Slight changes in potencies of the cyclic AMW3-130 and MTII compared to NDP-MSH could be observed at the Y182, W255 and F281 and Y182, F196, W255, and F281 receptor residues, respectively (Table 4-2). The I265 residue resulted in loss of ligand functional activity and binding affinity, only when mutated to Ser (Table 4-2, Table 4-5, Figu re 4-8 and Figure 4-9). This implicates this residue as part of the hydrophobic receptor bindi ng pocket and highlights the importance of the use of the hydrophilic Ser mutation strategy in addition to Ala scanning. Interestingly, the corresponding residue Y260 in the mouse MC4R was not implicated in ligand recognition

PAGE 119

119 aspects but rather into differentiation of a gonist versus antagonist activity of the SHU9119 antagonist ( 114 ). This observation provides further ev idence that the MC 3R and MC4R may possess overlapping but yet dis tinct ligand binding pockets. T267 is located at the extrac ellular end of TM6. Mutation of T267 to either Ala or Ser resulted in loss of functional activity as well as binding affinity, which indicates a putative involvement of this residue in ligand recogniti on aspects (Table 4-2, Ta ble 4-5, Figure 4-8 and Figure 4-9). Both mutant Flag-mMC3 receptors are expressed on the cell surface as illustrated in Figure 4-4 and Table 4-4. This observation was surprising since the corresponding residue in the MC4R actually is Ser. T267 is replaced by Leu in MC1R and Phe in MC2R as illustrated in the alignment of the melanocortin receptors, furthe r suggesting involvement of T267 in receptor subtype specificity (Figure 4-5). It can be speculated that the -methyl group of Thr might be crucial for direct involvement of ligand-MC3R molecular recognition, since this methyl group is the only difference between the amino acids Ser and Thr. These data further provide experimental evidence that Thr and Ser side ch ains are not equivalen t; although their hydrogen bonding capabilities are similar ( 151 ). Additionally, the Thr methyl group may play a defined structural role, differentiating in this case between MC3 and MC4 receptor subtypes. T267 has Pro in (i+2) position. Monte Carlo Simulation st udies have been used to simulate the conformations of socalled (S/TxP) motifs ( 152 ). Interestingly, based on these data significant differences in the conformation of SxP and TxP motifs were possible ( 152 ). It was suggested that due to steric clashes of the -methyl group, Thr preferably forms a hydrogen bond to the backbone of residue i-4, whereas Ser might hydrogen bond to the backbone of i-3 which was suggested to result into different bend angles in the Pro-kinked -helix ( 152 ). Mutation of T267 to Ala is lacking the -methyl group therefore completely a bolish the important conformational

PAGE 120

120 constrain introduced by Thr and lead to a stru ctural nonfunctional recept or. Ser however, also lacks the -methyl group and due to its ability to fo rm hydrogen bonds to the (i-3) residue which is L264 may induce a secondary conformational ch ange in TM6 which is unfavorable for ligand binding and/or receptor stimulation. Additionally, T267 is putatively located in close proximity to L264 and I265 (Table 4-2), potentially invol ved into receptor activat ion and ligand-receptor recognition, respectively. These two residues ar e substituted by the corresponding Phe and Tyr (Figure 4-5) in MC4R further i ndicating the differential regulation of this region in the MC3 and MC4 receptor subtypes. Receptor residues, putatively lining the hydr ophobic binding pocket, except for I265 and T267, were identified in this study by differences in potencies of structurally diverse ligands. Interestingly, a report of site -directed mutagenesis studies of the human MC3R only identified four receptor residues as part of the hydrophobic binding pocket ( 123 ). This might be due to the use of the highly potent NDP-MSH as the only ligand in co mbination with Ala scanning mutagenesis ( 123 ). Our study additionally employed the Ser mutation strategy, described in detail above which dramatically contributed to the identification of the hydrophobic binding pocket described herein. It might be also envision ed that ligand interactio ns occur with multiple subsites of the receptor, which is supported by mutagenesis studies of the human MC1R where mutagenesis of single aromatic residues to Ala did not significantly a ffect binding and potency, but double and to a higher extent triple aromatic mutants reduced binding affinities and potencies substantially ( 129 ). The higher binding affinity of the DPhe7 containing ligands might be due to correct orientation of the DPhe7 moiety allowing for either compensating or alternate interactions with the receptor, which might not be possible for the LPhe7 configuration. The use of multiple structurally distinct ligands in combination with Ala and Ser mutage nesis provided further

PAGE 121

121 considerable insight into the MC3R hydrophobi c binding pocket. The putative ligand binding pocket of the mMC3R is illustrated in Figure 4-10. Residues Important for Receptor Sign al Transduction and Structure Based upon the 2.6 crystal structure of bovine rhodopsin ( 86 ) it was suggested that some highly conserved residues among rhodopsin-like GPCRs are found to form binding sites for water molecules, including N56 (1.50) D84 ( 2.50), D295 (7.49) and Y299 (7.53) (mouse MC3R numbering). This water cluster is linked to D295, a key residue which is located at the initial position of the (N/D)PxxY motif commonly found in the helix VII of the rhodopsin-like GPCRs. D84 is potentially directly hydrogen-bonded to N56 (1.50) in TM1, and these two are frequently referred to as the ND pair in rhodopsin-like GPCRs. It is believed that the three highly conserved residues, N56, D84 and D295 (mous e MC3R numbering) constitute a hydrogenbonded chain in the inactive state structure of bovine rhodopsin and that disruption of these constraints may be involved in the receptor activation process ( 86 ). The close proximity of residue 2.50 (TM2) and 7.49 (TM7) is experimental ly supported by earlie r reciprocal mutation studies at these positions of the human serotonin receptor subtype (5HT2A) ( 153 ) and the mouse gonadotropin-releasing hormone receptor (GnRH) ( 154 ). Further proof was provided from a mutagenesis report of the mouse thyrotropin-re leasing hormone receptor (TRH) suggesting that the conserved residues 1.50, 2.50 and 7.49 are involved in specific intera ctions between TMs 1, 2, and 7 and that those interactions may be st ructurally important for all members of the rhodopsin receptor subfamily of GPCRs ( 155 ). Both Flag-mMC3R D84 and N56 residues resulted in loss of receptor func tional activity (Table 4-2), and due to their location in TM1 and TM2 it is not anticipated that these residues are directly involved in molecular recognition of melanocortin ligands. Mutation of the D84 residue re tained binding affinity values similar to the wild-type Flag-mMC3R whereas mu tation of N56 also resulted in loss of binding affinity to

PAGE 122

122 NDP-MSH (Table 4-2 and Table 4-5). This indica tes that D84 might be indeed involved in the receptor activation mechanisms, probably thr ough interaction with the DPxxY motif. N56 however might be contributing to the overall struct ural integrity of the receptor, which is also indicated by its high conserva tion throughout the whole family of rhodopsin-like GPCRs. That the N56 Flag-mMC3R is expressed on the cell surf ace is verified by Flow Cytometry (Figure 4-4 and Table 4-4). It may be envisioned that muta tion to Ala would abolish necessary participation in a receptor stabilizing H-bond network, and Ser may not have the right side chain length. D295 is located in TM7 within the highly conserved (D/N)PxxY motif, considered to be important for receptor activation. Both, D295A and D295S mutant receptors resulted in decreased agonist potency and partial agonist effi cacy, while retaining normal binding affinity to NDP-MSH (Table 4-2 and Table 4-5). These resu lts confirm the putative role of the DPxxY motif in receptor stimulation. The lower losses in binding affinities with the D295S as compared to the D295A mutant also support the hypothesis that this residues is inde ed participating in a hydrogen bond network, putatively formed by N56, D84 and D295 ( 86 ). A better knowledge about the determinants underlyi ng receptor activation is highly valuable for the generation of receptor models in the active conformation for the docking of agonist ligands for rational drug design. Residue L247 (6.40), when mutated to either Ser or Ala, resulted in loss of functional activity (Table 4-2 and Table 45). This observation was very su rprising, since the corresponding mutation L250A in the hMC4R resulted in full agonist activity (Figure 4-17) and mutation of L250 to Gln resulted in a constitutively active hMC4R ( 18 ). It was suggested th at the constitutive activity resulted from stabilization of the active stat e, rather from destabilization of the inactive state ( 18 ). A similar mechanism was proposed from st udies of constitutively active rhodopsin

PAGE 123

123 mutations at position 6.40 ( 156 ). Both studies suggested that the 6.40 residue may have an important structural role for GP CR activation by regula ting the position of TM 6, relative to TM3 and TM7. L247A in Flag-mMC3R was still able to bind the NDP-MSH ligand while The L247S mutation did not show binding affinity for NDP-MSH. The cell surface expression of both mutant receptors was fairly low (Table 4-4 and Figure 4-4), which could partly explain the loss of binding affinity of the L247S mutant. Howe ver, it might be also possible that L247 is interacting with nearby hydrophobic receptor resi dues, and then mutation to hydrophilic Ser would lead to repulsion, maybe causing a TM orie ntation which is unfavorable for both ligand binding and receptor signaling. Results from these studies suggest that L247 may play a role in receptor activation but this role might be distinct from the corresponding L250 hMC4R residue. The residue P257 (6.50) is cons erved in rhodopsin-like GPCRs and is therefore likely to play an important functional and st ructural role. It is generally pr edicted that Pro residues induce aflexible kink in the -helix of TM6 and rearrangements of the region around proline residues are important conformational changes accompanying receptor activation ( 151 ). This residue is situated in between a conserved aromatic cluste r of aromatic residues formed by F251 (6.44), W255 (6.48), F258 (6.51) and F259 (6.52). In the 2AR binding of agonist to a residue or residues in this aromatic cluster has been impli cated in either inducing or stabilizing an altered conformation of the side chains within this cl uster that may ultimately result in receptor activation. Such a concerted rearra ngement may be linked to a change in the configuration of the TM6 Pro (6.50) kink region and subsequent move ment of the cytoplasmic end of TM6 away from TM3 ( 151 ). However the relative contribution of each of those residues in ligand recognition and receptor activation of the Flag-m MC3R remains to be identified. Both P257A and P257S mutant Flag-mMC3 receptors resulted in loss of function, when stimulated with

PAGE 124

124 agonist ligands. Mutation to Ala resulted in loss in binding affinity, while P257S was still able to bind NDP-MSH (Table 4-2 and Table 4-5). These re sults underline the stru ctural and functional role of this conserved Pro residue in TM6. P 257A, due to its small size might destabilize the conformation around the Pro kink in TM6, thus causing a conformation unsuitable for ligand binding and/or stimulation. Intr oduction of a Ser residue into -helices may either cause a special bend in the helix ( 151 ) or may repulse surrounding residues, which might lead to a conformation of TM6 still allowing NDP-MSH binding but unable to couple to G-Protein. In summary, loss of receptor stimulation suggests that the conserved P257 (6.50), is crucial for regulating position of TM6, important for receptor activation. The F258 (6.51) residue, resulted in partial a gonist activity when mutated to Ala and in loss of function, when mutated to Ser (Table 4-2 and Table 4-5). However, both mMC3R mutations retained their binding a ffinity to NDP-MSH. These result s indicate that this receptor residue may not be involved in ligand binding bu t may be playing a key role in ligand induced receptor activation and confor mational changes that accompany receptor activation. The complete loss of function upon Ser makes sense c onsidering that this residue is located in a highly hydrophobic environment which might be disturbed by hydrophilic Ser. The F259 (6.52) residue resulted in loss of function, when mutated to Ala, however displayed full agonist activity upon mutati on to Ser, with reduced potencies for -, -MSH and JRH887-9 (Table 4-2 and Table 4-5). This residue might not interact dire ctly with the agonist Phe7 ligand residue since mutation to hydrophobic Al a abolished receptor stimulation, whereas mutation to hydrophilic Ser retained receptor stimulation. However, it may be possible that this residue regulates the positioning of adjacent re sidues because deletion of the bulky hydrophobic aromatic ring upon space generating Ala substituti on resulted in loss of receptor activation. Ser

PAGE 125

125 substitution might repulse surrounding aromatic re sidues in this region, maybe forcing them into positioning more suitable for receptor stimulation. Based upon models of the active and inactive state of the human MC4R residues of TM3 (L128 and I132) and TM6 (W255, F258 and F259) are situated in close proximity ( 128, 147 ), however the relative contributi on of each of these residues in ligand molecular recognition, receptor activation and differentiatio n of agonist versus antagonist activity at the MC3R remains to be identified. Results presented herein led to the hypothesis that the ligand DPhe7 residue is directly interacting with W 255 and I132 residues, whereas L128, F258 and F259 may important for receptor activation. The L128 (3.36) residue is located in TM3 a nd when mutated to Ala and Ser resulted in reduced receptor activation by a gonists but did not affect bindi ng affinity to NDP-MSH. This residue at position 3.36 is Gly in bovine rhodopsin and was implicated to play an important role for rhodopsin function ( 157, 158 ). Mutation of Gly to either Ala or Ser did not differ significantly from the wild-typ e rhodopsin receptor; however mutation to Leu resulted in conversion of the inverse agonist 11 -cis retinal into a partial agonist and this effect was directly related to the side ch ain size, supporting the role of this residue in receptor activation. Additionally, this effect could be reversed by simultaneous substitution of a residue in TM6, indicating the close proximity of this region to TM6 ( 157, 158 ). The reduction in efficacy of the agonists observed at the L128A and Ser Flag-mMC 3 receptors could be explained with a similar mechanism. Mutation of 3.36 to Ala or Ser in MC 3R causes partial agonism (inverse agonism in rhodopsin) whereas L3.36 in MC3R is important fo r full agonist activity (causes partial positive agonism in rhodopsin). In the Flag-mMC3R the si de chain of Leu might be therefore important for full agonist activity to occur.

PAGE 126

126 W255 (6.48) is located in TM6 and mutation to Ala and to higher extend Ser resulted in decreased agonist potencies (Table 4-2 and Table 4-5), suggesting th at this residue is directly involved in ligand molecular recognition. Using specific tryptophan UV-Absorbance changes it was suggested that changes in Trp rotamer c onfiguration are putatively accompanying rhodopsin receptor activation ( 159 ). Studies of the 2AR suggested that a cluster of hi ghly conserved aromatic residues including F6.44, W6.48, F6.51, and F6.52 face the bindi ng-site and may act as rotamer toggle switch ( 151, 160 ). Binding of agonist to a residue or residu es in this aromatic cluster has been hypothesized to induce or st abilize different configur ations of the side chai ns within this cluster which would induce receptor activation ( 151, 160 ). Another study of th e cannabinoid (CB1) receptor suggested that interaction of F3.36 a nd W6.48 act as toggle switch for this receptor, where F3.36 directs orientation of W6.48, switching from the inactive to the active receptor state ( 161 ). Based upon our results and modeling studies of the hMC4R ( 128, 147 ) it is predicted that residues L128 (3.36) and I132 (3.40) of TM3 are in close proximity to F258 (6.51), F259 (6.52) and W255 (6.48) of TM6 (Figure 4-11). Due to the bulkiness of these ami no acid side chains it could be speculated that movement of either of the residues may lead to a subsequent change in configuration of adjacent side chains to avoid ster ic clashes. Furthermore these residues seem to regulate ligand binding and subse quent receptor activation in a concerted manner. I132 and W255 may be important for liga nd molecular recognition, whereas L128 and F258 may facilitate receptor stabilization in its active state, since muta tion of either of these residues resulted in only partial agonist activity. In the inactive state of the hMC4R as well as the 2AR the side chain of W255 is hypothesized to exist in gauche + rotamer configuration (Figure 4-11). The space around W255 could be then occupied by L3.36 and F6.52 which would direct W6.48 in its

PAGE 127

127 inactive gauche + conformation. Figure 4-11 shows an example of how poten tial receptor side chain residue rearrangements coul d occur in this region. Because a model of the mouse MC3R is not available to date, this Figure 4-11 was ge nerated based upon the hMC4R active and inactive state models ( 128, 147 ) and may not represent the exact na ture of the MC3R. It could be envisioned that agonist-induced conformational changes might force F259 and L128 to change their orientation, allowing inte raction of L128 with F258, maybe along with movement of helices 3 and 6, and to avoid steric clashes W255 could ch ange to its trans rotame r, allowing interaction of agonist ligands hydrophobic amino acids wi th W255 and I132. This mechanism remains speculative and requires fu rther identification. The L264 residue (6.57), when mutated to Al a, resulted in a pharmacological profile, similar to the wild-type receptor, however when mutated to Ser loss of functional activity was observed, while retaining binding affinity to NDP -MSH (Table 4-2 and Table 4-5). Therefore, this residue might be important for receptor ac tivation. The corresponding residue F259S in the mouse MC4R did not result into loss of function and was implic ated into differentiation of agonist versus antagonist activity by conversi on of the SHU9119 competitive antagonist into a partial agonist ( 114 ). Thus, these results provide experi mental evidence that the L264 residue might play an important role in the distinct re gulation of receptor activa tion and differentiation of agonist/antagonist activity of the mMC3 compared to the mMC4 receptor. The extracellular loop 3 of the melanocortin receptors is a relatively short loop containing several Cys and Pro residues that are conserved in the melanocorti n receptor family (Figure 4-5). This strongly indicates that this extracellular loop 3 might fold in a very specific way. It has been suggested from Zn2+ binding ( 162, 163 ) and mutagenesis studies ( 147, 164 ) that C268 and C276 (mouse MC3R) residues within this loop form an intraloop disulfide bridge. Because of the

PAGE 128

128 spatial constraint that this disulfide bridge might impose upon the extracellular ends of TM6 and 7, it is very likely that these segments are kept closely together. It might be possible that this structurally and functionally important loop may influence positioning of TM6 and/or alter the conformation of the hydrophobic binding pocket. Mu tation of C268 to Ala resulted in loss of function as well as binding affinity (Table 4-2 and Table 4-5). Mutation of C268 to Ser resulted in loss of functional activity but slight binding affinity to NDP-MSH was retained. S268 might therefore still be able to hydr ogen bond to C272, thus causing slight stabili zation of the secondary structure of extrac ellular loop 3. These results unde rline the importance of the integrity of the disulfide bridge in extracellular lo op 3 and also highlight the possibility that this extracellular loop might be important for the c onformational changes that occur during receptor activation, maybe placing TM6 and TM7 into the ri ght conformation to inte ract with G-Protein. Studies using Zn2+ metal binding sites suggested that duri ng receptor activation, the extracellular end of TM6 might move towards TM3 and the ex tracellular end of TM 5 might be in close proximity to TM6. Additionally, the region in th e extracellular end of TM6 and extracellular loop 3 might be also involved in mela nocortin receptor subtype specificity. R302 is located at the extrac ellular end of TM7 neighboring a Ser residue which in the model of the human MC4R causes a bend connecti ng the intracellular sm all helix 8 to TM7 ( 128, 147 ). Both R302A and R302S Flag-mMC3 receptors resulted in partial agonist activity, which implicates these receptor residues and maybe the C-terminal helix 8 as important for receptor activation (Table 4-2 and Table 4-5). It may be sp eculated that this residue might interact with the conserved DPxxY motif, regul ating positioning of helix 8, as well as ligand induced signal transduction.

PAGE 129

129 I165 resulted in loss of functiona l activity when mutated to e ither Ala or Ser (Table 4-2 and Table 4-5). I165A was still ab le to retain binding affinity to NDP-MSH, however I165S did not show any binding affinity. This fact might be due to the extremely low cell surface expression of the I165S mutant (19% relative to wild-type). Another possibility, why binding affinity towards NDP-MSH was abolished could be that this Ile residue is participating in a network of hydrophobic amino acids, either stabiliz ing receptor structure or through involvement in yet unknown receptor activation mechanism of the MC3R. I165 corresponds to I170 in the hMC4R. A polymorphic receptor I170V was identifie d in obese patients, and when characterized pharmacologically in vitro did not differ significantly fr om the wild-type FlaghMC4R ( 95 ). That no differences in ligand binding and functional activity could be observed with this mutation might be attributed to the fact that the structure of Ile clearly resembles Val. Nonetheless, the I170V hMC4R mutation was found in an obese pa tient, but the molecular mechanisms by which this polymorphism causes a mal functioni ng receptor remain to be identified. The intracellular loops of GPCRs are potential binding sites for G-Prot eins and can contain phosphorylation sites that may be involved into signaling, internalization and receptor recycling ( 165 ). Potential proteinkinase A pho sphorylation sites in the intr acellular loops of the human MC1R are 142RYIS145 and 151RYHS154 ( 165 ). These sites correspond to 142RYVT145 and 151RYHS154 in the mMC3R. Mutation of T145 to Ala or Ser did not result into significant changes in agonist and inverse a gonist potencies (Table 4-2 and Ta ble 4-5). Interestingly, at the T145A mutant SHU9119 was converted into a competitive antagonist, devoid of any partial agonist activity while at the T145S mutation SHU9119 retained its partial agonist activity (Figure 4-12). This residue is putatively located at the interface of TM3 a nd the intracellular loop 2, in close proximity to the DRY motif. The c onserved DRY motif in rhodopsin-like GPCRs is

PAGE 130

130 potentially important fo r signal transduction ( 166 ). It might be envisioned that mutation of T145 may affect the conformation of side chai ns of the DRY motif, possibly through hydrogen bonding since mutation of T145 to Ser did not influence SHU9119 pharmacology, whereas mutation to the small hydrophobic Ala did. Based upon th ese data it could be speculated that this residue might be involved in orientation and/or positioning of TM3, thus maintaining a receptor structure suitable for partial agonism. Another possible explana tion could be that this Thr residue, due to its locatio n in the intracellu lar end of TM3 might be i nvolved in phosphorylation processes and/or inter action with G-Protein. Maybe intrace llular signaling reactions are necessary for partial agonist activ ity of SHU9119, which would be absent with mutation to Ala, leading to competitive antagonism. These explanati ons remain certainly speculative and need to be further verified. The corresponding mutati on T150I of the hMC4R resulted in reduced potencies of the agonists tested and no AGRP inverse agonist ac tivity was observed ( 95 ). The decreased potencies of the agoni sts were explained by the locati on of the T150 residue putatively causing a change towards the extracellular part of TM3 unf avorable for agonist-induced signal transduction ( 167 ). But again, the exact mechanism is not known and requires further experimental investigation. Mutation of Y152 to either Ala or Ser resulted in partial agon ist activity of all agonists tested. However, the antagonist potency of SH U9119 increased 16-fold for mutation to Ala and 8-fold for mutation to Ser (Table 4-2 and Table 45). This residue is located in the intracellular loop 2 and represents like T145 a potential phosphor ylation site. If this residue is involved in modifying TM3 and 4 to a conformation resembling an inactive receptor state, which would be supported by the fact that only partial agonist activity was observed and SHU9119 antagonist

PAGE 131

131 potency was increased or whether this Y152 residu e is involved into in tracellular interactions with effector proteins requi res further investigation. K160 is located in the end of intracellular loop 2, beginning of TM4. Arg and Lys residues are commonly predicted to lie at the cytoplasmi c end of TM helices, where they anchor the TM helix to the membrane through interaction wi th negatively charged phospholipid headgroups ( 151 ). Additionally, Arg/Lys groups at the cyt oplasmic ends of hydrophobic TM domains are important for the right membrane insertion of TM helices during protein processing in the ER. Mutation of K160 to hydrophobic Ala did not result into significant changes in agonist or antagonist potencies (Table 4-2 and Table 4-5). However, mutation to hy drophilic Ser resulted in loss of functional activity, but st ill allowed binding to NDP-MSH (T able 4-2 and Table 4-5). The cell surface expression of the K160S mutant recep tor was relatively low (Table 4-4 and Figure 44) which might be due to improper folding of the receptor followed by impaired processing to the cell membrane. This receptor might still have a conformation which allows for ligand binding but may be affecting orie ntation of intracellular loop 2 hindering receptor activation and/or interaction with G protei n. Again, this explanation remains speculative and remains to be experimentally verified. Differentiation of SHU9119 Antagon ist versus Agonist activity The difference between the agonist MTII and antagonist SHU9119 is the bulky DNal2 group in place of the agonist DPhe7 residue (Table 4-1). The hypot hesis that a bulky group at the DPhe7 position causes antagonism at the MC3 and MC4 receptors has been generated to explain the differential pharmacology of MTII and SHU9119 at these receptors ( 52 ). The structures of MTII and SHU9119 are illustrated in Figure 4-13. Little is known about the molecular mechanism of antagonism of SHU9119 at the MC3R. Several potential mechanisms might be possible: (i) SHU9119 and MTII co mpete for the same binding pocket (ii) SHU9119 binds to the

PAGE 132

132 inactive state and sterically prev ents rotation of side chains ne cessary for conformational changes during receptor activa tion and/or (iii) SHU9119 directly inte racts with residues, prevents them from undergoing conformational changes. As described above, residues of TM3 (L 128 and I132) and TM6 (W255, F258 and F259) may be located in close proximity and based upon the results presented he rein, it is hypothesized that the ligand DPhe7 residue is directly interacting with W255 and I132, whereas L128, F258 and F259 are important for receptor activation (Figur e 4-11). Mutation of either of these receptor amino acids resulted in conversion of SHU9119 antagonist to agonist activity. The L128 (3.36) residue is located in TM 3. The corresponding residues L165 hMC3R and L133 hMC4R have been implicated to be involve d in the differentiation of agonist versus antagonist activity of SHU9119 but the exact molecular mechanism remains speculative ( 123, 138 ). Using molecular homology models of the hMC4R in the active and inactive state it was suggested that the DNal2 residue could be eas ily accommodated into a cavity formed by L133 and W255 in the inactive receptor state, however prevented rotation of these residues important for receptor activation ( 128 ). In the study presented herein L128 was mutated to Ala and Ser. Both mutations resulted in re duced receptor activation by agonists but did not affect binding affinity to NDP-MSH (Table 4-2, Table 4-5 a nd Figure 4-14). Additiona lly, SHU9119 lost its competitive antagonist activity but retained simila r partial agonist activity as MTII (Table 4-2, Table 4-3, Table 4-5 and Figure 4-14). The I132 (3.40) residue is located in TM3 one helical turn below L128. Both mutations to Ala, as well as Ser resulted in potency losse s for the endogenous agonists as well as JRH887-9 and MTII (Table 4-2, Table 4-3, Table 4-5 and Fi gure 4-14). However, full agonist activity was retained. SHU9119 resulted in a full agonist at the I132A mutant recep tor and antagonist with

PAGE 133

133 increased partial agonist activity at the I132S mutant receptor (Table 4-2, Table 4-3, Table 4-5 and Figure 4-14). W255 (6.48) is located in TM6 and mutation to Ala and to higher extend Ser resulted in decreased agonist potencies, suggest ing that this residue is direct ly involved in ligand molecular recognition (Table 4-2, Table 4-3, Table 4-5 and Figure 4-14). Th e antagonist SHU9119 resulted in a full agonist at the W255 mutant receptors (T able 4-2, Table 4-3, Table 4-5 and Figure 4-14). F259 (6.52) is located one helical turn above W255 and mutation to Ala resulted in loss of function, whereas mutation to Ser resulted in full agonist activities, however reduced potencies, thus suggesting that this re sidue is not involved in mol ecular recognition of the hydrophobic receptor residues but rather in regulation of recepto r activity, as described earlier in this chapter (Table 4-2, Table 4-3, Table 4-5 and Figure 4-14). The antagonist SHU9119 resulted in a full agonist at the F259S mutant recep tor (Figure 4-14). F258 when muta ted to Ala resulted in partial agonist activity, and when SHU9119 was tested at this mutant receptor no antagonist activity was observed (Table 4-2, Table 4-3, Table 4-5 an d Figure 4-14). These results provide further experimental evidence that th e region around TM3 and TM6 may be regulated in a tightly fashion, since mutation of either of those rece ptor residues resulted in conversion of SHU9119 into an agonist. An explanation for this could be that a lack of one of these residues through space generating mutations to Ala and/or Ser re nders this region more flexible and more favorable for signal transduction. The DNal2 ring might either sterically interact with the aromatic cluster region, preventing necessary si de-chain rearrangements, or might directly interact through aromatic interactions with the Trp and Phe residues. Studies of the mMC4R suggested that SHU 9119 might interact with residues F254 and F259 (mouse MC4R numbering) through aromatic forces, preventing necessary rotation of

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134 TM6 for receptor activation, since mutation of eith er of these residues re sulted in conversion of the SHU9119 competitive antagonis t into partial agonism ( 168 ). F259 corresponds to Leu in mMC3R (L264) and MC1R. It was suggested that stronger interaction of SHU9119 with F259 results into competitive antagonism whereas pr esence of L264 might be responsible for the partial agonist activity observed for the MC 3R, or full agonist activity at the MC1R ( 168 ). This hypothesis might be confirmed by the mutagenesis results herein, since L264 was identified to not participate into differentia tion of agonist versus antagonis t activity, but rather might be important for ligand induced signal transduction. In MC1R the corresponding residue to L128 is Met and it has been demonstrated experimentally that mutation of Leu to Met resulted in SHU9119 partial agonist act ivity at the hMC4R ( 138 ), however it was not tested for SHU9119 competitive antagonism. When tested at the L 165M hMC3R SHU9119 resulted in agonism, with efficacy slightly less then MTII ( 123 ). These data provide the basi s to generate a new hypothesis whereby a combination of L3.36 and F6.57 mi ght be necessary for SHU9119 competitive antagonism at the MC4R, whereas a combinati on of L3.36 and L6.57 is important for SHU9119 competitive antagonism/partial agonism at th e MC3R and a combination of M3.36 and L6.57 might be responsible for SHU9119 MC1R agonism. This hypothesis could be further verified by creating the corresponding double mutations at each receptor subtype. The residue F179 is located in TM4 and did not result into potency changes when mutated to either Ala or Ser for the agonist ligands, wh ich does not implicate this residue in molecular recognition of agonists of the mMC3R (Table 4-2, Table 4-3, Table 4-5 and Figure 4-15). Interestingly, the corresponding residue in mous e MC4R resulted in lo ss of functional activity and binding for melanocortin agonists, but was st ill able to bind to the antagonist SHU9119. At the F179A mutant receptor SHU9119 showed sim ilar antagonist pharmaco logy to the wild-type

PAGE 135

135 receptor. However, at the F179S mutant recep tor SHU9119 possessed elevated efficacy, without any antagonist activity. F179 is lo cated in close proximity to Y182, implicated into agonist molecular recognition, and might re gulate positioning of this re sidue. SHU9119 might sterically interact with F179, preventing necessary conf ormational changes duri ng receptor activation, necessary for agonists to interact with Y182. M199 is located in TM5 at position 5.50 which corresponds to a conserved Pro residue in bovine rhodopsin (Figure 4-5). In terestingly, removal of this re sidue through muta tion to Ala and or Ser causes increases in potenci es of all ligands tested, with the most pronounced for JRH887-9 (Table 4-2, Table 4-3, Table 4-5 and Figure 4-15). Convers ely, the inverse agonist AGRP resulted in decreased potency at the M199A mutant. These obse rvations indicate that M199 might be involved in constraining the receptor st ructure in its inactive configuration. SHU9119 is converted into agonist at the M199S and has in creased partial agonist activity at the M199A mutant receptor. M199 is located one helical tu rn above M195 and also close to F196 and F197. An arylsulfur interaction has been proposed to be a favorable interaction in proteins because of the observed proximity of cysteine (Cys) and methi onine (Met) to aromatic side chains in protein crystal structures, although the exact driving force for this interaction is still under investigation ( 169 ). M199 might be involved in maintaining the inactiv e state and direct orientation of F196 and/or M195 into the ligand binding pocket. Anot her possible mechanism could be that M199 is engaged in a hydrogen bond network, which migh t be involved in maintaining an inactive receptor conformation. M199 is located in clos e in between two Met side chains, M195 and M204. Additionally, the side chain of M199 of TM 5 might be also located close to hydrogen bond donating residues in TM3, C131, S133 and N136. One might speculate that a network of hydrogen bonds in this area may be stabilizing an inactive receptor conformation, and especially

PAGE 136

136 when considering the putative involvement of TM3 in receptor activation, and the close proximity of C131, S133 and N136 to L 128 and I132, discussed in detail above. T270 is located in extrace llular loop 3 connecting TM6 with TM7. The potential involvement of this loop into receptor activati on and structural integrity has been discussed above. The Thr residue might be involved in a hydrogen bond network, which might change during receptor activation mechanisms, regulating positioning of TM6 and 7. This explanation is supported by experimental evidence since SHU9119 is converted into a full agonist at the T270A mutation but still retained antagoni st activity at the T270S mutant receptor (Table 4-2, Table 4-3, Table 4-5 and Figure 4-15). Thus, the Ser residue might still be able to from the necessary Hydrogen-bonds for retaining proper mMC3R function. Intere stingly, the T270 residue is replaced by Gln in MC4, MC5R and mouse MC1R Asn in mouse MC2R, Ser in human MC2R and Glu in human MC1R. Given the fact of the high variability of this residue together with its involvement in differentiation of agonist versus antagonist activity, T270 ma y play a key role in receptor specific activation/inhibition mechanis ms which might be of interest for further experimental investigations. F281 is located in TM7 and seems to be i nvolved in the hydrophobic network of receptor amino acids participating in the putative liga nd binding pocket. The drop in potency of the inverse agonist AGRP might indicate that this lig and is interacting with this residue in the inactive receptor state. The melanocortin base d antagonist SHU9119 is converted into a full agonist at the F281A and F281S Flag-mMC3 re ceptors (Table 4-2, Ta ble 4-3, Table 4-5 and Figure 4-15). Based upon these results it might be speculated that the DNal2 ligand moiety is interacting directly with the F281 side chai n, and either prevent the agonist MTII from

PAGE 137

137 interacting with this residue a nd/or may hinder sterically the necessary conformational changes of TM6 and 7 during receptor activation mechanisms. The original hypothesis regarding the mechan ism of antagonism of SHU9119 at the MC3 and MC4 receptor is attributed to the addition of a bulky moiety at the 7 position of the MTII agonist ( 52 ). This former hypothesis was modified for the mMC4R suggesting an alternative hypothesis for the mechanism of SHU9119 anta gonism, where both the stereochemistry and bulky naphthyl ring position (DNal1 versus DNal2) are impor tant for positioning of the ligand Arg8 residue interactions with the corr esponding hydrophilic am ino acids E94, D117, and/or D121 (mMC3R numbering) ( 114 ). This might be explained by putative cationinteraction between the positively charged guanidi ne moiety and the electron rich naphtyl ring. This study provides further experimental eviden ce supporting this hypothesis. D117 is putatively interacting with Arg8 and SHU9119 possessed enhanced par tial agonist activity at the D117A and D117S mutant receptors, devoid of comp etitive antagonism (Figure 4-16). E94S did not result into changes is SHU9119 potencies a nd is maybe not involved in SHU9119 molecular recognition (Table 4-3 and Figure 4-16) At the D121 mutant receptors a pA2 value could not be determined at up to 1 M antagonist concentration, sugge sting reduced SHU9119 potency (Figure 4-16). The relative contribution of D121 and E94 at the mMC3R, however, requires further investigation. The above mentioned residues were identified to differentiate SHU9119 antagonism versus agonism, by enhancing partial agonist activit y or converting SHU9119 into a full agonist. Interestingly, two mutations resulted in d ecreasing SHU9119 partial a gonist activity resulting into SHU9119 competitive antagonism. These Flag -mMC3R mutant receptors are N91A (Figure 4-16) and T145A (Figure 4-12). As discussed above, N91 may be putatively interacting with

PAGE 138

138 acidic residues of TM2 and TM3. It can be ther efore speculated that the Asn side chain is directing positioning of D117 and D121 identifi ed as being involved in SHU9119 antagonist activity through inte raction with Arg8. Mutation to Ala may abolish th is interaction, thus leading to SHU9119 competitive antagonism, further s upporting the hypothesis that intramolecular cationinteractions contribute to SHU9119 an tagonist activity. The molecular mechanism underlying the T145A mutation are less obvious and as mentioned earlier possible explanations include, interaction with the conserved DRY motif, modification of TM3 positioning and/or involvement into intracellu lar signaling processes. In summary, this study revealed multiple dete rminants involved in the differentiation of SHU9119 antagonist versus agonist activity. We fu rther provided experimental evidence that SHU9119 antagonism is not only due to steric hi ndrance of a bulky group at the ligand position 7 of the hydrophobic binding pocket but additi onally dependent on positioning of Arg8 and interaction with the hydrophilic ligand binding pocket. Moreover, re sidues have been identified as either directly and/or sterically interacting with the SHU9119 ligand (I132, W255, F281, L128, F179 and F259). Residues M199, T270, N91 and T 145 may not be directly in contact with SHU9119 but may be modifying the receptor st ructure favorable for SHU9119 antagonism. Additionally a new hypothesis has been proposed that the right combination of side chains of residues 3.36 and 6.57 might determin e SHU9119 antagonism or agonism. Determinants for AGRP Antagonist Activity Several studies have provided experimental evidence for AGRP possessing inverse agonist activity in addition to competitive antagonism of -MSH ( 15, 17, 18, 114 ). Attempts have been undertaken to map and model the inte ractions of AGRP with the MC4R ( 105, 114, 143, 147, 170 ), but determinants of AGRP-MC3R ligand-recep tor complexes have not been reported. In this study, selected Flag-mMC3 mutant receptors were functionally characterized with the AGRP

PAGE 139

139 C-terminal fragment hAGRP(87-132). Interestingl y, similar to a report characterizing 40 hMC4R polymorphisms AGRP was relatively inert to single receptor mutations ( 95 ). The highest decreases in antagonist potencies were observe d at the M199A, L264A, F281A and F281S. It is hypothesized, based upon the extended ternary complex model of rece ptor activation that inverse agonists may bind to either active or inactive rece ptor state and shift the equilibrium towards the inactive receptor population ( 90 ). M199 mutant receptors resulted in increased agonist potencies and consistent with this obs ervation, AGRP inverse agonist potency was reduced, further implicating this residue in maintaining the MC3R in its inactive form (Table 4-3). L264 was identified as participating in receptor activa tion mechanisms and the decrease in AGRP potency may confirm these results (Table 4-3). The corresponding residue to F281 in the hM C4R was suggested from homology modeling studies as directly intera cting with one of the antagonist core sequence Arg111-Phe112-Phe113 phenylalanine residues. F281A and to a higher extent F281S Flag-mMC3R mutations showed significantly decreased AGRP potency (Table 4-3), suggesting a si milar interaction of the AGRP Phe112 or Phe113 residues with this re ceptor amino acid. Residues F120 and H261 resulted in increases in AGRP potencies (T able 4-3) which might impli cate these side chains into intramolecular receptor interactions for mainta ining the active state. The N91A Flag-mMC3R was the only mutant possessing obser vable inverse agonist activity. Mutation of N91 to Ala converted the partia l agonist SHU9119 at the wild-type receptor into a competitive null antagonist and AGRP was converted into an inve rse agonist (Table 4-3 and Figure 4-17). The observati on is quite intriguing since fu ll agonists did not loose their efficacy, however the two antagonists tested both resulted in loss in efficacy. This might provide experimental evidence that antagoni sts/partial agonists and antagoni sts/inverse agonists interact

PAGE 140

140 distinct from full agonists with the mouse MC3R. If this effect is mediated by the residues of hydrophilic binding pocket remains to be identified. One might also speculate, that the inverse agonist activity of AGRP is obs erved because the N91A mutant might possess a certain degree of constitutive activation, which is often nece ssary to observe inverse agonist activity ( 16, 104 ). However, receptor theories proposed that comp etitive null antagonist s result into partial agonists at constitutively active receptors ( 104 ). Indeed, the constitutively active mouse MC4R mutation M192F converted the MC4R null anta gonist SHU9119 into a partial agonist, while retaining competitive antagonism ( 16 ). That we observed the comp lete opposite effect in the study presented herein which changed the MC 3R partial agonist SHU9119 into a null antagonist clearly excludes this possibility, th at the N91A Flag-mMC3R might be constitutively active. Transient transf ection assays where HEK293 cells we re transfected with increasing amounts of wild-type and N91A Flag-mMC3R plasmid confirm that N91A is not constitutively active (Figure 4-18). One possibili ty why N91A could have highe r basal activity might be the observation that the N91A muta nt seems to possess higher cell surface expression, as compared to the wild-type Flag-mMC3R (Figure 4-4, Table 4-4). Functional character ization of AGRP at the Flag-mMC3R was beneficial and led to the identification of receptor residues important for maintaining the active as well as the inactive receptor states. We provided additional evidence for AGRP inverse agonist activity at the mMC3 R and one residue was identified which is putatively interacting with one of the core Phe112 or Phe113 antagonist residues. Residues Involved In Differentiat ion between MC3R and MC4R The residues which might be involved in the differentiation of the MC3R versus the MC4R subtypes have been mostly mentioned during th e discussion above. Due to the vast amount of data discussed in this chapter this paragraph has the purpose to point out the importance of these residues more concisely.

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141 The F179S (6.40) Flag-mMC3R was identified herein as important for the differentiation of SHU9119 antagonist/agonist activity. This mutant receptor resulted in full agonist activity of all the agonist tested. Of in terest, the correspond ing F176S mouse MC4R mutation lost any functional activity when stimulated by agonist ligands, however reta ined reduced binding affinity to SHU9119. It was also suggested from these studies that this residue might be important for functional antagonism of AGRP ( 114 ). Homology modeling studies of the mMC4R and hMC4R further implicated this residue as inter acting with the antagoni st core sequence Phe112 and Phe113 ( 143, 147 ). If this residue is also involved in molecular recognition of the AGRP-mMC3R receptor complex is not conclusive from these studies and remains to be identified. The L264S (6.57) Flag-mMC3R resulted in loss of functional activity, but retained binding affinity to the NDP-MSH ligand, which would impli cate this residue as important for receptor activation. The corresponding muta tion F259S of mouse MC4R reta ined full agonist activity and was identified as important for the differentiatio n of agonist/versus anta gonist activity at the MC4R subtype. Hereby, the aromatic character of the Phe side was suggested to be involved in strong interactions with the anta gonist DNal2 moiety. The presence of Phe instead of Leu was used to explain the absence of SHU 9119 partial agonist activity at the MC4R ( 114 ). In the mMC3R residue 6.57 was shown to play a differe nt role which might confirm the before mentioned hypothesis. Mutation of T267 (6.60) to either Ala or Ser resulted in loss of functional as well as binding affinity, which might implicate this position as important for ligand molecular recognition of the mMC3R. Interestingly, the co rresponding amino acid in the MC4R actually is Ser. This observation clearly indicates that this Thr residue might play a key role in differentiation of the two receptor subtypes.

PAGE 142

142 Residue L247 (6.40) of mMC3R corresponds to L250 in the hMC4R. Chapter 6 will extensively investigate this pos ition in the hMC4R which was identified to participate into switching the hMC4R from the inactive to the active confor mation. The L247 mMC3R residue might be regulated in a diffe rent way since mutation to Ala and Ser abolished signal transduction, whereas the L250A hMC4R mutant resulted in full agonist activity (Figure 4-19). This study provided experimental evidence that residue 6.40 of mMC3R may be involved in receptor activation but with a mechanism distin ct from the hMC4R, which remains to be identified. Conclusion In summary, 78 Flag-mMC3R receptor mutations have been generated and pharmacologically characterized using several st ructurally diverse a gonist and antagonist ligands. This study identified residues partic ipating in the hydrophi lic and hydrophobic binding pocket of melanocortin ligands, residues that ar e important for receptor activation, key residues involved in differentiation of agonist/antagonist activity as well as differentiation of MC3R/MC4R subtypes. Results can be potenti ally useful for generating MC3R homology models in the active, as well as inactive state as template for structure-based drug design. Additionally, these results can be used to genera te testable working hypotheses for further SAR studies and site-directed mutagene sis studies and thus contribute substantially to the iterative drug design process. Ultimately, the discovery of MC3R selective ligands will aid in elucidating the myriad of physiological functions of the MC3R in vivo which may lead to the identification of new treatment options for obesity and related diseases.

PAGE 143

143 Figure 4.1. Two-dimensional repr esentation of the Flag-mMC3R, showing the residues that were mutated to Ala and Ser. The Flag-tag was inserted in between the Met start codon and the first receptor amino acid. Figure 4-2. Proposed agonist resi dues DPhe-Arg-Trp interactio ns with the mMC4R, modified from Ref ( 114 ). The putative ligand binding pocke t consists of a network of hydrophobic and acidic amino acids.

PAGE 144

144 Table 4-1. Primary sequence of the peptides used for pharmacological characterization of the wildtype and mutant Flag-mMC3 receptors. Peptide Primary Sequence AMW3-130 Tyr-c[CysHis-DPhe-Arg-Trp -Asn-Ala-Phe-Cys]-Tyr-NH2 -MSH Ac Ser-Tyr-Ser-Met-GluHis-Phe-Arg-TrpGly-Lys-Pro-Val -NH2 -MSH Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg-Met-GluHis-Phe-Arg-TrpGly-Ser-Pro-ProLys-Asp 2-MSH Tyr-Val-Met-GlyHis-Phe-Arg-TrpAsp-Arg-Phe-Gly NDP-MSH Ac-Ser-Tyr-Ser-Nle-GluHis-DPhe-Arg-TrpGly-Lys-Pro-Val-NH2 JRH887-9 AcHis-DPhe-Arg-Trp -NH2 MTII Ac-Nle-c[AspHis-DPhe-Arg-TrpLys]-NH2 SHU9119 Ac-Nle-c[AspHis-DNal(2)-Arg-Trp -Lys]-NH2 hAGRP (89-132) CVRLHESCLGQQVPCCDPCATCYC RFF NAFCYCRKLGTAMNPCSRT

PAGE 145

145 Table 4-2. Functional Activities of the endoge nous and synthetic agonists at the wild-type and mutant Flag-mMC3 receptors. TM NDP-MSH EC50(nM) Fold Diff. AMW3-130 EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. JRH887-9 EC50(nM) Fold Diff. MTII EC50(nM) Fold Diff. mMC3R 0.14.014 1 0.72.060 1 16.05.14 1 1.65.15 1 29.22.38 1 1114 1 0.11.010 1 S52A TM1 0.340.033 2 1.120.13 2 22.6710.16 1 4.151.30 3 70.0016.24 2 392170 2 0.220.063 2 N56A TM1 >1000 >1000 >1000 >1000 >1000 >10000 >1000 N56S TM2 >1000 >1000 >1000 >1000 >1000 >10000 >1000 D84A TM2 >1000 >1000 >1000 >1000 >1000 >10000 >1000 D84S TM2 >1000 >1000 >1000 >1000 >1000 >10000 >1000 N91A TM2 0.300.055 2 1.630.33 2 21.775.11 1 6.901.26 4 11025.40 4 22482.54 2 0.320.067 3 N91S TM2 >1000 >1000 >1000 >1000 >1000 >10000 >1000 E94A TM2 >1000 >1000 >1000 >1000 >1000 >10000 >1000 E94S TM2 0.680.10 5 10.430.80 14 >1000 50% @ 1 M >1000 50% @ 10 M 0.500.15 5 I96A TM2 0.160.026 1 0.620.060 1 8.215.02 -2 2.61.04 2 27.319.25 1 143.5232.11 1 0.0700.023 1 I96S TM2 0.110.010 pA 1 0.550.042 pA 1 12.771.06 pA 1 2.350.30 pA 1 63.3416.29 pA 2 425138 pA 4 0.130.023 pA 1 D117A TM3 0.570.18 4 19.376.90 27 20%@1 M 50%@1 M 20% @ 1 M 50% @ 10 M 1.220.28 11 D117S TM3 0.690.14 5 34.304.03 48 20%@1 M 70%@1 M 25% @ 1M 70% @ 10 M 1.550.38 14 F120A 0.0980.020 pA 1 0.390.075 pA 1 pA 2.270.15 pA 1 pA 398.5192.32 pA 4 0.100.009 pA 1 F120S TM3 0.15.022 1 1.41.38 2 50% @ 1 M 8.59.71 pA 5 50% @ 1 M 60% @ 10 M 0.41.13 4 D121A TM3 12.99.90 pA 93 pA >1000 >1000 >1000 >10000 pA D121S TM3 6.84.58 pA 49 pA >1000 >1000 >1000 >10000 pA L128A TM3 pA pA pA pA pA pA pA L128S TM3 pA pA pA pA pA pA pA I132A TM3 0.32.042 2 2.13.16 3 35.22.16 2 6.15.42 4 1038 4 124377 11 0.39.091 4 I132S TM3 0.43.13 3 2.35.59 3 80% @ 1 M 14.40 1.17 9 80% @ 1 M 80% @ 10 M 0.74.15 7

PAGE 146

146 Table 4-2. continued. NDP-MSH EC50(nM) Fold Diff. AMW3-130 EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. JRH887-9 EC50(nM) Fold Diff. MTII EC50(nM) Fold Diff. mMC3R 0.14.014 1 0.72.060 1 16.05.14 1 1.65.15 1 29.22.38 1 1114 1 0.11.010 1 T145A IL2 0.43.16 3 2.36.61 3 74.736.69 5 6.97.45 4 1587 5 5124 5 0.44.063 4 T145S IL2 0.070.019 -2 0.452.041 -2 9.21.36 1 0. 94.16 -2 13.52.89 -2 29.54.51 -4 0.097.047 1 Y152A IL2 0.20.14 pA 1 0.37.076 pA -2 13.07.6 pA 1 1.53.40 pA 1 86.788.94 pA 3 32481 pA 3 0.088.017 pA 1 Y152S IL2 0.35.084 pA 3 1.33.087 pA 2 34.521.03 pA 2 12.07.13 pA 7 1712 pA 6 1052281 pA 9 0.20.06 pA 2 K160A TM4 0.13.041 1 0.66.11 1 11.95.69 1 2.90.76 2 41.446.41 1 1175 1 0.11.27 1 K160S TM4 >1000 >1000 >1000 >1000 >1000 >10000 >1000 I165A TM4 >1000 >1000 >1000 >1000 >1000 >10000 >1000 I165S TM4 >1000 >1000 >1000 >1000 >1000 >10000 >1000 C175A TM4 0.13.034 pA 1 0.55.070 pA 1 17.79.67 pA 1 2.15.52 pA 1 61.467.12 pA 2 1176 pA 1 0.11.022 pA 1 C175S* TM4 0.08.033 pA -2 0.74.073 pA 1 74.989.81 pA 5 3.11.13 pA 2 69.300.95 pA 2 4790 pA 4 0.073.0012 pA -2 F179A TM4 0.17.022 1 0.63.11 1 26.903.12 2 8. 33.00 5 36.421.84 1 94.682.71 1 0.090.014 1 F179S TM4 0.056.012 -3 0.44.11 1 11.09.07 1 1. 17.22 1 19.99.79 1 39.250.28 -3 0.063.0041 1 Y182A TM4 0.18.015 pA 1 2.26.61 pA 3 pA pA pA pA 0.70.061 pA 6 Y182S TM4 0.23.037 2 6.70.46 9 60% @ 1 M 16. 67.04 10 60% @ 1 M 60% @ 1 M 1.01.12 9 E184A EL2 0.15.27 pA 1 1.09.14 pA 2 23.78.26 pA 1 5.19.43 pA 3 68.253.82 pA 2 1038144 pA 9 0.17.013 pA 2 E184S EL2 0.12.017 pA 1 0.87.15 pA 1 17.72.67 pA 1 8.17.33 pA 5 62.845.26 pA 2 69273 pA 6 0.34.073 pA 3 M195A TM5 0.18.041 1 1.55.47 2 23.67.15 1 4.20.32 3 1031 4 68407 6 0.13.017 1 M195S TM5 0.21.070 2 0.91.18 1 60% @ 1 M 4. 32.51 3 70% @ 1 M 70% @ 1 M 0.14.012 1 F196A TM5 0.17.021 1 1.23.14 2 21.29.92 1 3.59.67 2 86.850.74 3 1965827 18 0.25.0067 2 F196S TM5 0.34.11 2 2.45.78 3 50% @ 1 M 6.52.81 4 50% @ 1 M 1793323 16 0.50.15 5

PAGE 147

147 Table 4-2. continued. TM NDP-MSH EC50(nM) Fold Diff. AMW3-130 EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. JRH887-9 EC50(nM) Fold Diff. MTII EC50(nM) Fold Diff. mMC3R 0.14.014 1 0.72.060 1 16.05.14 1 1.65.15 1 29.22.38 1 1114 1 0.11.010 1 F197A TM5 0.12.031 1 0.75.098 1 12.38.53 1 2.11.60 1 38.873.68 1 49389 4 0.14.013 1 F197S TM5 0.012.033 -12 0.63.11 1 8.21.66 1 1.16.11 1 35.01.26 1 2386 2 0.16.027 1 M199A TM5 0.078.025 -2 0.38.050 -2 2.74.32 -6 0.85.26 -2 7.52.23 -4 3.01.45 -37 0.057.021 -2 M199S TM5 0.035.0072 -4 0.31.095 -2 4.95.18 -3 0.40.15 -4 5.28.50 -6 0.94.35 -97 0.033.0043 -3 L247A TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 L247S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 W255A TM6 0.18.032 1 1.79.21 3 65.951. 53 4 9.28.67 6 1473 5 1230298 11 0.79.22 7 W255S TM6 0.23.070 2 4.14.67 6 60% @ 1 M 13. 07.25 8 60% @ 1 M 60% @ 10 M 1.14.22 10 P257A TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 P257S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 F258A TM6 pA pA pA pA pA pA pA F258S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 F259A TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 F259S TM6 0.15.023 1 1.57.26 2 70% 6.17.99 4 70% 70% 0.50.25 5 H261A TM6 0.24.12 pA 2 0.58.20 pA 1 pA 13.45.16 pA 8 pA 1636 pA 1 0.23.11 pA 2 H261S TM6 0.15.018 pA 1 0.84.21 pA 1 pA 15.32.26 pA 9 pA 50300 pA 5 0.30.06 pA 3 L264A TM6 0.18.090 pA 1 0.42.094 pA 1 13.36.82 pA 1 3.19.34 pA 2 77.923.10 pA 3 1256 pA 1 0.11.015 pA 1 L264S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 I265A TM6 0.24.046 2 2.13.10 3 60% @ 1 M 28.500.04 17 60% @ 1 M 89086 8 0.16.27 1 I265S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000

PAGE 148

148 Table 4-2. continued. NDP-MSH EC50(nM) Fold Diff. AMW3-130 EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. -MSH EC50(nM) Fold Diff. JRH887-9 EC50(nM) Fold Diff. MTII EC50(nM) Fold Diff. mMC3R 0.14.014 1 0.72.060 1 16.05.14 1 1.65.15 1 29.22.38 1 1114 1 0.11.010 1 T267A TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 T267S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 C268A TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 C268S TM6 >1000 >1000 >1000 >1000 >1000 >10000 >1000 T270A EL3 0.079.018 -2 0.54.26 1 3.30.89 -5 0.81 0.32 -2 9.36.86 -3 20.90.50 -5 0.033.018 -3 T270S EL3 0.036.0018 -4 0.46.15 -2 8.54.38 -2 0.59 0.083 -3 18.82.25 -2 24.59.45 -5 0.035.0097 -3 T278A EL3 0.18.034 1 0.72.087 1 13.11.28 1 2.62.54 2 43.051.85 1 1392 1 0.13.026 1 T278S EL3 0.17.043 1 0.70.17 1 7.17.04 -2 1. 30.29 1 44.753.61 1 54.894.50 -2 0.14.063 1 N282A TM7 0.14.038 pA 1 1.91.61 pA 3 pA pA pA pA 0.22.042 pA 2 N282S TM7 0.21.029 2 0.91.055 1 70% @ 1 M 9.14.09 6 70% @ 1 M 48846 4 0.12.021 1 F281A TM7 0.14.049 1 2.58.78 4 69.337.66 4 4.05.64 2 2195 8 25.99.74 -4 0.24.17 2 F281S TM7 0.46.091 3 2.81.34 4 60% @ 1 M 6. 96.57 4 60% @ 1 M 18.61.58 -6 0.15.020 1 D295A TM7 1.38.52 pA 10 7.02.99 pA 10 pA 53.502.93 pA 32 pA pA 2.11.17 pA 19 D295S TM7 0.42.10 pA 3 2.21.34 pA 3 pA 38.11.43 pA 23 pA 1428155 pA 13 0.8.02 pA 7 R302A TM7 0.67.12 pA 5 3.54.075 pA 5 pA 13.20.56 pA 8 pA 1425 pA 1 0.71.010 pA 6 R302S TM7 0.54.077 pA 4 2.50.78 pA 8 pA 14.49.99 pA 9 pA 5226 pA 5 0.30.08 pA 3 The indicated errors represent the standard error of the mean from at least three i ndependent experiments done in duplicate. *I ndicates that this experiment was done two times in duplicate and the error is de rived from Standard Deviation. Compounds possessing >1000 or >100 00 denotes EC50 values were not found to possess agonist or antagonist activity at up to 1 M or 10 M concentrations. pA denotes partial agon ist.

PAGE 149

149 Table 4-3. Functional antagonist pharmacology at the wild-type a nd mutant Flag-mMC3 receptors. SHU9119 hAGRP(87-132) pA2 values Pharmacology Ki values (nM) Fold Diff. pA2 values Ki values (nM) Fold Diff. wildtype 8.960.13 partial agonist 1.1018.870.24 1.351 S52A TM1 9.820.29 partial agonist 0.15-79.300.085 0.50-3 N91A TM2 8.320.092 competitive antagonist 4.794 8.510.22 inverse agonist 3.092 E94S TM2 8.960.18 partial agonist 1.1019.070.14 0.85-2 I96A TM2 9.710.13 partial agonist 0.19-6 I96S TM2 9.700.097 partial agonist 0.20-59.130.18 0.74-2 D117A TM3 partial agonist 7.350.88 78.860.24 1.381 D117S TM3 partial agonist 12.562.46 119.180.035 0.66-2 F120A TM3 9.420.034 slight agonist 0.38-3 F120S TM3 9.170.034 partial agonist 0.68-29.920.22 0.12-11 S122A TM3 9.410.13 partial agonist 0.39-38.950.30 1.121 L128A TM3 partial agonist L128S TM3 partial agonist 8.760.35 1.741 I132A TM3 0.960.13 full agonist 18.330.064 4.683 I132S TM3 8.650.07 1.680.43 partial agonist 2.2428.590.34 2.572

PAGE 150

150 Table 4-3. continued. SHU9119 hAGRP(87-132) pA2 values Pharmacology Ki values (nM) Fold Diff. pA2 values Ki values (nM) Fold Diff. wildtype 8.960.13 partial agonist1.1018.870.24 1.351 T145A IL2 8.400.12 competitive antagonist 3.9848.890.35 1.291 T145S IL2 8.270.17 partial agonist5.3758.350.53 4.473 Y152A IL2 10.170.10 slight agonist0.07-169.40.27 0.40-3 Y152S IL2 9.890.11 slight agonist0.13-98.810.53 1.551 K160A TM4 9.280.23 partial agonist0.52-28.830.26 1.481 C175A TM4 9.490.16 partial agonist0.32-39.100.20 0.79-2 C175S* TM4 9.810.0041 partial agonist0.15-7 F179A TM4 9.310.18 partial agonist0.49-29.030.16 0.931 F179S TM4 0.350.015 partial agonist -38.970.064 1.071 Y182A TM4 9.230.15 slight agonist0.59-29.260.15 0.55-2 Y182S TM4 8.720.015 slight agonist1.9118.550.36 2.822 E184A EL2 9.650.11 slight agonist0.22-58.800.14 1.581 E184S EL2 9.400.096 slight agonist 0.40-39.150.58 0.71-2 M195A TM5 8.910.05 partial agonist1.2318.230.25 5.894 M195S TM5 8.910.29 partial agonist1.2318.970.13 1.071 F196A TM5 8.460.053 partial agonist3.4738.570.33 2.692 F196S TM5 8.270.10 partial agonist5.3758.410.53 3.863 F197A TM5 9.210.40 partial agonist0.62-29.470.28 0.34-4 F197S TM5 9.100.047 partial agonist0.7918.790.36 1.621

PAGE 151

151 Table 4-3. continued. SHU9119 hAGRP(87-132) pA2 values Pharmacology Ki values (nM) Fold Diff. pA2 values Ki values (nM) Fold Diff. wildtype 8.960.13 partial agonist1.1018.870.24 1.351 M199A TM5 9.520.019 0.230.026 partial agonist 0.3047.870.47 13.4910 M199S TM5 0.440.16 full agonist -38.650.17 2.242 W255A TM6 1.560.79 full agonist 19.000.028 1.001 W255S TM6 1.510.24 full agonist 18.960.021 1.101 F259S TM6 ag 0.640.060 full agonist -28.980.51 1.051 H261A TM6 9.160.35 0.580.14 partial agonist 0.69 -210.870.18 0.01-100 H261S TM6 9.130.24 partial agonist0.741 L264A TM6 9.270.31 partial agonist0.54-28.110.22 7.766 I265A TM6 9.480.096 partial agonist 0.33-39.490.0071 0.32-4 T270A EL3 0.620.28 full agonist -28.290.10 5.134 T270S EL3 9.470.32 0.530.15 partial agonist 0.32-38.520.76 3.022 T278A EL3 9.270.15 partial agonist0.54-29.070.56 0.85-2 T278S EL3 9.540.37 partial agonist0.29-48.750.71 1.781

PAGE 152

152 Table 4-3. continued. SHU9119 hAGRP(87-132) pA2 values Pharmacology Ki values (nM) Fold Diff. pA2 values Ki values (nM) Fold Diff. wildtype 8.960.13 partial agonist1.1018.870.24 1.351 N282A TM7 9.720.025 0.19-68.890.46 1.291 N282S TM7 9.210.24 0.62-28.840.36 1.451 F281A TM7 0.41+0.056 full agonist -37.690.50 20.4215 F281S TM7 0.670.061 full agonist -27.160.23 69.1851 D295A TM7 9.600.096 0.25-48.020.18 9.557 D295S TM7 9.520.27 0.30-48.710.0071 1.951 R302A TM7 9.130.076 slight agonist0.7418.890.078 1.291 R302S TM7 10.080.22 slight agonist0.08-139.190.30 0.65-2The indicated pA2 values of SHU9119 represent the average of at least three independent experiments done in duplicate with their standard errors of the mean. *Indicates that this experiment was done twice in duplicate and the error represents the standard deviation. The indicated pA2 values of hAGRP(87-132) represent the average of at least two independent experiments done in dublicate with their standard deviation. To calculate the fold difference compared to the wild-type Flag-mMC3R, the pA2 values were converted into their corresponding Ki values with Ki = log pA2.

PAGE 153

153 Figure 4-3. Pharmacology curves of agonists and antagonists at the wild-type Flag-mMC3R. (Top Left) Functional agonist curv es. (Top Right) Binding affinity IC50 dose-response curve. (Bottom Left) Functional SHU 9119 antagonist curves. (Bottom Right) Functional AGRP antagonist curves.

PAGE 154

154 Table 4-4. Comparison of the wild-type and mutant mMC3R cell surface and total receptor expression levels us ing Flow Cytometry. Cell Surface Receptor Expression relative to the wild-type receptor (%) Total Cell Receptor Expression relative to the wild-type receptor (%) wild-type 100.0100.0 S52A 89.03.189.32.5 N56A 25.78.058.018.0 N56S 70.09.881.013.9 D84A 76.712.289.05.3 D84S 91.04.692.06.1 N91A 127.312.9117.016.5 N91S 99.05.594.018.7 E94A 74.016.486.37.5 E94S 119.35.7107.06.6 I96A 116.34.699.32.1 I96S 48.711.752.319.0 D117A 87.317.096.726.3 D117S 73.04.078.77.4 F120A 39.71.963.72.1 F120S 92.37.193.714.4 D121A 53.714.365.735.9 D121S 75.01.081.32.1 S122A 94.08.589.324.7 L128A 54.78.678.311.0 L128S 66.38.869.010.4 I132A 98.014.988.314.6 I132S 44.017.841.728.9 T145A 129.022.3100.018.1 T145S 113.018.5104.327.0 Y152A 36.32.754.35.1 Y152S 39.76.754.730.6 K160A 106.713.993.711.8 K160S 40.79.158.712.6 I165A 79.33.778.72.1 I165S 19.010.146.321.6 C175A 98.38.298.311.0 C175S 93.04.292.311.9 F179A 83.311.892.014.5 F179S 120.714.7109.717.5 Y182A 66.715.860.76.7 Y182S 86.36.997.03.6 E184A 36.310.248.316.7 E184S 55.78.058.731.2 M195A 105.78.588.72.1 M195S 68.01.085.79.7 F196A 70.39.370.320.1 F196S 61.33.872.79.1 Table 4-4. continued.

PAGE 155

155 Cell Surface Receptor Expression relative to the wild-type receptor (%) Total Cell Receptor Expression relative to the wild-type receptor (%) wild-type 100.0100.0 F197A 54.76.976.06.9 F197S 54.09.858.73.5 M199A 87.34.495.75.1 M199S 76.73.595.04.6 L247A 38.711.875.326.4 L247S 54.36.275.79.5 W255A 62.72.871.311.4 W255S 78.310.381.33.1 P257A 60.74.168.015.6 P257S 56.04.576.712.0 F258A 44.711.864.013.7 F258S 59.05.975.010.5 F259A 46.05.155.76.0 F259S 74.03.687.79.6 H261A 59.39.077.75.5 H261S 56.07.261.722.1 L264A 72.05.383.713.9 L264S 21.72.744.010.5 I265A 64.33.782.711.7 I265S 59.02.573.010.8 T267A 65.33.885.013.1 T267S 58.08.179.76.0 C268A 93.77.0101.711.6 C268S 22.79.244.332.6 T270A 126.310.7116.318.6 T270S 78.713.596.34.2 T278A 82.09.689.76.8 T278S 59.710.959.021.6 N282A 42.014.056.732.4 N282S 80.715.290.08.5 F281A 79.36.992.73.2 F281S 103.712.7100.78.3 D295A 92.315.096.725.3 D295S 50.09.064.724.4 R302A 83.010.683.06.2 R302S 86.07.694.76.1 Cell surface and total receptor expression of th e wild-type mMC3R were defined as 100% for comparative purposes. The values listed indicate th e average of the mean from three independent experiments and the error represents the standard error of the mean.

PAGE 156

156 Figure 4-4. Relative receptor cell surface and tota l expression of wild-type and mutant FlagmMC3 receptors determinted by flow cytometry. For comparative purposes receptor cell surface and total expressi on of the wild-type Flag-mMC3R is set to 100%. Data represents the mean fluorescence measured fr om 10000 cells/sample, run in triplicate.

PAGE 157

157 Table 4-5. Summary of the synthetic agoni st NDP-MSH competitive displacement binding affinity studies (IC50) and functional (EC50) studies of the wild-type and mutant Flag-mMC3 receptors. TM NDP-MSH IC50(nM) Fold Difference NDP-MSH EC50(nM) Fold Difference wild-type 10.11.5410.140.014 1 S52A TM1 11.13.910.340.033 2 N56A TM2 > 1000> 1000 N56S TM2 > 1000> 1000 D84A TM2 13.33.51> 1000 D84S TM2 19.317.12> 1000 N91A TM2 42.51.740.300.055 2 N91S TM2 31.115.53> 1000 E94A TM2 5.45.4-2> 1000 E94S TM2 4.04.2-30.680.10 5 I96A TM2 10.62.310.160.026 1 I96S TM2 7.01.11 0.110.010 pA 1 D117A TM3 27.415.930.570.18 4 D117S TM3 26.76.730.690.14 5 F120A TM3 17.47.22 0.0980.020 pA 1 F120S TM3 3.082.0-30.150.022 1 D121A TM3 > 1000 12.992.90 pA 93 D121S TM3 1.10.97-9 6.842.58 pA 49 S122A TM3 5.00.76-2 0.150.055 pA 1 L128A TM3 3.42.9-3pA L128S TM3 15.911.42pA I132A TM3 21.18.520.320.042 2 I132S TM3 27.315.430.430.13 3 T145A IL2 5.30.04-20.430.16 3 T145S IL2 7.40.8610.0700.019 -2 Y152A IL2 17.211.52 0.200.14 pA 1 Y152S IL2 9.94.01 0.350.084 pA 3 K160A TM4 13.51.310.130.041 1 K160S TM4 9.73.61> 1000 I165A TM4 26.99.33> 1000 I165S TM4 > 1000> 1000 C175A TM4 3.12.9-3 0.130.034 pA 1 C175S TM4 35.432.24 0.0800.033 pA -2

PAGE 158

158 Table 4-5. continued TM NDP-MSH IC50(nM) Fold Difference NDP-MSH EC50(nM) Fold Difference wild-type 10.11.5410.140.014 1 F179A TM4 18.22.820.170.022 1 F179S TM4 15.310.020.0560.012 -3 Y182A TM4 1645216 0.180.015 pA 1 Y182S TM4 32.913.730.230.037 2 E184A EL2 12.48.010.150.27 1 E184S EL2 4.60.1-20.120.017 1 M195A TM5 30.34.230.180.041 1 M195S TM5 6.27.3-20.210.070 2 F196A TM5 19.08.420.170.021 1 F196S TM5 15.15.010.340.11 2 F197A TM5 1.050.25-100.120.031 1 F197S TM5 8.40.6510.0120.033 -12 M199A TM5 10.21.210.0780.025 -2 M199S TM5 3.61.3-30.0350.0072 -4 L247A TM6 84.173.48> 1000 L247S TM6 > 1000> 1000 W255A TM6 46.126.050.190.037 1 W255S TM6 6.43.920.230.070 2 P257A TM6 > 1000> 1000 P257S TM6 30.625.23> 1000 F258A TM6 61.643.46pA F258S TM6 4.52.2-2> 1000 F259A TM6 12.110.01> 1000 F259S TM6 3.63.3-30.150.023 1 H261A TM6 9.58.01 0.240.12 pA 2 H261S TM6 16.57.72 0.150.018 pA 1 L264A TM6 14.19.410.180.090 1 L264S TM6 6.81.41> 1000 I265A TM6 12.44.010.240.046 2 I265S TM6 > 1000> 1000 T267A TM6 > 1000> 1000 T267S TM6 > 1000> 1000 C268A TM6 > 1000> 1000 C268S TM6 2707327> 1000 T270A EL3 5.052.8-20.0790.018 -2 T270S EL3 15.09.310.0360.0018 -4

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159 Table 4-5. continued TM NDP-MSH IC50(nM) Fold Difference NDP-MSH EC50(nM) Fold Difference wild-type 10.11.5410.140.014 1 T278A TM7 7.614.7010.180.034 1 T278S TM7 3.31.1-30.170.043 1 F281A TM7 23.510.020.140.038 1 F281S TM7 6.80.9610.210.029 2 N282A TM7 36.924.440.150.059 1 N282S TM7 6.00.56-20.460.091 3 D295A TM7 18.313.52 1.380.52 pA 10 D295S TM7 13.78.92 0.420.10 pA 3 R302A TM7 8.14.91 0.670.12 pA 5 R302S TM7 10.32.61 0.540.077 pA 4I125 NDP-MSH was used to competitively displace nonlabeled NDP-MSH in a doseresponse fashion. >1000 indicates that an IC50 and/or EC50 could not be determined at up to 1000 nM concentration or no competitive bindin g or cAMP stimulation was observed at up to 1000 nM concentration. EC50 values represent the standard error of the mean from at least 3 independent experiments done in duplicate, IC50 values represent either the standard deviation of 2 independent experiments done in duplicate or the standard error of the mean of 3 independent experiments done in duplicate.

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160 TM1 IL1 Rhodopsin EPWQFSMLAAYMFLLIMLGFPINFLTLYVTVQ HKKLRT hMC1R CL-EVSISDGLFLSLGLVSLVENALVVATIAK NRNLHS mMC1R CL-YVSIPDGLFLSLGLVSLVENVLVVIAITK NRNLHS hMC2R CP-RVVLPEEIFFTISIVGVLENLIVLLAVFK NKNLQA mMC2R CP-DVVLPEEIFFTISVIGILENLIVLLAVIK NKNLQS hMC3R CE-QVFIKPEVFLSLGIVSLLENILVILAVVR NGNLHS mMC3R CE-QVFIKPEVFLALGIV S LME N ILVILAVVR NGNLHS hMC4R CYEQLFVSPEVFVTLGVISLLENILVIVAIAK NKNLHS mMC4R CYEQLFVSPEVFVTLGVISLLENILVIVAIAK NKNLHS hMC5R CE-DMGIAVEVFLTLGVISLLENILVIGAIVK NKNLHS mMC5R CE-EMGIAVEVFLTLGLVSLLENILVIGAIVK NKNLHS TM2 EL1 Rhodopsin PLNYILLNLAVADLFMVFGGFTTTLYTSLH GYFVF-------GPT hMC1R PMYCFICCLALSDLLVSGSNVLETAVIL LLEAGALVARAAVLQQL mMC1R PMYYFICCLALSDLMVSVSIVLETTIIL LLEVGILVARVALVQQL hMC2R PMYFFICSLAISDMLGSLYKILENILII LRNMGYLKPRGSFETTA mMC2R PMYFFICSLAISDMLGSLYKILENILIM FRNMGYLKPRGSFESTA hMC3R PMYFFLCSLAVADMLVSVSNALETIMIA IVHSDYLTFEDQFIQHM mMC3R PMYFFLCSLAAA D MLVSLS N SL E T I MIA VINSDSLTLEDQFIQHM hMC4R PMYFFICSLAVADMLVSVSNGSETIVIT LLNSTDTD-AQSFTVNI mMC4R PMYFFICSLAVADMLVSVSNGSETIVIT LLNSTDTD-AQSFTVNI hMC5R PMYFFVCSLAVADMLVSMSSAWETITIY LLNNKHLVIADAFVRHI mMC5R PMYFFVGSLAVADMLVSMSNAWETVTIY LLNNKHLVIADTFVRHI TM3 IL2 Rhodopsin CNLEGFFATLGGEIALWSLVVLAIERYVVVC KPMSNFRFGhMC1R DNVIDVITCSSMLSSLCFLGAIAVDRYISIF YALRYHSIVT mMC1R DNLIDVLICGSMVSSLCFLGIIAIDRYISIF YALRYHSIVT hMC2R DNVIDVITCSSMLSSLCFLGAIAVDRYISIF YALRYHSIVT mMC2R DNLIDVLICGSMVSSLCFLGIIAIDRYISIF YALRYHSIVT hMC3R DNIFDSMICISLVASICNLLAIAVDRYVTIF YALRYHSIMT mMC3R D NI FDS MICIS L VAS I CNLLAIAIDRYV T IF YALR Y HSIMT hMC4R DNVIDSVICSSLLASICSLLSIAVDRYFTIF YALQYHNIMT mMC4R DNVIDSVICSSLLASICSLLSIAVDRYFTIF YALQYHNIMT hMC5R DNVFDSMICISVVASMCSLLAIAVDRYVTIF YALRYHHIMT mMC5R DNVFDSMICISVVASMCSLLAIAVDRYITIF YALRYHHIMT TM4 EL2 Rhodopsin ENHAIMGVAFTWVMALACAAPPLV GWSRYIPEGMQCSCGIDYYTPHEETN hMC1R LPRARRAVAAIWVASVVFSTLFIA YY mMC1R LPRARRAVVGIWMVSIVSSTLFIT YY hMC2R MRRTVVVLTVIWILD-TYPEFCTGTGITMVI FS mMC2R MRRTIITLTIIWMFCTGSGITMVI FS hMC3R VRKALTLIVAIWVCCGVCGVVFIV YS mMC3R VR K ALTL I GVIWVCCGI C GVM F II Y S hMC4R VKRVGIIISCIWAACTVSGILFII YS mMC4R VRRVGIIISCIWAACTVSGVLFII YS hMC5R ARRSGAIIAGIWAFCTGCGIVFIL YS mMC5R ARRSGVIIACIWILD-TYPEFCISCGIVFII YY

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161 TM5 IL3 Rhodopsin NESFVIYMFVVHFIIPLIVIFFCYGQLVFTVKEA AAQQQE-----hMC1R DHVAVLLCLVVFFLAMLVLMAVLYVHMLARACQHAQGI ARLHKRQmMC1R KHTAVLLCLVTFFLAMLALMAILYAHMFTRACQHVQGI AQLHKRRhMC2R HHVPTVITFTSLFPLMLVFILCLYVHMFLLARSHTRKI -------mMC2R HHIPTVLTFTSLFPLMLVFILCLYIHMFLLARSHARKI -------hMC3R ESKMVIVCLITMFFAMMLLMGTLYVHMFLFARLHVKRI AALPPADG mMC3R E SKMVIVCLIT MFF A M VLLMGTLYIHMFLFARLHVQRI AVLPPAGV hMC4R DSSAVIICLITMFFTMLALMASLYVHMFLMARLHIKRI AVLPGT-mMC4R DSSAVIICLISMFFTMLVLMASLYVHMFLMARLHIKRI AVLPGT-hMC5R ESTYVILCLISMFFAMLFLLVSLYIHMFLLARTHVKRI AALP-GAmMC5R ESKYVIICLISMFFTMLFFMVSLYIHMFLLARNHVKRI AASP-RY* TM6 EL3 Rhodopsin SATTQKAEKEVTRMVIIMVIAFLICWLPYAGVAFYIFTH Q---GSDFGP hMC1R RPVHQGFGLKGAVTLTILLGIFFLCWGPFFLHLTLIVLC PEHPTCGCIF mMC1R RSIRQGFCLKGAATLTILLGIFFLCWGPFFLHLLLIVLC PQHPTCSCIF hMC2R -STLPRANMKGAITLTILLGVFIFCWAPFVLHVLLMTFC PSNPYCACYM mMC2R -STLPRTNMKGAMTLTILLGVFIFCWAPFVLHVLLMTFC PNNPYCVCYM hMC3R VAPQQHSCMKGAVTITILLGVFIFCWAPFFLHLVLIITC PTNPYCICYT mMC3R VAPQQHSCMKGAVTITILLGVFIFC W A PFF L H LV LI I TC P T NPYCICY T hMC4R GAIRQGANMKGAITLTILIGVFVVCWAPFFLHLIFYISC PQNPYCVCFM mMC4R GTIRQGTNMKGAITLTILIGVFVVCWAPFFLHLLFYISC PQNPYCVCFM hMC5R SSARQRTSMQGAVTVTMLLGVFTVCWAPFFLHLTLMLSC PQNLYCSRFM mMC5R NSVRQRTSMKGAITLTMLLGIFIVCWSPFFLHLILMISC PQNVYCSCFM TM7 IL4 Rhodopsin IFMTIPAFFAKTSAVYNPVIYIMM NKQFRNCMVTTLC CGK hMC1R KNFNLFLALIICNAIIDPLIYAFH SQELRRTLKEVLT CSW mMC1R KNFNLFLLLIVLSSTVDPLIYAFR SQELRMTLKEVLL CSW hMC2R SLFQVNGMLIMCNAVIDPFIYAFR SPELRDAFKKMIF CSR mMC2R SLFQVNGMLIMCNAVIDPFIYAFR SPELRDAFKRML FCNR hMC3R AHFNTYLVLIMCNSVIDPLIYAFR SLELRNTFREIL CGCN mMC3R AH FN TYLVLIMCNSVI D PLIYAF R SLELRNTFKEIL CGCN hMC4R SHFNLYLILIMCNSIIDPLIYALR SQELRKTFKEII CCYP mMC4R SHFNLYLILIMCNAVIDPLIYALR SQELRKTFKEII CFYP hMC5R SHFNMYLILIMCNSVMDPLIYAFR SQEMRKTFKEII C-CRG mMC5R SYFNMYLILIMCNSVIDPLIYALR SQEMRRTFKEIV C-CHG Figure 4-5. Alignment of the mouse and human melanocortin receptors and bovine rhodopsin. The mMC3R residues mutated in this study are highlighted in blue. Residues conserved throughout the GPCR family A are marked by asterisk. The alignment of transmembrane domains was performed based on a previous report ( 147 ).

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162 Figure 4-6. Agonist pharmacology curves at mu tant Flag-mMC3 receptors putatively involved in the hydrophilic binding pocket. Figure 4-7. Explanation why no binding affinity value could be determined for the D121A. Flag-mMC3R. The graph on the left shows the total specific c ounts at the NDP-MSH concentration of 10-11M of the D121A, D 12S and wild-type Flag-mMC3 receptors. The graph on the left illustrates the re lative cell surface expr ession of the D121A, D12S and wild-type Flag-mMC3 receptors.

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163 Figure 4-8. Pharmacology curves of TM6 residu es putatively particip ating in the hydrophobic binding pocket of the Flag-mMC3R Figure 4-9. Binding affinities to NDP-MSH of TM6 residues putatively participating in the hydrophobic binding pocket of the Flag-mMC3R.

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164 Figure 4-10. Schematic represen tation of the putative DPhe-Ar g-Trp interaction with the mMC3R. The hydrophilic region is highlight ed in blue, the hydrophilic region is highlighted in red. Figure 4-11. Schematic represen tation of possible side chain arrangements in the hydrophobic region around TM3 and TM6. For the genera tion of this graphic models of the hMC4R in the active and inactive state were used ( 128, 147 ). Based on these models it might be envisioned that the inactive state is maintained by interaction of L3.36 and F6.52, whereas the active state might be maintained by interaction of L3.36 and F6.51. Residues in this region are bulky and it is hypothesized that movement of one residue might lead to a concerted movement of adjacent residues in this hydrophobic cluster.

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165 Figure 4-12. Agonist and SHU 9119 antagonist pharmacology curves at the T145A and T145S Flag-mMC3 receptors. Figure 4-13. Structure of the agonist MT II (left) and the anta gonist SHU9119 (right).

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166 Figure 4-14. Antagonist pharmacology curves showing conversion of SHU9119 antagonist to agonism at TM3 and TM6 Flag-mMC3R residues.

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167 Figure 4-15. Antagonist pharmacology curves of SHU9119 resulting into conversion of antagonist to agonist activity at re sidues from TM4, TM5, el3 and TM7.

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168 Figure 4-16. Antagonist pharmacology curves of SHU9119 at the mutant E94, D117 and D121 Flag-mMC3 receptors. Figure 4-17. Pharmacology curves of the anta gonists AGRP and SHU9119 at the N91A FlagmMC3 receptors.

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169 Figure 4-18. Comparison of agonist pharmacol ogy curves of L247A Flag-mMC3R and L250A FlaghMC4R. This figure depict s that the L247 residue is i nvolved in differentiation of MC3R and MC4R subtypes. Figure 4-19. Transient transfect ion assay. The wild-typ e and N91A Flag-mMC3R plasmids were transiently transfected into HEK293cells, using increasing amounts of DNA. Neither wild-type not N91A were identified to be constitutively active.

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170 CHAPTER 5 A COMBINATION OF TETRAPEPTIDE SAR AND RECEPTOR MUTAGENESIS All peptides were designed, synthesized, purif ied and analytically characterized by Bettina Proneth under the supervision of Dr. Carrie Ha skell-Luevano, except for JRH887-12 which was synthesized by Ryan Holder, a former graduate student of the HaskellLuevano laboratory. The functional assays at the wild type and mutant flag-tagged mouse melanocortin receptors were carried out by Bettina Proneth. Data analysis was performed by Dr. Carrie Haskell-Luevano. Introduction High-throughput screening methods and classi cal ligand structure-act ivity studies (SAR) often result in identification of useful lead co mpounds for subsequent optimization efforts in the classical chemistrydriven drug discovery process ( 171 ). Nevertheless, a se lective agonist or antagonist ligand (> 500 fold selective) for the melanocortin-3 receptor (MC3R) subtype, to our knowledge, has not yet been iden tified. It is hypothesized that detailed knowledge of the molecular mechanism of activation and inactiva tion of the MC3R versus the MC4R will be valuable for future drug discovery efforts in the melanocortin receptor family. The tetrapeptide template Ac-His-DPhe-Arg-Trp-NH2 was used to investigate the basis of agonism versus antagonism at the melanocortin-3 receptor (MC3 R). As shown in Chapter 3, the ligand DPhe position was identified as functional switch in discriminating agonist a nd antagonist activity at the MC3R subtype. Site-directed mutagenesis st udies of the mouse MC3R, done in Chapter 4, revealed several key residues that when mutated to either Alanine (Ala) and/or (Ser) shifted antagonist to agonist function. In the absence of cr ystal structures of GPCR, the structural basis and the molecular mechanism of receptor activation and ligand-receptor interactions need to be addressed by using less direct methods, like ligand structure-ac tivity relationship (SAR) studies and receptor site-directed mutagenesis. He rein a dual pronged approach was undertaken,

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171 combining both chemical and biological techniqu es in attempts to identify substructural determinants of the ligand as well as the recepto r involved in receptor activation or inhibition of the MC3R. Data, obtained from these studies cont ribute to the rational drug design process in that they are valuable for the generation of 3D GPCR homology models and identification of MC3R selective ligands. Results The structures of the tetrapeptid e ligands used in this study are illustrated in Figure 5-1 and a two-dimensional representation of the Flag-mMC 3R showing residues, mu tated in this study, is depicted in Figure 5-2. The result s of the functional activities of the tetrapeptide ligands at the wild-type and mutant Flag-mMC3 receptors are summarized in Table 5-1. The rationale for choosing Ala and Ser for receptor mutagenesis wa s explained in detail in chapter 4 of this dissertation. The Flag-mMC3R an tagonist ligands have been re ported previously (BP7-27 and JRH887-12) ( 108 ) or have been discovered from SAR st udies discussed in Chapter 3 (BP7-26, BP7-30 and BP7-147). Antagonist pharmacology cu rves at the wild-type Flag-mMC3R are illustrated in Figure 5-3. The Flag-mMC3R agoni st ligands (MTII, BP6-120 and BP6-121) have been reported previously ( 52, 108 ) and incorporated in this study for comparative purposes. Each compound was tested at selected Flag-mMC3 mutant receptors in attempts to investigate the structural basis for agonism versus antagonism at this receptor. Due to th e inherent experimental error of the CRE/ -galactosidase reporter gene assay used, changes in potency up to 3-fold were considered as equipotent, 4-9 fold changes were considered as slightly decreased and changes of 10-fold and more were considered as signifi cantly decreased in pot ency. MTII, BP6-120 and BP6-121 will be referred to as MC3R tetrap eptide agonists and BP7-26, BP7-27, BP7-30, BP7147 and JRH887-12 will be te rmed MC3R antagonists th roughout this chapter.

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172 Functional Characterizati on of MC3R Agonists MTII is the agonist of choice, used in our laboratory, for antagonist competitive displacement assays and was include d herein for the reason to be ab le to directly compare results derived from this study with ot her results from our laboratory ( 95, 108, 114 ). The cyclic peptide agonist MTII possessed subnanomolar activity with an EC50 value of 0.097 nM at the wild-type Flag-mMC3R and was equipotent at the F179A, F179S, M199A, M199S, F259S, T270A, T270S, F281A and F281S Flag-mMC3 receptors, as compared to the Wild-type (wild-type) receptor. At the I132A, I132S and W255A Fl ag-mMC3 receptors MTII possessed slightly decreased potency (5-, 4-, and 7-fold, respectiv ely) and at the W255S Flag-mMC3 receptor MTII possessed significantly reduced potency (20-fold), compared to wild-type. Of all the agonists tested MTII was least prone to potency changes at the mutant Flag-mMC3 receptors compared to the short linear tetrapeptides. An explanation fo r that might be that cy clization and the presence of Nle at the N-terminus of MTII may be modi fying the secondary structure of the core HisDPhe-Arg-Trp region and interfer e with how this ligand might in teract with the MC3R putative binding pocket. The unsubstituted tetrapeptide BP6-120 was publis hed previously and was included in this study as control compound ( 121 ). BP6-120 displayed a different pharmacological profile compared to MTII at the Flag-mMC3 receptors. C onsistent with previous reports it possessed an EC50 value of 184 nM at the wild-type Flag-mMC3R ( 108 ), within experimental errors. BP6-120 was almost equipotent at the F179A, T270S and F281S Flag-mMC3 receptors but showed significantly reduced potencie s at the I132A, I132S, W255A W255S and F259S mutant receptors (30-, 31-, 18-, 54and 21-fold respectiv ely), compared to the wild-type receptor. Of special interest was the slight gain of f unction at the F179S, T270A and F281A Flag-mMC3 receptors 5-, 4and 4-fold, respectively) and th e remarkable gain of function at the M199A and

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173 M199S Flag-mMC3 receptors (29and 66fold respectively), as compared to the wild-type receptor. The tetrapeptide BP6-121, where the DPhe phenyl ring is substituted in para position with the hydrophilic OH group was reported previous ly as well as in Chapter 3 to interact unfavorably with the putative hydro phobic receptor-ligand binding pocket ( 108 ) and was included to investigate possible loss and gain of potencies at the hydr ophobic alanine and/or hydrophilic serine substituted mutant Fl ag-mMC3 receptors. BP6-121 possessed an EC50 value of 9700 nM at the wild-type Flag-mMC3R and wa s unable to fully stimulate the I132A. I132S, W255A, and F259S Flag-mMC3 receptors and at the W255S Flag-mMC3R neither agonist nor antagonist activity was observed at up to 100 M concentration. The F179A, F179S, M199A, T270A, T270S, F281A and F281S Flag-mMC3 receptors possessed almost equipotency towards BP6-121 as compared to the wild-type receptor. Interestingly, at the M199S Flag-mMC3R, BP6121 resulted in potency gain of 15-fold, compared to the wild-type receptor. Functional Characterizati on of MC3R Antagonists The trifluoromethyl group -CF3 is comparable in size to -Cl, which was found in Chapter 3 to be a full agonist at the mouse MC3R, whereas -CF3 possessed partial agonist/antagonist activity. However substitution with -CF3 increases lipophilicity and decreases the electron density of the DPhe phenyl ring through electron attracting effect s. Additionally, fluorination has been shown to result into increased folding st ability in protein, proba bly thourgh increase in hydrophobicity compared to hydrocarbons ( 135, 136 ). This tetrapeptide antagonist may differ from the other tetrapeptide antagonists in that the CF3 group is thought to be unable to participate in electrostatic charge-transfer in teractions, while the B r, -I and -diCl are ( 120 ). BP7-26 possessed antagonist with partial agonist activity at the wild-type and F179A FlagmMC3 receptors with equipotent antagonist Ki values of 150 and 107 nM, respectively. At the

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174 remaining mutant Flag-mMC3 receptors tested, BP726 was converted from an antagonist to full agonist, but varying potency. BP7-26 was equi potent at the M199A, M199S, T270A, F281A and F281S mutant Flag-mMC3 receptors and possessed slightly reduced potency at the F179S and T270S Flag-mMC3 receptors, as compared to wild-type. Interestingly, at the I132A, I132S, W255A, W255S and F259S Flag-mMC3Rs, the CF3 containing tetrapeptide possessed significantly decreased potency (39-, 23-, 39-, 84and 56, respectively). BP7-30 contains a Br substitution at the DPhe phenyl ring. Brand CF3possess almost the same degree of lipophilicity, while they di ffer in their electronic and steric properties. Bromine is less electron withdraw ing and significantly bigger in size then a trifluoromethyl group. Additionally, -Br allows for charge-transfer interactions, described in Chapter 3. BP7-30 resulted in a potent antagonist/p artial agonist at both the wild -type (Figure 5-3) and F179A receptor with equipotent Ki antagonist values of 16 and 28 nM, respectively. Like the CF3containing tetrapeptide, this compound possessed full agonist activity at the remaining FlagmMC3 receptors, tested in this study. BP7-30 possessed equipotency at the F179S, T270A, and F281A Flag-mMC3 receptors and significantly decreased potency at the I132A, I132S, W255A, W255S and F259S mutant receptor s (45-, 39-, 96-, 63-, 138and 89-fold respectively). At the M199A and M199S Flag-mMC3R this compound result ed into slight potency increases of 4and 5-fold respectively). The I containing tetrapeptide BP7-27 was reported previously as antagonist/partial agonist at the wild -type mouse MC3R ( 108 ) (Figure 5-3) and possessed a Ki potency value of 35 nM in this study. Isubstitution differ from B r-substitution through increased size, lipophilicity, and enhanced charge-transfer capabilities. The el ectron withdrawing property however is higher with bromine. Surprisingly, BP7-27 was the onl y compound tested showing antagonist activity at

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175 two mutant Flag-mMC3 receptors, I132A in addition to F179A (Figure 5-4). Ki potency values were equipotent at the F179A and slightly decr eased (5-fold) at the I 132A Flag-mMC3 receptors, compared to wild-type. BP7-27 was a full agoni st at all remaining mutant receptors. This tetrapeptide resulted in equipotency at th e F179S, T270S and F281A Flag-mMC3 receptors, slightly reduced potency at the F281S Flag-mMC 3R (6-fold) and signifi cantly reduced potency at the I132S, W255A, W255S, F259S and T270S Fl ag-mMC3 receptors (66-, 38-, 118-, 111and 14-fold), compared to wild-type. Additionall y, BP7-27, similar to the Brcontaining BP7-30 resulted in slight increases in potency of 7-fold at both the M199A and M199S Flag-mMC3 receptors. In BP7-147 the DPhe phenyl ring is disubstitu ted with Clin para and meta position. Double substitution resulted in mMC3R antagonism /partial agonism (Figure 5-3), while single substitution at para or meta pos ition alone, led to full agonist ac tivity. This observation might be to an increase in lipophilicity, electron withdraw ing and/or charge-transfer capacity of the two chlorine substituents, compared to single ch lorine substitution. BP7-147 resulted in antagonism/partial agonism at the wild-type and F179A Flag-mMC3 r eceptors with similar potency (105 nM and 58 nM, respectively). At the F179S, T270A and F281A, this tetrapeptide retained equipotency, within the experimental er ror, slightly decreased potency was observed at the T270S and F281S Flag-mMC3 receptors and a significant decrease in potency was observed at the I132A, I132S, W255A, W255 S and F259S Flag-mMC3 receptors compared to the wildtype control. Substitution of M199 with Ala resulted in 4-fold increased and substitution with Ser resulted in 14-fold increased potency, as compared to the wild-type receptor. The DNal2 containing JRH887-12 is unique in th at it is the only co mpound that switches agonist activity in the tetrapeptide template to antagonist activity at the MC3 as well as the MC4

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176 receptor ( 108 ). It was hypothesized previously, based upon substitution of DPhe of the cyclic agonist MTII with DNal2 (SHU9119) that bulky aromatic amino acid substitutions confer antagonist activity at the MC3 and MC4 receptors ( 52 ). JRH887-12 may possess a similar degree of lipophilicity compared to the halogen compari ng tetrapeptides, when looking at the k values obtained by HPLC (Chapter 2), how ever differs in the topography of DNal2 compared to DPhe. Additionally, the DNal2 ring might be very elect ron rich compared to the electron poor phenyl rings of halogen containing peptides. JRH887-12 re sulted into antagonist/p artial agonist activity at the wild-type and F179A Flag-mMC3 receptors with almost equipotent Ki values of 220 and 224 nM, respectively. At the remaining mutant receptors, the DNal2 containing tetrapeptide possessed full agonist activity, with the surp rising exception of the T270S where JRH887-12 resulted into a partial agonist. Potencies at the M199A, T270A and F281A Flag-mMC3 receptors were similar to the wild-type Flag-mMC3R At the F179S, M199S and F281S Flag-mMC3 receptors JRH887-12 possessed slightly decreased potencies and at the I132A, I132S W255A, W255S and F259S Flag-mMC3 receptors, this te trapeptide resulted in significantly reduced potencies, compared to the wild-type receptor. Unexpectedly, at the T270S Flag-mMC3R partial agonist activity was observed with decreases in potency of 28-fold when compared to wild-type (Figure 5-5). BP7-26, BP7-27, BP7-30, BP7-147 and JRH887-12 possessed antagonist with partial agonist activity at the wild-type Flag-mMC3R, as s hown in Chapter 3 of this dissertation, with Ki values of 150, 35, 16, 105 and 220 nM, respectively (F igure 5-3). The antagoni st potencies at the wild-type receptor theref ore increased in the series DNal2 < (p-CF3DPhe) < (3,4diClDPhe) < (p-IDPhe) (p-BrDPhe), with BP7-30 being 13-fold more potent then JRH887-12, 9-fold more potent as BP7-26, 7-fold more potent as BP7147 and almost equipotent to BP7-27. Antagonist

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177 pA2 values derived from Schild analysis were converted into the corresponding potency Ki values (Ki = log pA2) for a better comparison with potency EC50 values. At the F179A FlagmMC3R the tetrapeptides BP7-26, BP7-27, BP7-30, BP7-147 and JRH887-12 retained their antagonist activity and possessed equipotent Ki values, compared to the wild-type receptor, within the experimental error. However, when comparing the maximum cAMP stimulation at 104M ligand concentration the efficacy of BP 7-30, BP7-27 and BP7-147 was significantly increased and with BP7-26 a trend towards incr eased efficacy could be observed, but JRH887-12 did not show any change compared to wild-type (Figure 5-6). Interest ingly, BP7-27, where DPhe is substituted with Iodine in para position was the only tetrapeptide tested in this study where antagonist/partial agonis t activity was observed at the I132A Flag-mMC3R, as demonstrated by Schild analysis (Figure 5-4). However consiste nt with the decreased potency of the FlagmMC3R agonists at this mutation the antagonist pot ency of BP7-27 was also decreased (5-fold), as compared to the wild-type receptor. JRH88712 was the only tetrapep tide possessing partial agonist activity at the T270S Flag-mMC3R (Figure 5-5), however was devoid of antagonist activity as opposed to the DNal2 containing SHU9119 (Chapter 4). Discussion Chapter 3 describes how sma ll changes at the ligand DPhe7 position resulted into a functional switch by shifting agonist to antagon ist activity at the mouse MC3R. Chapter 4 investigated how selected singl e amino acid substitutions within the receptor sequence resulted into swapped functions of the antagonist SHU9119 to a full or partial agonist. In this study a combined approach of modifications of the me lanocortin core tetrapep tide Ac-His-DPhe-ArgTrp-NH2 and site-directed mutagenesis of the receptor were undertaken to unravel the molecular basis of the differentiation of agonism versus antagonism of both, the ligand and the receptor.

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178 Previous SAR studies of the tetrapep tide template Ac-His-DPhe-Arg-Trp-NH2 led to the discovery of Ac-His-(pI)DPhe-Arg-Trp-NH2, which resulted into pa rtial agonist/antagonist activity at the MC3R, while retaining full agoni st activity at the MC1, MC4 and MC5 receptors ( 108 ). Further SAR studies were undertaken in attemp ts to investigate the effect of DPhe phenyl ring substitutions on functional ac tivity, as discussed in detail in Chapter 3. These studies revealed that certain substituents (p-CF3, p-Br, p-I and 3,4-diCl) are able to switch agonist to antagonist function at the MC3R, while retaining full agonist act ivity at the MC1, MC4 and MC5 receptors (Chapter 3). To achieve antagonist activity at both MC3 and MC4 receptor, substitution of DPhe7 with the bulky aromatic am ino acid DNal2 was necessary ( 52, 108 ). These data suggested that minor st ructural changes at the DPhe7 phenyl ring mark this position as functional switch to differentiate agonist/a ntagonist activity at the MC3R subtype and indicated distinct receptor act ivation mechanisms of the MC3 and MC4 receptors. In addition, the peptide SAR studies implicated that there mi ght be differences in the molecular mechanism of antagonist action of halogen containing peptid es and DNal2 containing peptides (SAR). During systematic mutational mapping studies of the MC3R (Chapter 4) seven key receptor residues were identified, that when muta ted to either Ala and/or Ser converted the DNal2 containing antagonist SHU9119 into a part ial or full agonist, devoid of antagonist activity. These residues are located in TM3 (I132), TM4 (F179), TM5 (M199), TM6 (W255, F259), el3 (T270) and TM7 (F281) of the Flag -mMC3R. The molecular mechanism and the involvement of each of these residues in switchi ng the function from antagonist to agonist are not yet completely understood. If they do so by stabilizing the inactiv e receptor conformation through intramolecular interacti on and preventing conformati onal changes during receptor activation, by direct interaction with the liga nd DPhe residue, by steric hindrance of by a

PAGE 179

179 combination of multiple effects remains to be identified. The possible mechanisms have been discussed in detail in Chapter 4. Residues have been identified th at may be directly interacting with the DPhe7 and/or DNal2 moiety (I132, W255, F281, L128, F179, F259) as well as residues that may be involved in discriminating recepto r function and/or maintaining the active or inactive state (M199 and T270). Residue L128, when mutated to Ala or Ser, only resulted in partial agonist activity and was not included in the study herein. Residues I132 (TM3), W255 (TM6) and F259 (TM6), when mutated to Ala or Ser resulted into a decrease in potency for both BP6-120 and BP6-121. The aromatic cluster containing W255, and F259 was indicated as being involved in ligand recognition and signal transduction ( 151 ). The mutagenesis studies su ggested that W255 was important for molecular recognition of melanocortin ligands, whereas F259 might be im portant for ligand induced receptor activation, maybe regulating in concert with L128 positio ning of W255 and/or I132. Mutation of W255 resulted into significant decreases in functional activity of the tetrapeptide agonist ligand BP6120, BP121 and the tetrapeptide antagonist ligands wh ich resulted into full agonist activity at these mutant receptors. This might further underli ne a direct involvement of this residue in molecular recognition of the ligand postion 7. The close proximity of W255 to the ligand DPhe phenyl ring is further demonstrated by the obser vation that all ligands tested showed higher potencies when W255 was mutated to the hydrop hobic Ala and lower potencies when W255 was substituted with the hydrophilic Se r. This might indicate that the -OH group of Ser interacts unfavorably with the aromatic DPhe ligand re sidues, maybe through repulsive effects. The potencies of the tetrapeptide antagonists which are full agonist at this mutant receptor do not differ considerably between each other and are similar to that observed with BP6-120. This

PAGE 180

180 observation provides further expe rimental evidence that agonist s and antagonists may share a common binding pocket. The F259 residue is part of the conserved arom atic cluster in TM6 a nd situated in close proximity to W255. The F259S mutant receptor re sulted in decreased lig and potencies, compared to the wild-type receptor. Interestingly, while th e F259S mutant was able to produce cAMP when stimulated with ligands, the co rresponding F259A mutant resulted in complete loss of function. However this mutant receptor as shown in Ch apter 4 was still able to result in high binding affinity to NDP-MSH, which renders the F259 a key residue important for receptor activation. That F259 residue might not be directly involved in ligand rec ognition is further supported by the observation that ligands were able to activ ate the hydrophilic Ser containing receptor, while unable to stimulate the hydrophobic Ala containing receptor. Ligands tested at the F259S mutant receptor showed a similar pharmacological profil e to that observed with the W255 mutations, with generally remarkably decreases in poten cies and not significant differences when comparing antagonist with BP6-120 potency. The I132 residue is located in TM3 of th e Flag-mMC3R one helic al turn below L128. Experimental evidence from Flag -mMC3R mutagenesis studies done in Chapter 4 implicated the L128 residue as important for ligand induced rece ptor stimulation and di fferentiation of agonist versus antagonist activity. The hydrophobic I132 Flag-mMC3R residue might be directly interacting with the ligand DPhe residue, sin ce substitution with the space generating Ala and Ser resulted into signific ant decreases in potencies of the te trapeptide agonists and antagonists, which were converted to full agonists at these mutant receptors. Unexpectedly, the para Iodo containing tetrapeptide was the only tetrapeptide tested that result ed into antagonist activity at the I132A mutant Flag-mMC3R (Figure 5-4). Iodi ne, due to its size and highest charge-transfer

PAGE 181

181 capability might be still able to keep weak contact with TM3 Ala or other residues of TM3, further suggesting the close proximity of DPhe. It might be also envisioned that steric effects play a role still hindering adjacent residues from changing side chain conformation necessary for receptor activation. That I132 is still involved in to differentiation of agonist versus antagonist activity of the pI containing pe ptide BP7-27 is supported by th e observation that the partial agonist activity is significantly elevated compared to the wild-type Flag-mMC3R. Results from Schild analysis are shown in Figure 5-4 to ve rify competitive antagonism. Similar to W255 and F259, no significant differences in antagonist po tencies compared to BP6-120 were observed. The lack of either of these residues I132, W 255 and F259 resulted in conversion of antagonism to agonist activity, which might further indicate that receptor ac tivation is regulated through a concerted movement of these residues from TM 3 and TM6. Mutation of either one of these receptor amino acids might result into higher fl exibility which might allow for conformational changes during signal transduction. In chapter 4 it was hypothesized that SHU9119 DNal2 may interact with W255 and F259 through aromatic interactions, preventing necessary side-chain and TM rotations for receptor activation. It may be envisioned that the halogen containing tetrapeptides interact with these residues in a similar way through aromatic-aromatic and chargetransfer interactions. These in teractions may be tighter with Br and I compared to CF3 which does not exert charge-transfer interactions but might still be able to interact with the electron rich ring of Trp through its electron poo r phenyl ring and high lipophilicit y, as reflected in the higher antagonist potencies of BP7-27 and BP7-30 compared to BP7-26 at the wild -type receptor. These observations remain speculative and molecular m odeling may aid in further investigating this possibility.

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182 The residue F179 is locate d in TM4 of the Flag-mMC3R. The corresponding mMC4R residue F176 was implicated in direct intera ction with the melanocortin ligand DPhe residue since mutation with serine dire ctly abolished ligand induced signal transduction as well as binding to NDP-MSH, whereas binding affinity was retained to SHU9119 ( 114 ). This residue therefore was identified as key residue in th e differentiation of MC3 and MC4 receptor subtypes (Chapter 4). When the correspond ing residue F179 of the Flag-m MC3R was mutated to Ser, full agonist activity was observed of all antagonists tested and potencies remained similar to the wild-type receptor. Another intere sting aspect is that tetrapep tide antagonists retained their antagonist activity with similar antagonist potencies at the F179A compared to the wild-type receptor. The observation that tetrapeptide agonist s and antagonists showed similar potencies as compared to the wild-type receptors strongly s uggests that the F179 residue is not directly involved in molecular recognition of DP he containing tetrapeptide agonists. The most significant observation at the F179A and S mutant receptors is that antagonist activity of the tetrapeptid e antagonists is retained at the Al a mutant, whereas full agonist activity was gained at the F179S mutant. Neither an tagonist nor agonist lig and potencies changed significantly at both, the F179A and F179S Flag -mMC3 receptors compared to the wild-type receptor. However, when comparing the maximum cAMP stimulation at 10-4 M ligand concentration at the wild-type and F179A, a clear trend in increasing efficacy can be observed for the DPhe phenyl substituted tetrapeptides but not for the DNal2 containing JRH887-12 (Figure 5-6). These results indicate that the F179 residue might be involved in the differentiation of halogen containing versus DNa l2 containing antagonists. In ch apter 3 it was suggested that halogen containing tetrapeptide agonists might in teract through charge-t ransfer in addition to aromatic interactions with the receptor. F179 is located in close proximity to Y182, which was

PAGE 183

183 identified in Chapter 4 as important for a gonist molecular recognition. Halogen containing tetrapeptides might interact with the electron rich Tyr in addi tion to F179, which might explain the increase of partial agonist activity. Mutati on of F179 to Ala may render the Y182 more flexible. Mutation of F179 to Ser, may either allo w the tetrapeptides to freely interact with Y182, or might force Y182 into the agonist binding pocket through repulsion. It might be envisioned that the halogen containing tetrapeptides might be engaged in arom atic charge-transfer interactions with the electron ri ch Tyr. JRH887-12 seems to interact distinct from the halogens, since mutation to Ala does not increase part ial agonist activity of JRH887-12. Additionally, JRH887-12 is the only tetrapeptide antagonist subs tituted at the DPhe position which was able to antagonize both, MC3 and MC4 rece ptors; the remaining tetrapep tide agonists only antagonize the MC3R while retaining full pot ent agonist activity at the MC 4R. These observations provide further experimental evidence that the ligands may interact differentially with these receptor subtypes and also that these receptors might follow different mechanisms upon ligand induced stimulation and inhibition. At the wild-type Fl ag-mMC3R JRH887-12 is equipotent compared to BP6-120, however at the F179S receptor JRH887-12 is 28-fold less potent as the parent peptide. Also BP7-26 is almost equipotent to BP6-120 at the wild-type Flag-mMC3R but 17-fold less potent at the F179 mutant. BP7-26 differs from the other halogen containing peptides in its inability to form charge-transfer complexes and this is also reflected in the less pronounced increase in partial agonist activity at the F179A mutant (Figure 5-6). T270 is located in the extrace llular loop 3 and resulted when mutated to Ala and Ser into conversion of antagonist to agonist activity. Ser and Thr, show similar hydrogen bonding patterns, but differ in Thrs C methyl group which might have a defined structural and/or functional role within the receptor sequence ( 151 ). The corresponding residue in el3 of the

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184 hMC4R is Q273 (MC4R numbering). All antagonists tested at the T270A mutant were converted into full agonists at this mutant receptor. Tetr apeptides containing haloge ns retained equipotency to BP6-120, however JRH887-12 lost potency. Mutati on to Ser still maintains ability to exert hydrogen bonds like Thr. A ll tetrapeptide anta gonists were converted in to agonists, with BP7-30 almost equipotent to BP6-120, smaller decreases in potencies for BP 7-26, BP7-27 and BP7-147 but 107-fold decreased potency for JRH887-12. In addition, JRH887-12 resulted into only partial agonist activity at this T270S mutant Flag-mMC 3R, further suggesting distinct mechanism of action of JRH887-12 (Figure 5-6). Interestingly, the SHU9119 antagoni st resulted into increased partial agonist activity at this receptor mutant however retained competitive antagonism. This observations might be attributed to the cyclizat ion and the additional Cand N-terminal amino acids of SHU9119 which may either be influenci ng positioning of extracellular loop 3 or modify the secondary structure of the His-DNal2-Arg -Trp antagonist sequenc e of how the ligand interacts with the MC3R. This provides furthe r experimental evidence that the region around extracellular loop 3 is involved in the differentiation of agonist versus antagonist activity and receptor activation, maybe by regulating the activ e/inactive receptor state equilibrium through Hydrogen-bonding with receptor residues that still have to be identified. This explanation is experimentally supported by the f act that antagonist s except for BP7-30 resulted into higher potency changes at the T270S mutant receptor then the agonists tested. F281 is located in TM7 and resulted when mutate d to Ser or Ala into agonist activity of the tetrapeptide antagonists. Mutation did not significantly affect agoni st potencies, suggesting that this residue might not be crucial for DPhe cont aining agonist molecular r ecognition but rather in differentiation of agonist versus antagonist activity. Although as s hown in chapter 4 the endogenous LPhe containing agonists resulted into decreased poten cies, suggesting this residue

PAGE 185

185 as part of the putative MC3R binding pocket. An explanation might be that the DPhe residue is still able to engage into alternate ligand-recepto r interactions, which migh t not be possible for the LPhe containing ligands. The tetrap eptide antagonists might interact directly with this residue, preventing rotation of maybe TM6 or TM7 that is necessary for receptor activation. This residue might be also involved in maintaining the inac tive receptor state, as mutation resulted into significant decreases in potencie s for the inverse agonist AGRP (Chapter 4). The tetrapeptide antagonists might therefore interact directly with the F281 residue in th e inactive state and/or sterically interfere with n ecessary side chain/and or TM 7 rotation necessary for signal transduction to occur. This might be experiment ally verified by the f act that tetrapeptide antagonists resulted into decrea ses in potencies compared to the agonist BP6-120 (Table 5-1), further suggesting a direct intera ction of antagonists wi th this residue in the inactive state. Mutation of M199 resulted into significantly increased potencies of the agonists BP6-120 and BP6-121 and the tetrapeptide antagonists were converted into full agoni sts at these mutant receptors. This observation might indicate that in the wild-type receptor M199 is keeping the receptor in a conformational stat e, less favorable for agonist i nduced receptor activation. Possible mechanisms for this were suggested in chap ter 4. Interestingly, the halogen containing tetrapeptides resulted into generally increased pot encies at the M199 mutant receptors, with the exception of the DNal2 containing JRH887-12 wh ich possessed equipotency at the M199A and 6-fold decreased potency at the M199S Fl ag-mMC3R. These results provide further experimental evidence that JRH 887-12 interacts distinct from th e halogen containing tetrapeptide agonists with the MC3R. Conclusion In conclusion, this study revealed fundamental insights into the mol ecular basis of ligand and receptor involved in the diffe rentiation of agonist and antagoni st activity. Receptor residues

PAGE 186

186 identified from chapter 4 as important for SHU9119 antagonist activity have been further verified in switching antagonist into agonist activity. The pr oposed mechanism of halogen containing tetrapeptide antagonism consisting of a combination of charge-transfer and aromatic interaction has been further suppor ted. Residues have been identifie d that might engage in this interaction (F259, W255, F179, Y182). In additi on residues have been identified that may discriminate between halogen and DNal2 c ontaining tetrapeptides (F179, T270, M199). The latter residues might also be the basis w hy JRH887-12 is an MC3R/MC4R antagonist while halogen containing peptides are only MC3R anta gonists. These findings are further useful for rational drug design of MC3R selective ligands.

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187 Figure 5-1. Structures of the mMC3R agonists (red) and antagonist s (blue) used in this study.

PAGE 188

188 Figure 5-2. Two-dimensional re presentation of the Flag-mMC3R. The residues which were mutated to either Ala or Se r are highlighted in red.

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189 Table 5-1. Functional activiti es of the ligands tested at the Wild -type and mutant Flag-mMC3 receptors. TM MTII Fold Diff. BP7-26 Fold Diff. BP7-27 Fold Diff. BP7-30 Fold Diff. BP7-147 Fold Diff. JRH887-12 Fold Diff. BP6-120 Fold Diff. BP6-121 Fold Diff. wildtype 0.0970.009 1 pA2=6.820.10 Partial Agonist (Ki~150) 1 pA2=7.460.15 Partial Agonist (Ki~35) 1 pA2=7.800.28 Partial Agonist (Ki~16) 1 pA2=6.990.18 Partial Agonist (Ki~105) 1 pA2=6.660.18 Partial Agonist (Ki~220) 1 18434 1 97002180 1 I132A 3 0.500.06* 5 58501400 39 pA2=6.740.58 Partial Agonist 1130 32 720160 45 1460180 14 42501290 19 5450810 30 25% @ 100M I132S 3 0.390.05 4 34001900 23 23001380 66 630270 39 18401140 18 5780+2300 26 57704270 31 50% @ 100M F179A 5 0.0830.013 1 pA2=6.970.05 Partial Agonist pA2=7.550.13 Partial Agonist pA2=7.550.35 Partial Agonist 96 pA2=7.240.03 pA2=6.650.18 530250 3 157002400 2 F179S 5 0.0450.010 -2 650350 4 77.621.3 2 41.311.0 3 13645 1 1080580 5 38.46.8 -5 3760605 -3 M199A 5 0.0530.014 1 88.320.4 1 5.400.83 -7 4.501.20 -4 26.712.0 -4 210140 1 6.372.42 -29 31301350 -3 M199S 5 0.0280.009 -3 52.323.1 -3 4.892.46 -7 3.201.53 -5 7.691.33 -14 37.510.3 6 2.800.7 -66 65015.3 -15 W255A 6 0.660.013 7 59101230 39 1320460 38 1000430 63 41602300 40 66803100 3 33101700 18 25% @ 100M W255S 6 1.970.27 20 126203520 84 41401170 118 2210970 138 95003200 90 153404080 70 99701940 54 >100M F259A 6 loss of function F259S 6 0.310.034 -3 83602100 56 38701480 111 1420350 89 3560690 34 48401560 22 38101840 21 30% @ 100M T270A El3 0.0610.016 -2 19784.5 1 48.716.2 1 24.814.3 2 69.818.4 2 630260 3 46.226.6 -4 48502400 -2 T270S El3 0.0780.005 1 1020300 7 500400 14 33.97.83 2 600220 6 Partial Agonist 61700 28 57.715.7 -3 79003860 1 F281A 7 0.0420.006 -2 13043.9 1 37.0513.9 1 31.617.5 2 11035.1 1 424126 2 43.914.7 -4 60901640 -2 F281S 7 0.0880.022 1 420126 3 210146 6 45.321.8 3 640360 6 1190520 5 75.721.6 -2 108303120 1 The indicated errors represent the sta ndard error of the mean from at least three independent experiments. > 100 EC50 values denotes that compounds did not possess agonist or antagonist activity at up to 100 M ligand concentrations. The compounds possessing antago nist activity at the wild-type receptor were tested for an tagonist activity at each mutant Flag-mMC3R. When antagonist activity was lost, potenc y is denoted in agonist EC50 values. Antagonist potency is denoted by pA2 and Ki values, whereas agonist potency is denoted by EC50 values. Ki=-log pA2; pA=partial agonist. At the F259A Flag-mMC3R, no functional activit y was observed at 1M ligand conc entration (compare Chapter 4 ).

PAGE 190

190 Figure 5-3. Antagonist pharmacology curves of te trapeptide ligands at the wild-type FlagmMC3R.

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191 Figure 5-4. Antagonist pharmacology curves of BP7-27 at the I132A Flag-mMC3R. Schild Analysis was performed to verify antagonist activity of this compound. Figure 5-5. Pharmacology curves of tetrapepti de ligands at the T 270S Flag-mMC3R. JRH88712 possesses partial agonist activity, whereas the remaining ligands resulted into full agonists.

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192 Figure 5-6. Comparision of cAMP activities at 10-4M concentration of the tetrapeptide antagonists at the wild-type and F179A Flag -mMC3 receptors. Student t-test has been performed comparing the cAMP activities of the wild-type an d F179A Flag-mMC3 receptors of each ligand. (* = p<0.05, ** = p<0.005).

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193 CHAPTER 6 MOLECULAR MECHANISM OF CONSTI TUTIVE ACTIVATION OF A HUMAN MELANOCORTIN-4 RECEPTOR POLYMORPHISM Receptor mutagenesis and generation of stab le cell lines has been performed by Bettina Proneth. Determination of cell receptor expressi on by flow cytometry has been performed by Bettina Proneth, in the laboratory of Dr. Sally Litherland, Department of Pathology, Immunology and Laboratory Medicine. Functional and binding assays have been performed by Zhimin Xiang, biological scientist of the Haskell-Luevano la boratory. Radiolabelled I125-NDP-MSH and I125AGRP was synthesized and purified by Dr. Amanda Shaw, former graduate student of Dr. William Millard, Department of Pharmacodynami cs. The model of the human MC4R in the active state has been generated by Dr. Irina Pogozheva in the la boratory of Dr. Henry Mosberg, Department of Medicinal Chemistry at the Univer sity of Michigan. Data analysis was performed by Dr. Carrie Haskell-Luevano. Introduction The L250Q human Melanocortin-4 Receptor (h MC4R) polymorphism has been discovered in an extremely obese French female patient ( 59 ). This receptor mutation, when characterized pharmacologically resulted in the discovery of the first constitutively active hMC4R ( 90, 172 ). Investigation of the molecular mechanisms of constitutively active polymorphic GPCRs may contribute to the rational drug design approaches in two different ways. First, constitutively active receptors result into increased basal cAMP levels without being stimulated by agonist ligands ( 59 ). According to the extended ternary complex model it is hypothesized that agonists bind pref erentially to the active recept or state, allowing interaction with G-Protein and signal transduction ( 90 ). Constitutively active receptors potentially mimic the agonist induced receptor conformation and are postula ted to be able to bind to G-Protein in the absence of agonist ligands, explaining the elevated basal activity ( 90 ). The currently available

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194 crystal structures of bovine rhodopsin which ar e used as template for GPCR homology modeling represent inactive state conformations ( 86-88 ). Modeling of GPCRs in the active state, for the docking of agonists, has been ve ry challenging so far, and is based mainly on experimental constraints and theoretical com putational methods. Investigation of the molecular mechanism of a constitutively active receptor by mutagenesi s studies could therefore provide important insights, into receptor activation mechanisms and thus aid in the development of receptor models of the active state. Second, another important objective in ra tional drug design targeting polymorphic receptors is to gain knowledge about the putativ e disease causing physiological functions of the constitutively active receptor, in attempts to design and synthesize compounds that might correct for this malfunctioning receptor. This is especially important in the case of the L250Q patient, which represents a physiological contradiction, since chronic activati on and high basal cAMP levels of the MC4R should theoretically resu lt into a lean or normal phenotype. Although, melanocortin agonists were still able to fully stimulate the L250Q receptor in vitro treatment with agonists might not be an e ffective option in this case. In the study presented in this chapter, we combined approach es of receptor mutagenesis and pharmacology in attempts to identify the molecular mechanism by which modification of L250 results into constitutive activation of th e MC4R, as well as the physiological mechanism contributing to obesity development in the L250Q patient. Results Figure 6-1 represents a two-dimensional model of the hMC4R highlighting the L250 residue in TM6 ( 18 ). In vitro site-directed mutagenesis was performed by mutating residue Leu250 (6.40) in of the hMC4R to Ala, Glu, Phe, Lys, Asn, Arg, and Gln in attempts to identify requirements at this position for constitutive activation (Figure 6-1). The amino acids for

PAGE 195

195 receptor mutagenesis were selected based upon thei r side-chain properties which differ in charge, hydrophilic, hydrophobic and aromatic characteristics, length and bulk (Figure 6-1). Table 6-1 contains the structures of th e endogenous melanocortin agonists -MSH, -MSH, 2-MSH, and ACTH(1) ( 9, 10 ), the synthetic melanoc ortin agonists NDP-MSH ( 141 ), MTII ( 51 ) and JRH887 ( 108 ) and the endogenous MC4R antagonist hAGRP(87) ( 14 ) used for the pharmacological characterization of wild-type an d mutant Flag-hMC4 receptors. The rationale for choosing structurally diverse endogenous as well as synthetic agonists and antagonist ligands was to identify potential differences in ligand receptor interactions resulting from these structurally distinct compounds. Competitive Binding Assay The binding affinity (IC50) values of agonist NDP-MSH and the antagonist/inverse agonist hAGRP(87) at wild-type and mutant hMC4 r eceptors are summarized in Table 6-2. It is hypothesized that NDP-MSH and AGRP might not occupy ligand binding pockets in the receptor slightly distinct from each other. Fo r this reason for each radiolabeled peptide [125INDP-MSH and 125I-hAGRP(87)] the respective non-iodinat ed peptide was used to displace its corresponding labeled compound in a competitive manner. Mutation of the L250 (6.40) residue did not result into significant differences in NDP -MSH or hAGRP(87)-binding affinity IC50 values, compared to the wild-type hM C4R, with the excep tion of hAGRP(87) binding affinity at the L250N hMC4R. The fact that the ligand binding af finity values of NDPMSH and hAGRP(87) at the muta nt receptors were almost e quipotent to the WT hMC4R, provides experimental evidence that the L250 (6 .40) residue of TM6 may not be directly involved in molecular recogniti on aspects of these agonist or antagonist peptide ligands.

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196 Agonist Functional Assays The agonist EC50 values are summarized in Table 63 and the agonist pharmacology curves of the wild-type and mutant Flag-hMC4 receptors are shown in Figure 6-2. The endogenous agonists -MSH, -MSH, and ACTH(1) and the synt hetic tetrapeptide JRH887 possessed equipotent activity, within the inherent experimental error at the wild-type Flag-hMC4R, whereas the endogenous agonist 2-MSH agonist resulted in appr oximately 200-fold decreased potency when compared ti -MSH. The synthetic linear NDP-M SH and cyclic MTII agonist ligands possessed potencies in the sub nM range at the wild-type Flag-hMC4R. The -MSH ligand was essentially equipotent at the L250N hMC4R, while possessing slightly decreased potency at the L250F, L250K, L250E, and L250Q hMC4 receptors, and significantly reduced potencies at the L250A (39-fold) and L250R (65-fold) hMC4Rs, as compared to the wild-type receptor. The -MSH endogenous ligand possessed 15to 33-fold reduced potencies at the L250F, L250Q, and L250N Flag-hMC4 receptors and significantly reduced potencies at the L250A (43-fold), L250R (165-fold), L250K (70-fold), and L250E (41fold) Flag-hMC4 receptors, when compared to the wild-type control. Interestingly, the endogenous 2-MSH agonist possessed almost equipot ency within all Flag-hMC4 receptors examined in this study. The ACTH(1-24) agonist led to possessed 14to 27-fold potency decreases at the L250F, L250E, L250Q, and L250N Flag-hMC4 receptor, 55to 58-fold decreases at the L250A and L250K hMC4Rs, respectively, and 220-fold decr eases in agonist potencies at the L250R FlaghM4R. The cyclic synthetic MTII peptide agonist resu lted in nearly equi potent activity at the L250F, L250E, L250Q, and L250N, 14to 18-fold reduced potency at the L250A and L250K and 39-fold decreased potency at the L250R Fl ag-hMC4 receptors. The synthetic linear NDP-

PAGE 197

197 MSH agonist possessed 5to 7 fold decreases in potencies at the L 250F and L250Q, 21to 33fold reduced potency at the L250A, L250E, and L250N and 44to 78-fold reduced potency at the L250K and L250R Flag-hMC4 receptors, resp ectively. The syntheti c tetrapeptide JRH887 agonist possessed essentially equi potent activity at the L250A, L250Q, and L250N, and slightly decreased potency at the L250F, L250R, L250K, and L250E Flag-hMC4 receptors. In summary, the use of structurally divers e endogenous and synthetic melanocortin agonist ligands was beneficial in that it resulted into di stinct pharmacologic profil es at the wild-type and mutant Flag-hMC4 receptors. Constitutive Activation It was reported in several studies th at the L250Q hMC4R polymorphism possesses constitutive activity, resulting into increased ba sal cAMP activity in th e absence of agonist ligands ( 59, 63, 67 ). The basal cAMP activities of wild -type and mutant Flag-hMC4 receptors, stably expressed in HEK293 cells are represen ted in Figure 6-3. The L250F and L250A FlaghMC4 receptors possessed reduced and similar ba sal activity relative to the wild-type receptor, respectively, whereas the L250R, L250K, L250E, and L250N resulted into increased basal cAMP activity. To determine if the increased basal activity correlates also with constitutive activity a transient transfection assay have been used. The results of the transient transfection assays are illustrated in Figure 6-4. Experime ntal evidence were provided that L250E, L250Q, and L250N Flag-hMC4 receptors are indeed constitutively active GPCRs, L250K hMC4R possessed moderate constitutive activity and th e wild-type, L250F, L250A, and L250R FlaghMC4 receptors may either possess no or low constitutive activity. Receptor Cell Surface Expression The wild-type and mutant Flag-hMC4 receptors contain a Flag-tag inserted between the Methionine start codon and th e first receptor amino acid (F igure 6-1) to allow for

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198 immunocytochemical detection and localization of the receptor within the cell, using a fluorescently conjugated antibody and flow cytometr y. Previous studies suggested a decrease in relative receptor cell surface expr ession as mechanism for malf unctioning of the L250Q hMC4R polymorphic receptor ( 63, 67 ). To investigate this hypothesis we determined the relative cell surface and total cell receptor expression of wild-t ype and mutant Flag-hMC4 receptors, stably expressed in HEK293 cells. Figure 6-5 summarizes the flow cytometry results. Hereby, the wildtype receptor cell surface and tota l receptor expression were defined as 100%. The not or slight constitutively active L250A, L250F, and L250R Flag-hMC4 receptors possessed receptor cell surface expression levels of 57%, 46%, and 52%, relative to the wild-type Flag-hMC4R. The L250K Flag-hMC4R which result ed into intermediate consti tutive activation possessed cell surface expression levels of 58% relative to the wild-type Flag-hMC4R. The constitutively active L250E, L250N, and L250Q mutant hMC4Rs posse ssed cell surface expression levels of 66%, 54% and 72%, respectively, relative to the WT Fl ag-hMC4R. To investigate if there is a direct relationship between recept or cell surface expression, -MSH functional activity and degree of constitutive activation, we compar ed these parameters in Table 6-4. However, we were not able to identify a correlation be tween the endogenous agonist -MSH potency and receptor cell surface expression levels. Antagonist/Inverse Agonist Functional Assay The endogenous ligand hAGRP(87) was demons trated in previous studies to possess inverse agonist activity in addition to competitive antagonism at the MC4R ( 15-17 ), which is consistent with the result s presented herein. The pA2 antagonist values are summarized in Table 6-5 and the inverse agonist/antagonist pharmacol ogy curves of hAGRP(87) are illustrated in Figure 6-6. The effect of the inverse agonist activity of hA GRP(87) varied at the L250K, L250E, L250Q, and L250N Flag-hMC4 receptors. The highest degree of inverse agonist activity

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199 was observed at the L250N Flag-hMC4 receptors. These data provide experimental evidence further suggesting that these latte r Flag-hMC4 receptors are indeed constitutively active (Figures 6-3 and 6-4). At the L250A, L250F and L250R Flag-hMC4 receptors, very low or no inverse agonist activity could be observed, which might be consistent with the lack of constitutive activation at these receptors. The hAGRP(87) antagonist/inverse agonist was essentially equipotent at all the Flag-hMC4 receptors examined within the inherent experimental error. Discussion The study presented in this ch apter was undertaken to inves tigate the molecular mechanism and structural requirements of constitutive activation of th e L250Q hMC4R polymorphism. The MC4R, plays a major physiologic role in obesit y, appetite control and energy homeostasis in both humans and rodents ( 29-31, 48 ). Since the first discovery of naturally occurring mutations in the hMC4R in severely obese patients in 1998 ( 29, 30 ), more then 70 polymorphisms have been discovered in morbidly obese children a nd adults. However, the molecular mechanisms underlying these obesity causing mutations are not fully understood. Several hypotheses have been generated upon experimental evidence, incl uding modified endogenous agonist potencies, reduced cell surface expression, intr acellular retention in the ER through truncated or misfolded receptors. However, none of the above proposed hypotheses alone could explain the mechanism by which the constitutively active L250Q hMC4 R may cause obesity in humans. The binding affinities of the agonist NDP-M SH and the antagonist/ inverse agonist hAGRP(87) to the L250Q hMC4R were essentially equipotent to th e wild-type hMC4R. This observation provides experimental evidence that the L250 (6.40) resi due may not be important for receptor ligand binding or molecular recognition. P oor receptor surface expression or intracellular retention have been previously suggested as basis for rece ptor malfunction of the mutant L250Q hMC4R ( 63,

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200 67 ). However this observation alone as illustra ted in Figure 6-5 and Table 6-4 might not be sufficient to explain the obesity developm ent in the morbidly obese L250Q patient. AGRP appears to act as inverse agonist at this constitutively active L250Q hMC4R, consequently decreasing the high basal cAMP le vels. In the study presented herein we have indeed demonstrated that hAGRP(87) can dos e-dependently suppress basal activity of the L250Q hMC4R to levels similar to the basal activity of the wild-type hMC4R in vitro (Figure 66). Upon identification of AGRP as inverse agonist ( 15-17 ) it was suggested that AGRP could have its own separate physiol ogic functions, by acting both inde pendently and in concert with the endogenous agonist -MSH. AGRP is expressed in neurons of the ARC in the hypothalamus of the brain and is implicated in the regulation of energy homeostasis ( 14, 53 ). AGRP-containing neurons are situated near the POMC-containi ng ARC neurons that re lease the melanocortin receptor agonist -MSH ( 173-177 ). Generally, it is assumed th at energy homeostasis and food intake is regulated by the concerted action of food intake stimulating AGRP neurons and satiety inducing POMC neurons. Experiment al evidence provided by studies of AGRP mRNA levels show increased expression during chronic food restriction, ( 14, 53 ) while POMC or MSH mRNA levels are decr eased in the hypothalamus ( 178, 179 ). In addition, it has been proposed that MC4R signaling in the hypothalamu s could be regulated primarily by differences in the orexigenic peptide AGRP levels, as opposed to POMC, which is the precursor of -MSH ( 174 ). Based upon these previously published reports we generated the hypothesis that the increased basal activity and s ubsequent high basal cAMP leve ls of the L250Q hMC4R might mimic a situation in the body, similar to starva tion or chronic food de privation. This might trigger increased AGRP releases from closely located AGRP neurons in order to decrease the

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201 high basal activity of the receptor by AGRP inverse agonism in attempts to restore energy homeostastis. However, in the case of the L 250Q patient this counter regulatory action may result in increased appetite a nd hyperphagia which in turn may ha ve led to the development of obesity in this patient. Furthermore, it has b een experimentally demonstrated that AGRP can cause long-term orexigenic effects, blocking the anorectic effect of the MTII agonist ( 55 ), which would support the hyperphagia and obesity obs erved in the L250Q patient. A possible physiological mechanism is illustrated in Figur e 6-7. This putative physiological mechanism remains speculative and the extent by which AG RP contributes to obes ity development in the L250Q phenotype remains to be verified using in vivo physiologic studies, maybe by generating a L250Q hMC4R knock-in mouse. The second objective of this study was to inve stigate the determinants by which the L250Q hMC4R human polymorphism results in a constit utively active GPCR on a molecular basis. Thus, the L250 residue was mutated to a series of amino acids Ala, Glu, Phe, Lys, Asn, Arg, and Gln in order to shed light on the requirements at this position necessary for hMC4R constitutive activation. The rationale for the selection of th ese amino acids was described in detail in the results section. Based upon the results and receptor homology models of the hMC4R in the active and inactive state ( 128, 147 ), we hypothesized that this L250 hMC4R may be involved in the differentiation between the putative 'active' and 'inactive' receptor conformations of the GPCR. Mutations L250N, L250Q, and L250E hM C4Rs possessed significantly increased basal and constitutive activity, compared to the wild -type receptor, with L250N > L250Q > L250E (Figure 6-3). Based upon the transient transfectio n experiment (Figure 6-4), the L250K hMC4R possessed intermediate constitutive activation an d the L250F, L250A, and L250R hMC4Rs were not constitutively active. Constitutively active re ceptors are often used to proof the inverse

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202 agonist activity of antagonists which is ge nerally more pronounced at constitutively active GPCRs ( 146, 180 ). Inverse agonist activity of th e hAGRP(87) was observed at the constitutively active L250E, L250Q, and L250N hMC4Rs (Figure 6-6). The non-constitutively active L250F, L250A, and L250R mutant hMC4Rs, hAGRP(87) resulted in normal competitive antagonist activity with low or no observable inverse agonist activity. The hMC4R mutagenesis results of the L250 re sidue presented in th is study might be explained using theoretical thr ee-dimensional (3D) homology mo lecular models of an active (agonist-bound) and an inactive (antagonistbound) conformation of the hMC4R. Several independent studies have provides experimental evidence that rece ptor activation is accompanied by conformational changes, such as rearrangement of side chains, disruption of intramolecular constraints and/or rotation of TM helices ( 86, 89, 181 ). Based upon the homology models of the hMC4R it might be envisioned that L250 substituti on by polar side chains of Asn, Gln, and Glu (but not Lys and Arg) may be involved in the formation of hydrogen bonds with R147 (3.49) and Y212 (5.58) which may mimic a conformation appropr iate for the 'active' receptor state (Figure 6-8). Thus, hydrogen bond interactions that stabili ze the 'active' receptor state may be responsible for the increased constitutive activity of the L250N, L250Q, and L250E Flag-hMC4 receptors. A similar mechanism was proposed for the constitu tive activity of mutations at the corresponding residue M257 (6.40) in bovine rhodopsin ( 156 ). Results from this study in addition to previous reports of constitutive activity of corr esponding M257 (6.40) mutants in rhodopsin ( 156 ) indicate that residue 6.40 may have an impor tant structural role for GPCR activation and/or regulating of positioning of TM6 relative to TM3 and TM7. The L250N, L250Q, and L250E Flag-hMC4 recept or mainly differ in the length of their side chains which is reflected in their different levels of in creased basal activity and agonist

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203 potency changes. L250N, possessing the highest de gree of constitutive activity might have a side chain length more favorable for the formati on of a hydrogen bond with R147 (3.50) and maybe placement of TM6 that provides most, resulting in the most effective activation of G-protein. The presence of the polar, basic side chai ns of K250 or R250 might not provide the necessary hydrogen bonding interactions to stabilize the 'active' receptor state. This is also reflected in the low basal activity of the L 250K and L250R Flag-hMC4 receptors. Additionally, the R250 side chain may form alternative hydrogen bonds with S136 (3.39), D298 (7.49), and Y302 (7.53), favoring the 'inactive' rather then for the 'active' receptor conformation. This could explain the low level of constitu tive activation and the decreased agonist potencies of the L250R Flag-hMC4R. The small side chain of the L 250 to Ala substitution might destabilizes both 'active' and 'inactive' states and this might al so be the reason for the decreased potency of the agonists at this mutant receptor. The bulky aroma tic L250F side chain might be involved in the reinforcement of TM6 interactions and cons equently hinder the se paration of these TM domains putatively accompanying receptor activation, thus favoring the inactive state. This is also in agreement with the decreased potency of agonists at the L250F Flag-hMC4R. The smaller hydrophobic and aliphatic side chain of L250 in the wild-type Flag-hMC 4R may stabilize the inactive conformation in a lesse r extent compared to F250. Interpretations of the results in this study by theoretical homology modeling certainly remain to be experimentally verified. Howeve r they provide testable working hypotheses and guidelines for GPCR activation mechanisms and s upport the hypothesis that the L250 residue is putatively involved in the switching of active to inac tive receptor conformation. Conclusion In conclusion, by generating seven hMC4R mu tations at position 250 (6.40), we identified crucial sub structural requirements for recept or activation of the hMC4R. Hereby the residue

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204 L250 might play a key role in switching from th e active to the in active receptor state, underlining the involvement of TM6 and its residues in th e receptor activation mechanism of GPCRs. These findings and can be applied to other GPCR sy stems to aid in the rational drug design and creation of homology models in the active state. Additionally, we have provided experimental evidence that AGRP functions as an in vitro inverse agonist at the constitutively active L250Q hMC4R. Moreover we have generated a hypothesi s, supporting additional functional roles for AGRP besides a competitive antagonist in the brain as basis to explain the physiological mechanism of the unanticipated obese phenotype a ssociated with the constitutively active L250Q hMC4R polymorphism.

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205 Figure 6-1. Two-dimensional representation of the Flag-h MC4R. The L250 residue is highlighted in red. A Flag-tag is inserted at the N-terminus for immunocytochemical detection, depicted in a blue box. The ami no acid side chains for mutagenesis are illustrated at the bottom.

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206 Table 6-1. Primary sequences of endogenous/synthetic agonists and the endogenous antagonist/ inverse agonist hAGRP(87-132) used in this study. Peptide Primary Sequence ACTH (1-24) Ser-Tyr-Ser-Met-GluHis-Phe-Arg-Trp Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-Pro-Val-Lys-Val-Tyr-Pro -MSH Ac Ser-Tyr-Ser-Met-GluHis-Phe-Arg-Trp Gly-Lys-Pro-Val NH2 -MSH Ala-Glu-Lys-Lys-Asp-Glu-Gly-Pro-Tyr-Arg-Met-GluHis-Phe-Arg-Tr p Gly-Ser-Pro-Pro-Lys-Asp 2-MSH Tyr-Val-Met-GlyHis-Phe-Arg-Trp Asp-Arg-Phe-Gly MTII Ac-Nle-c[AspHis-DPhe-Arg-Trp Lys]-NH2 NDP-MSH Ac-Ser-Tyr-Ser-Nle-GluHis-DPhe-Arg-Trp Gly-Lys-Pro-Val-NH2 JRH 887-9 AcHis-DPhe-Arg-Trp -NH2 hAGRP (87-132) This table was modified from Ref. ( 18 ) with permission of Blackwell Publishing. CVRLHESCLGQQVPCCDPCATCYCRFFNAFCYCRKLGTAMNPCSRT

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207 Table 6-2. Competitive binding affinity IC50 results at the wild-type and mutant Flag-hMC4 receptors Binding Affinity IC50 (nM) Values NDP-MSH hAGRP (87-132) hMC4R 5.200.59 24.712.6 L250E 7.138.30 14.012.4 L250R 5.800.52 34.032.0 L250N 5.380.34 42.815.5 L250K 4.490.87 14.36.91 L250Q 4.170.83 5.980.53 L250F 5.792.14 4.560.75 L250A 4.850.87 5.680.96 Noniodinated NDP-MSH and hAGRP(87-132) were used to competitively displace the corresponding 125Iradiolabeled peptide. The i ndicated errors represent the standard deviation of the mean from at least two independent experiments done in duplicate. This table was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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208 Table 6-3. Functional activity of melanocortin ligands at the w ild-type and mutant Flag-hMC4 t Potency EC50 (nM) Values -MSH -MSH 2-MSH ACTH(1-24) MTII JRH887-9 hMC4R 0.880.11 0.240.071704.800.860.040.0170.0070.720.12 L250E 11.83.17 9.840.9991356023.511.90.140.0353.760.98 L250R 57.616.3 39.55.321480370190180.670.00710.30.95 L250N 1.551.92 8.000.911936313.23.480.0540.0520.920.31 L250K 11.96.94 16.72.905302049.924.30.300.056.930.98 L250Q 4.321.10 3.551.67442243.630.820.0340.0201.090.29 L250F 12.02.58 6.661.887258816.62.740.0340.02011.13.57 L250A 34.512.7 10.33.4497020746.912.00.230.066.253.26 EC50 values S.E. are derived from at least two independent experiments and each was performed in duplicate. The fold difference is calculated relative to the wild-type hMC4R. This table was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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209 Figure 6-2. Agonist pharmacology curves at the wild-type and mutant Flag-hMC4 receptors. This figure was modified from Ref. ( 18 ) with permission of Blackwell Publishing.

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210 Figure 6-3. Basal cAMP activity of the wild-type and the mutant Flag-hMC4 receptors stably expressed in HEK293 cells. Figure 6-4. Transient transfection assay. Incr easing amounts of wild-t ype and mutant FlaghMC4 receptors were transiently transfect ed into HEK293 cells. This figure was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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211 Figure 6-5. Relative cell surface and total cell expression of th e wild-type and mutant FlaghMC4 receptors. This was determined using Flow Cytometry. This figure was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing. Table 6-4. Comparison of recep tor cell surface expression with the functional activity of the endogenous ligand MSH and the level of constitutive activation of mutant and wild-type Flag-hMC4 receptors. Receptor Cell Surface Expression (%) -MSH Potency (EC50 in nM) Fold Diff. Constitutive Activation hMC4R 1000.880.111No L250F 4612.02.5814No L250A 5734.512.739No L250R 5257.616.365No L250K 5811.96.9413Moderate L250E 6611.83.1713Yes L250Q 724.321.105Yes L250N 541.551.922Yes Receptor cell surface expression of the wild-type hMC4R was set to 100%. EC50 values (nM) S.E.M. are derived from at least 3 experiments done in duplicate. The fold difference in EC50 values is calculated relative to the wild-type hMC4R. This table was reprinted from Ref. ( 18 ) with p ermission of Blackwell Publishin g

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212 Table 6-5. Functional activity of the antagonist hAGRP (87-132) at the wild-type and mutant Flag-hMC4 receptors. hAGRP (87-132) antagonist pA2 values (nM) hMC4R 8.200.007 L250E 8.270.50 L250R 7.640.35 L250N 9.130.97 L250K 8.240.008 L250Q 8.530.64 L250F 8.640.04 L250A 8.120.22 pA2 values S.D. are derived from at least two independent experiments done in duplicate. This table was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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213 Figure 6-6. Antagonist pharmacology curves of the antagonist/inverse agonist AGRP. This figure was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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214 Figure 6-7. Possible physiological mechanism by which AGRP might contribute to the obese L250Q phenotype through a regulatory feedb ack mechanism. Constitutive activity of the L250Q hMC4R might signal to the brain a situation which is similar to starvation. To maintain energy homeostasis it might be envisioned that in a regulatory feedback mechanism APRG is released resulting in hyperphagia and incr eased food intake.

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215 Figure 6-8. Comparison of wild -type hMC4R and L250Q hMC4R in the putative inactive and active receptor states. This fi gure was reprinted from Ref. ( 18 ) with permission of Blackwell Publishing.

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216 CHAPTER 7 INTRACELLULAR EXPRESSION OF AN ENDOGENOUS AGONIST IN VITRO Generation of the Met-MSH plasmid was performe d by Bettina Proneth. The -MSH peptide derivatives were synthesized by Bettina Proneth. The wild-type and mutant Flag-hMC4 receptors expressed in stable cell lines were c onstructed by Zhimin Xiang, biological scientist in the Haskell-Luevano laboratory. Flow cytometr y and deconvolution microscopy were done in collaboration with Sally Litherland, Depart ment of Pathology, Immunology and Laboratory Medicine. The fluorescent ligand DTAF-NDP-MSH ([N-chlorotriazinylaminofluorescein-Ser1, Nle4, DPhe7]-MSH) was synthesized by Dr. Andrzej Wil czynski, of the Haskell-Luevano laboratory. Introduction Since the first discovery of polymorphism in the human MC4R (hMC4R) in 1998 ( 29, 30 ), more than 70 different polymorphisms of the MC 4R gene have been discovered and associated with early and adult onset obesity ( 29, 30, 48, 58-76 ). Several hypotheses for impaired functions of hMC4 receptors containing these polymorphisms have been proposed from these studies. They include a modified potency of the endoge nous melanocortin agoni sts or antagonist AGRP at those receptors, truncated or structurally non-functional receptors, a nd decreased cell surface expression and intracellular retention of r eceptor mutants in the endoplasmic reticulum. However, several polymorphisms have been discovered in obese in dividuals, where the above proposed hypotheses are not sufficient to explain the mechanism by which those mutations lead to obesity development in human s. The objective of ratio nal drug design targeting polymorphic receptors is to (i) identify the phenot ype of a specific receptor mutation and then to (ii) design and synthesize ligands that might co rrect or rescue the ma lfunctioning polymorphism. In attempts to identify new molecular mechanis ms underlying mutations in the hMC4R that are

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217 associated with obesity we hypothesized that mutant hMC4 receptors might show different melanocortin receptor-ligand kinetics compared to the wild-type receptor. This rationale is based upon a report investigating peptide disso ciation rates at the human MC1R ( 182 ). These studies revealed that -MSH remained 25% bound, NDP-MSH 65% bound, and MT-II 86% bound 6h after the ligand had been rem oved from the assay medium ( 182 ). The relative dissociation rate of MT-II was 4 times slower than that for -MSH and 2 times slower than that for NDP-MSH, which was 2 times slower than that for -MSH. These data suggested that slow dissociation kinetics (hours) may contribute to the prolonge d biological activities ob served for both NDPMSH and MT-II peptides in vitro and in vivo ( 182 ). Vise versa, it might be envisioned that a receptor polymorphism could influence the ligand di ssociation kinetics of natural endogenous, as well as synthetic ligands to the hMC4R. The goal of this study was therefore to develop an assay that can be used to screen mutant hMC4 receptors for differences in receptor-ligand interactions. A better knowledge of mechanisms underlying polymorphic receptors could be used for new therapeutic approaches, where drug treatment could be conducted ac cording to a persons individual genetic makeup to select the mo st appropriate drug for a patients needs. Results and Discussion In our laboratory we have deve loped a plasmid, Met(Methionine)-MSH/pCDNA3, that when transfected into HEK293 ce lls stably expressing wild-type or mutant Flag-hMC4 receptors is able to not only cause a decrease in receptor cell surface expression but is also able to influence the kinetics by which a fluorescently labeled NDP-MSH analog interacts with the wildtype Flag-hMC4R. In vivo -MSH is generated thr ough posttranslational proces sing of POMC, followed by amidation and N-acetylation ( 39 ). Since -MSH will be encoded by a plasmid in vitro these

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218 posttranslational modifications might not occur. Additionally, it was necessary to insert the Methionine start codon, at the N-terminus upstream the -MSH coding sequence. For the construction of Met-MSH/pCDNA3, the -MSH coding region, containing the additional Methionine as start codon at the -MSH N-terminus was cloned into the HindIII/XbaI sites of the mammalian expression vector pCDNA3. To assure that Met-MSH has similar pharmacology than endogenous -MSH, it we synthesized Peptides BP2-38 and BP2-67 (Table 7-1). Both peptides contained the additional Met residue at the N-terminus; however they differ in their C-termini. BP2-38 is carboxy amidated, while BP2-67 contains the free carboxylic acid to mimic both possible scenarios of po sttranslational modifications of Met-MSH. These peptides have been synthesized using sta ndard Fmoc chemistry and pharmacologically characterized at the Flag-hMC4R with a -galactosidase/CRE reporter gene assay. The EC50 values and structures are shown in Table 7-1. Both peptides possessed EC50 values similar to endogenous -MSH within the experimental errors, implying that potentially generated MetMSH in vitro should have comparable functiona l activity, compar ed to natural -MSH, despite the modified N-terminus and either the amidat ed or free carboxy terminus. For the following assays HEK293 cells stably expressing wild-t ype or mutant Flag-hMC4 receptors were transiently transfected with Met-MSH/pCDNA3 or pCDNA3 as control. Analysis was performed 48 hrs after transfecti on. Flow cytometric analysis was performed to assess the impact of Met-MSH/pCDNA3 transfection on the cell surface ex pression of wild-type and mutant Flag-hMC4 receptors. A schematic representation of the Flag-hMC4R, de picting the receptor mutations is illustrated in Figure 7-1. The recepto r mutants for this experiment were chosen based on -MSH potency at these receptors (Table 2, adapted from ( 95 ). Figure 7-2 summarizes the percentage of relative cell surface and total receptor expressi on of the wild-type and mutant

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219 hMC4 receptors stably expressed in HEK 293 cells and transfected with either MetMSH/pCDNA3 or pCDNA3 (control). Wild-type and R18C Fl ag-hMC4 receptors, which possess essentially equipotency towards -MSH in the functional assay, re sulted when transfected with Met-MSH/pCDNA3 into a significant reduction in cel l surface expression relative to their corresponding control. The N97D hMC4 recepto r showing no functional response at up to 10 M, when stimulated with -MSH, does not possess changes in cell surface expression when transfected with Met-MSH/pCDNA3, relative to the control. Th ese results suggest that MetMSH when produced inside the cell is interacting with the wild-type and R18C but not the N97D Flag-hMC4 receptors and is leading to a decreas e in cell surface expression of the wild-type and R18C Flag-hMC4 receptors. To monitor if transfection with -MSH/pCDNA3 would infl uence receptor-ligand dissociation kinetics, live ce ll flow cytometry was performe d. DTAF labeled NDP-MSH ([Nalpha-chlorotriazinylaminofluorescein-Ser1, Nle4, DPhe7]-MSH) was used as the fluorescent ligand (Figure 7-3) ( 183 ). DTAF-NDP-MSH possessed almost equipotency to NDP-MSH as illustrated in Table 7-3. Cells stably expressing Flag-hMC4R were incubated for 40 min at 37C with Anti-Flag-APC (allophycocyanin) to st ain for receptor ce ll surface expression. Subsequently, DTAF-NDP-MSH was added and cells were fixed with formaldehyde after 1, 2, 5, 8 and 12min. Figure 7-4 shows the percentage of DTAF-NDP-MSH on the cell surface that is associated with the Flag-hMC4R after different time points. Following flow cytometry cells were cytospinned and used for deconvolution microsco py to visualize results obtained by live cell flow cytometry (Figure 7-4). Differences in how the ligand DTAF-NDP-MSH interacts with the Flag-hMC4R can be seen at the 2 min time point. While in control cells 50 -60% of the ligand are colocalized with the receptor a nd dissociation occurs slowly, in cells transfected with the Met-

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220 MSH/pCDNA3 plasmid only a total of approximately 50% of the ligand is associated with the receptor at 1 min, then 25% of the ligand disso ciate, which leaves only 25% DTAF-NDP-MSH at the cell surface at 2min. Those results correlate with the deconvolution microscopy pictures. Transfection with plasmid MetMSH/pCDNA3 can change the cell su rface expression of the wild-type and mutant Flag-hMC4 receptors, which show normal -MSH potency, suggesting that Met-MSH is interacting with the receptors inside the cell. MetMSH/pCDNA3 is also able to change the kinetics of how long the li gand DTAF-NDP-MSH interacts with the wild-type Flag-hMC4R. Those changes could be successfu lly monitored by using flow cytometry and deconvolution microscopy. The assay presented here in can be applied to screen mutant hMC4 receptors for differences in melanocortin receptorligand dissociation kinetics. This might lead to the identification of a new mechanism that contri butes to the development of obesity in humans. Future Directions Based on the results presented in this chapter it can be assumed that Met-MSH is generated and present in HEK293 cells. However, Met-MSH could not be detected by Western Blotting using an anti-MSH antibody. A possible reason for this might be that antibodies which are commercially available for -MSH are directed towards the amidated C-terminus to differentiate between -MSH and ACTH. In ACTH and -MSH the first amino acids are identical; however ACTH does not po ssess the amidated C-terminus. Met-MSH generated in HEK293 cells might therefore exist with a free carboxyl at the C-terminal end. To further verify the presence of Met-MSH it might be necessary using an N-terminal epitope tag, which is distinct from Flag, used already for imm unocytochemical detection of the hMC4R. One possibility would be the use of an enc odable His-tag. In addition His-tagged Met-MSH-OH has to be synthesized and pharmacologically ch aracterized to verify if this new compound possesses similar pharmacology then endogenous -MSH. Certainly, flow cytometric and

PAGE 221

221 deconvolution microscopy analyses would have to be repeated with the newly constructed Histagged Met-MSH-OH.

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222 Table 7-1. Functional Activity of -MSH, BP2-38 and BP2-67 at the wt Flag-hMC4R Peptide Sequence Potency EC50(nM) -MSH Ac -Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val NH2 0.690.014 BP2-38 Met -Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val NH2 2.120.014 BP2-67 Met -Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val OH 2.460.10 EC50 values S.E. are derived from at least three independent experiments and each was performed in duplicate. Figure 7-1. Two-dimensional repr esentation of the Flag-hMC4R s howing the receptor mutations used for this study. The N-terminal Flag -tag is highlighted in a dashed box.

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223 Table 7-2. Functional Activity of the melanocortin ligand MSH at the wild-type and mutant Flag-hMC4 receptors Mutation -MSH Potency EC50(nM) wild-type 0.650.19 R18C 0.690.32 N97D >10 EC50 values S.E. are derived from at least three independent experiments and each was performed in duplicate. >10 indicate that an EC50 value was not reportable at up to 10 M ligand concentration Figure 7-2. Cell surface and total receptor expres sion of wild-type (WT) and mutant Flag-hMC4 receptor after transfection with pCDNA3 (-) control and Met-MSH/pCDNA3(+). Statistical analysis (paired t-test) was performed by comparing the receptor cell surface expression of WT+ with WTand R18C + with R18C-. denotes a p-value of < 0.05.

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224 Figure 7-3. Structure of DT AF-NDP-MSH compared to -MSH and NDP-MSH. Table 7-3. Functional activity of NDPMSH and DTAF-NDP-MSH at the mouse MC4R. Peptide Potency EC50(nM) NDP-MSH 0.19 0.20 DTAF-NDP-MSH 0.61 0.29 EC50 values S.E. are derived from at least three independent experiments and each was performed in duplicate. DTAF-NDP-MSH: [N-chlorotriazinylaminofluorescein-Ser1, Nle4, DPhe7]-MSH.

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225 Figure 7-4. Live cell flow cytometry and dec onvolution microscopy. The percentage of DTAFNDP-MSH associated with Flag-hMC4R on the cell surface over time using HEK293 cells stably expressing the Flag-hMC4R transiently transfected with pCDNA3 (control) or Met-MSH/pCDNA3 is illustrated above. The corresponding deconvolution microscope images obtained afte r flow cytometry are illustrated below. DTAF-NDP-MSH (green), Flag-hMC4R (red) and nucleus (blue). These experiments have been repeated twice, however we re not able to re produce the results.

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226 CHAPTER 8 FUTURE DIRECTIONS This chapter has the purpose to suggest additional experiments to further experimentally verify the hypotheses or the putative mo lecular mechanisms underlying ligand-MC3R interactions and MC3R signali ng processes proposed in chapters 3-6. These experiments may provide further insights into th e determinants underlying agonist and antagonist activity at the mMC3R as well as the requirements fo r MC3R/MC4R subtype specificity. Experiment 1 The relative contribution of the individual am ino acids of the ligand His-Phe-Arg-Trp melanocortin message sequence towards the formati on of ligand receptor complexes is still under investigation. His7 and Arg8 are putatively part of the acidi c receptor binding pocket whereas Phe7 and Trp9 are potentially engaged in to interactions with a hydrophobic network of receptor amino acids. In our laboratory, Ala scans have been performed using NDP-MSH, -MSH and JRH887-9. The functional characterization of these peptides at the mMC3R is illustrated in Table 8-1. Ala scan of NDP-MSH resulted in less dramatic changes in ligand potencies as compared to -MSH and JRH887-9 (Table 8-1). Consistent with these results, NDP-MSH also resulted in less potency changes when tested at the mutant Flag-mMC3 receptors, as opposed to -MSH and JRH887-9 (Chapter 4). It is therefore hypothe sized that by testing Ala substituted NDP-MSH peptides at selected Flag-mMC3 receptors putat ively involved in ligand receptor recognition, it might be possible to obtain higher changes in potency EC50 values and to determine the relative contribution of each of the melanocortin liga nd core sequence amino acids to ligand-mMC3R recognition and ligand functional activity. The Flag -mMC3R mutations that may be useful for pharmacological characterization with the Ala6-9-NDP-MSH analogs are summarized in Table 82.

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227 Experiment 2 The difference between the synthetic ligands NDP-MSH and JRH887-9 are the additional Nand C-terminal amino acids. Several Flag-m MC3 receptors resulted in differences in potencies between these two liga nds. Truncation studies of NDP-MSH revealed that the minimal fragment necessary for essentially equipotent mMC3R functional activity is Ac-Nle-Glu-HisDPhe-Arg-Trp-NH2 ( 121 ). In addition, when JRH887-9 was extended with the C-terminus of NDP-MSH, this peptide resulted in less decreas e in potency; compared to NDP-MSH, further suggesting importance of C-terminal amino acids ( 121 ). It is hypothesized that by pharmacologically characterizing selected NDP-M SH peptide fragments at mutant Flag-mMC3 receptors, differentiating between NDP-MSH and JRH887-9 it is possible to further investigate the importance of Nand C-terminal extensions of the melanocortin message sequence. The proposed NDP-MSH peptide fragments for this expe riment are summarized in Table 8-3 and the mutant Flag-mMC3 receptors are represented in Table 8-4. Experiment 3 Studies of the mMC4R suggested that S HU9119 might interact with mMC4R residues F254 (6.52)and F259 (6.57) (mouse MC4R numbering) through aromatic forces, preventing necessary rotation of TM6 for receptor activati on, since mutation of eith er of these residues resulted in conversion of the SHU9119 comp etitive antagonist into partial agonism ( 168 ). F259 corresponds to Leu (6.57) in mMC3 R and MC1R. It was suggested that stronger interaction of SHU9119 with F259 (6.57) results into competitive antagonism whereas presence of L264 (6.57) might be responsible for the partial agonist activity observed for the MC3R, or full agonist activity at the MC1R ( 168 ). In chapter 4, the corresponding mMC3R L264 (6.57) residue was identified to not participate into differentiation of agonist versus antagonist activit y, but rather to

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228 be important for ligand induced signal trans duction, further supporting the before mentioned hypothesis. In MC1R the corresponding residue to L128 (3.36) is Met (3.36) and it has been demonstrated experimentally that mutation of Leu to Met resulted in SHU9119 partial agonist activity at the hMC4R ( 138 ), however this study did not investigate SHU9119 for competitive antagonism ( 138 ). When pharmacologically character ized at the L165M (3.36) hMC3R, SHU9119 resulted in agonist activity, with efficacy slightly less then MTII ( 123 ). It is therefore hypothesized that a combination of L3.36 a nd F6.57 might be necessary for SHU9119 competitive antagonism at the MC 4R, whereas a combination of L3.36 and L6.57 is important for SHU9119 competitive antagonism/partial agonism at the MC3R and a combination of M3.36 and L6.57 might be responsible for SHU9119 MC 1R agonism. To test this hypothesis the following single and double mutations could be generated (Table 8-5) and pharmacologically characterized. Experiment 4 In the Flag-mMC3R mutagenesis studies, resi dues were mutated to Ala as well as to Ser and pharmacologically characterized using several structurally diverse, endogenous and synthetic agonist and antagonist ligands. Due to limited struct ural data of GPCRs a nd the lack of a MC3R Homology model, the molecular mechanisms underlying mutant Flag-mMC3 receptors are difficult to interpret and based on assumptions. To further investigate the role of specific receptor residues, in ligand recognition, ligand induced signal transduction, MC3R/MC4R specificity and differentiation of agonist/antagoni st activity, the following recepto r mutations can be generated as illustrated in Table 8-6. Hereby, the amino acids for receptor mutagenesis were selected based upon (i) their side-chain propert ies which differ in charge, hy drophilic, hydrophobic, aliphatic and aromatic characteristics, length and bulk, (ii) genetic studies of the hMC4R and (iii)

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229 differences between the melanocortin receptor s ubtypes. It is hypothesized that by introducing multiple mutations at one specific receptor site and pharmacologically characterizing the resulting mutant receptors with the agonist/antag onist ligands from chapter 4, it is possible to elucidate the role of this specific resi due in the above mentioned processes.

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230 Table 8-1. Ala scan of NDP-MSH, -MSH and JRH887-9 Compound Structure mMC3R EC50(nM) RDP3-21-4 Ac-Ser-Tyr-Ser-Nle-GluAla -D Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.55 RDP25-4 Ac-Ser-Tyr-Ser-Nle-Glu-HisAla -Arg-Trp-Gly-Lys-Pro-Val-NH2 260 RDP3-21-3 Ac-Ser-Tyr-Ser-Nle-Glu-His-D PheAla -Trp-Gly-Lys-Pro-Val-NH2 2.07 RDP3-21-2 Ac-Ser-Tyr-Ser-Nle-Glu-His-D Phe-ArgAla -Gly-Lys-Pro-Val-NH2 295 JRH3107-22 AcAla -DPhe-Arg-Trp-NH2 9000 JRH3107-24 Ac-HisAla -Arg-Trp-NH2 >100 000 JRH3107-29 Ac-His-DPheAla -Trp-NH2 42000 JRH3107-16 Ac-His-D-Phe-ArgAla -NH2 >100000 AT849-9 Ac-Ser-Tyr-SerMet -GluAla -Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2 4270 AT849-8 Ac-Ser-Tyr-SerMet -Glu-HisAla -Arg-Trp-Gly-Lys-Pro-Val-NH2 1660 AT849-7 Ac-Ser-Tyr-Ser-Met-Glu-His-PheAla -Trp-Gly-Lys-Pro-Val-NH2 670 AT849-6 Ac-Ser-Tyr-SerMet -Glu-His-Phe-ArgAla -Gly-Lys-Pro-Val-NH2 15700 An EC50 value of >100000 denotes that no stimulatory or inhibitory avtivity could be detected at up to 10-4M ligand concentration.

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231 Table 8-2. Proposed Flag-mMC3R mutations for Experiment 1. Flag-mMC3R N91A TM2 E94S TM2 D117A TM3 D117S TM3 F120A TM3 F120S TM3 D121A TM3 D121S TM3 L128A TM3 L128S TM3 I132A TM3 I132S TM3 F179A TM4 F179S TM4 Y182A TM4 Y182S TM4 M195A TM5 M195S TM5 F196A TM5 F196S TM5 W255A TM6 W255S TM6 F258A TM6 F259S TM6 I265A TM6 N282A TM7 N282S TM7 F281A TM7 F281S TM7

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232 Table 8-3. Proposed peptides for experiment 2. Peptide Structure mMC3R EC50(nM) NDP-MSH Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.73 JRH887-9 Ac-His-DPhe-Arg-Trp-NH2 195 1 Ac-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 0.11 2 Ac-Nle-Glu-His-DPhe-Arg-Trp-NH2 0.26 3 Ac-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 15.2 4 Ac-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH2 6.82 The EC50 values were taken from Ref. ( 121 ) Table 8-4. Proposed Flag-mMC3R mutations for experiment 2. Flag-mMC3R E94S TM2 D117A TM3 D117S TM3 F120A TM3 F120S TM3 D121A TM3 D121S TM3 I132A TM3 I132S TM3 Y182A TM4 Y182S TM4 W255A TM6 W255S TM6 F259S TM6 I265A TM6 N282A TM7 N282S TM7

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233 Table 8-5. Proposed muta tions for experiment 3. FlagmMC1R wild-type M3.36L L6.57F M3.36L/L6.57F Flag-mMC3R wild-type L3.36M L6.57F L3.36M/L6.57F FlagmMC4R wild-type L3.36M F6.57L L3.36M/F6.57L

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234 Table 8-6. Proposed mMC3R mu tations for experiment 4. mMC3R residue proposed amino acids for mutation N91 D, E, Q, K, R, I, F E94 D, E, Q, N, K, R, F D117 E, Q, N, K, R, F D121 E, Q, N, K, R, F F120 I, K L128 M, V, F, W, H, Y I132 M, L, F, W, H, Y T145A V, I, Y, D, N, K K160 R, Q, W, H F179 Y, W, H, K, L, Q, M Y182 F, W, H, K, L, Q, C E184 D, K, Q, N, Y, R M195 L, F, R, Q F196 L, Y, W, R, Q M199 P, F, K, R, Q. L L247 Q, F, K, N, W255 Y, H, W, F, L F258 Y, H, W, L, K, N F259 Y, H, W, L, K, N H261 Y, W, L, K, N L264 F, Y, H, W I265 F, Y, H, W T267 V, F, L, Y, C C268 Y, T, R, N, D, F T270 N, Q, C, Y, V N282 D, E, Q, K, R, I, F F281 Y, H, W, L, K, N

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235 APPENDIX A SEQUENCING FILES Appendix A contains all sequences of Wt and mutant FlagmMC3 and FlaghMC4 receptors. The first part of the page shows the receptor nucleotide sequences. On the second part the receptor nucleotide sequences are translat ed into the correspondi ng receptor amino acid sequences (open reading frames), with restriction enzymes sites included.

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236 FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGcTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGcTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCgAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

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237 S52A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCGCTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCGCTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGCGAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V A L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

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238 N56A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAGCCATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAGCCATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTCGGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E A I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 239

239 N56S FlagmMC3R/pCDNA3 ACAAGaACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCC GG AGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAGCATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACAT GC TGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCT GG TGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTG CT GCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGC CA GGCTCCACGTCCAGCGCATCGCAgTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCC TT TCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGC CT TCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | ACAAGaACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TGTTCtTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGCCGTCACC frame 2 > K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAGCATCCTGGTGATCCTGGCTGTGGTCAGGAAT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTCGTAGGACCACTAGGACCGACACCAGTCCTTA frame 2 > F C E Q V F I K P E V F L A L G I V S L M E S I L V I L A V V R N BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | GGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCGGC frame 2 > G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V BsaXb BsaXa BsaXa PflM1 | BsaB1 | | || | TGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCTCCATCTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 ACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGAGGTAGAC frame 2 > I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAACTAGCCC frame 2 > N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G Nco1 BsaB1 BsaB1 BseR1 | | | || GTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGG 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTACC frame 2 > V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F A M V BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | TGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAgTGCTGCCCCCTGCTGGCGTGGTGGCCCCACA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 ACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTcACGACGGGGGACGACCGCACCACCGGGGTGT frame 2 > L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Nar1 Kas1 | BseY1 BseY1 || | | || GCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGAGTAGTAG frame 2 > Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L I I BspM1 BfuA1 Aar1 | ACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 TGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGATGC frame 2 > T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A SpAcc Fsp1 | | CCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+971 GGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 2 > F R S L E L R N T F K E I L C G C N S M N L G

PAGE 240

240 D84A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGCCATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGCCATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCGGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A A M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCG ATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 241

241 D84S FlagmMC3R/pCDNA3 GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGT GA GCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGC AG CCTGGCTGCAGCCTCCATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGAC TC TATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTG AT CGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATAT AT CCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTT TT CATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTC AT CGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG Bbs1 SpAcc Ear1 | Pst1 | | | | GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGG frame 1 > D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | AGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACT frame 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | TCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCTCCATGCTGGTGAGCCTGTCCAACTCCCT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGAGGTACGACCACTCGGACAGGTTGAGGGA frame 1 > L A V V R N G N L H S P M Y F F L C S L A A A S M L V S L S N S L BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | GGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAG frame 1 > E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCT frame 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K SpDon BsaB1 BsaB1 | | | AAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTA frame 1 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGA frame 1 > T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || GCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGG frame 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L BspM1 BfuA1 Aar1 | TCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCA frame 1 > H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V SpAcc Fsp1 | | CATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+991 GTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 242

242 N91A FlagmMC3R/pCDNA3 ATGgactacaaGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTgaGCacCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCGCCTCCCTGGAGACCATcaTGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGC TAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGgactacaaGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTgaGCacCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACctgatgttCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGActCGtgGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 SpDon Bsg1 BfuA1 Pst1 Bsg1 Eci1 Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCGCCTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGCGGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S A S BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | CTGGAGACCATcaTGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAgtACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 243

243 N91S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAGCTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAGCTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTCGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S S S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 244

244 E94A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCtGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGCGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCtGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGaCACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGCGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCGCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L A T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

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245 E94S FlagmMC3R/pCDNA3 GGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTC TT CATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCT GC AGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGTCGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATT TG CATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTC AT CTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATG TT CCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTC TG CTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCC CT CATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa | | | | GGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCC frame 1 > D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R Bts1 BspM1 BsaXb BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | AGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGAC frame 1 > S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Sal1 | | | | | | TGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGTCGACCAT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 ACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACAGCTGGTA frame 1 > V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L S T I BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | CATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCAC frame 1 > M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | GCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 CGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGT frame 1 > A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T SpDon BsaB1 BsaB1 | | | CCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAA frame 1 > L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCAC frame 1 > F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V Nar1 Kas1 | BseY1 BseY1 || | | || GTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACC frame 1 > V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V BspM1 BfuA1 Aar1 | TCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGG frame 1 > L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P SpAcc Fsp1 | | CCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+-982 GGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 246

246 I96A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCGCCATGATCGCCGTGATCAAcAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCGCCATGATCGCCGTGATCAAcAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGCGGTACTAGCGGCACTAGTTgTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T A M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 247

247 I96S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCAGCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCAGCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTCGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T S M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 248

248 D117A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGCTAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCaTCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGCTAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCGATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M A N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCaTCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGtAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 249

249 D117S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGTCTAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCcTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTgGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa BspLU | BsaB1 | | | | | | | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGTCTAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACAGATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M S N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCcTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGgAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTgGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGAcCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 250

250 F120A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCGCCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCGCCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGCGGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I A D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 251

251 F120S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTCCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTCCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAGGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I S D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 252

252 D121A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG CC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGCCTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCGGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F A S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 253

253 D121S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCT CC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGtG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 |BseR1 BsaB1 | | | | || | | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCTCCTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGAGGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F S S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGtGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCaCAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L

PAGE 254

254 S122A FlagmMC3R/pCDNA3 GAcGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTaACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCA TC AAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAG CC GACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACGCTATGATTTGCA TC TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCT GG GTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCC TC TTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCT GG GCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCA TC TACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | GAcGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTaACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTgCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGAtTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGC orf 1 > M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCA orf 1 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | CAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTAC orf 1 > R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | ATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACGCTATGATTTGCATCTCCCTGGTGGCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGCGATACTAAACGTAGAGGGACCACCGGA orf 1 > I A V I N S D S L T L E D Q F I Q H M D N I F D A M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAA orf 1 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L BsaB1 BsaB1 | | GATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAG orf 1 > I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACC orf 1 > A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 Kas1 | BseY1 BseY1 || | | || CCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGA orf 1 > P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L BspM1 BfuA1 Aar1 | CATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAG orf 1 > I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L SpAcc Fsp1 | | ATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-------978 TAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 255

255 L128A FlagmMC3R/pCDNA3 CAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAG GT CTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAGCATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTG GC TGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATG AT TTGCATCTCCGCGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGG GT CATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCAC AT GTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATC TT CTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGAC CC CCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa | | | | CAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAAC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 GTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTG frame 1 > K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N Bts1 BspM1 BsaXb BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | CGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAGCATCCTGGTGATCCTGG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTCGTAGGACCACTAGGACC frame 1 > R S G S G F C E Q V F I K P E V F L A L G I V S L M E S I L V I L A BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | CTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGAC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTG frame 1 > V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 Sac2 | | | || | | CATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCGCG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGCGC frame 1 > I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S A Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | GTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCC 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 CACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGG frame 1 > V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L SpDon BsaB1 BsaB1 | | | TCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTA frame 1 > T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCG frame 1 > F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G Nar1 Kas1 | BseY1 BseY1 || | | || GTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGG frame 1 > V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L BspM1 BfuA1 Aar1 | TGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 ACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCT frame 1 > V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D SpAcc Fsp1 | | CCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+----985 GGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 256

256 L128S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCTCGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BsaB1 BstX1 BsaXb BsaXa BsaXa PflM1 | BseR1 | | | | | || | | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCTCGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGAGCCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S S V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 257

257 I132A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCGCCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 Eci1 BsrD1 Drd1 Acc65 | BspH1 | | | | | | TCTCCCTGGTGGCCTCCGCCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGCGGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S A C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 258

258 I132S FlagmMC3R/pCDNA3 AACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGG TC TTCCTGGCTCTGGGCATCgTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGG TG AGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGG CC TCCAGCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCG GC ATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGC TC CACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCT TC CTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTTCC GC AGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bts1 Bbs1 BspM1 Ear1 | Pst1 BsaXa BsrB1 BfuA1 | | | | | | AACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGCCGTCACCCAAGA frame 0 > N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C BsaXb Bbs1 | Bbs1 Bsg1 | | | | GTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCgTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGcAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGTCCTTACCGTT frame 0 > E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | CCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCGGCACTAG frame 0 > L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I BsaXb Bpm1 BsaXa BsaXa PflM1 | BsaB1 | Pvu2 | | || | | | AACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCTCCAGCTGCAACC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGAGGTCGACGTTGG frame 0 > N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S S C N L Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | TCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCAT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAACTAGCCCCAGTA frame 0 > L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I Nco1 BsaB1 BsaB1 BseR1 | | | || CTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTACCACGAG frame 0 > W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F A M V L BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | CTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGGGGTGTCGTCG frame 0 > L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Q H Nar1 Kas1 | BseY1 BseY1 || | | || ACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 TGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGAGTAGTAGTGGAC frame 0 > S C M K G A V T I T I L L G V F I F C W A P F F L H L V L I I T C BspM1 BfuA1 Aar1 | CCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGATGCGGAAG frame 0 > P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A F SpAcc Fsp1 | | CGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+-----966 GCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > R S L E L R N T F K E I L C G C N S M N L G

PAGE 259

259 T145A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGAtcgCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCGCCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACgTCCAGCgCATCGCAgTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAgCAgCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGcTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGAtcgCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTagcGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCGCCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGCGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V A I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACgTCCAGCgCATCGCAgTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGcAGGTCGcGTAGCGTcACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAgCAgCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTcGTcGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGcTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCgATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 260

260 T145S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCAGCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCAGCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTCGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V S I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 261

261 Y152A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGGCCCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Apa1 PspOM | BsrD1 Drd1 Bgl1 | | BspH1 | | || | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGGCCCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCCGGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R A H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 262

262 Y152S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTCCCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I BsrD1 Drd1 BspH1 | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTCCCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCAGGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R S H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 263

263 K160A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGGCAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GGCAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CCGTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > A A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 264

264 K160S FlagmMC3R/pCDNA3 GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAgCAACCgGAgCGGCAGTGGGTTCTGT GA GCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGC AG CCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGAC TC TATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAGCGCCCTCACCTTG AT CGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATAT AT CCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTT TT CATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTC AT CGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGG frame 1 > D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | AgCAACCgGAgCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TcGTTGGcCTcGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACT frame 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | TCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGA frame 1 > L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | GGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAG frame 1 > E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCT frame 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R S BseR1 SpDon| BsaB1 BsaB1 || | | GCGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTA frame 1 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGA frame 1 > T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || GCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGG frame 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L BspM1 BfuA1 Aar1 | TCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCA frame 1 > H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V SpAcc Fsp1 | | CATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+991 GTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 265

265 I165A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG GCCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGGCCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACCGGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L A G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 266

266 I165S FlagmMC3R/pCDNA3 ATGgacTAcAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG AGCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGgacTAcAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACctgATgTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsrB1 BpuE1 BsaB1 BsaB1 | | | | | GAAAGCCCTCACCTTGAGCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTCGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L S G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 267

267 C175A FlagmMC3R/pCDNA3 GACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCA TC AAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAG CC GACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA TC TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCT GG GTCTGCTGCGGCATCGCCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCC TC TTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCT GG GCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCA TC TACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | GACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGC frame 0 > D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCA frame 0 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | CAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTAC frame 0 > R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M BsaXb BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | || | ATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGA frame 0 > I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAA frame 0 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L Nae1 SgrA1 | BtgZ1 NgoM4 | BsaB1 BsaB1 | | | | | GATCGGGGTCATCTGGGTCTGCTGCGGCATCGCCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTAGCCCCAGTAGACCCAGACGACGCCGTAGCGGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAG frame 0 > I G V I W V C C G I A G V M F I I Y S E S K M V I V C L I T M F F Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACC frame 0 > A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 Kas1 | BseY1 BseY1 || | | || CCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGA frame 0 > P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L BspM1 BfuA1 Aar1 | CATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAG frame 0 > I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L SpAcc Fsp1 | | ATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-------978 TAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 268

268 C175S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCAGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC frame 0 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA frame 0 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG frame 0 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT frame 0 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC frame 0 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCAGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGTCGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG frame 0 > K A L T L I G V I W V C C G I S G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG frame 0 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA frame 0 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG frame 0 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 269

269 F179A FlagmMC3R/pCDNA3 CTTCCTGCTGCCTGTCTTCTGTTTCTCCgATGCTgCCTAACCTCTCTGAgCACCCTGCAGCCCCTCCTGCCAgCAACCgGAGcGgCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTT CC TGgCTCTgGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGcCTGGCTGCAGCCGACATGCTGGTGAG CC TGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCgACTCTATgATTTGCATCTCCCTGGTGGCCTC CA TCTGCAACCTCCTGgCCaTTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCAT CT GCGGCGTGATGGCCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCA CG TCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGtCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCT CC ACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTTCCGCAG CC TGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bts1 BspM1 Bbs1 Pst1 BsaXa BsrB1 BfuA1 Bbs1 | | | | | | CTTCCTGCTGCCTGTCTTCTGTTTCTCCgATGCTgCCTAACCTCTCTGAgCACCCTGCAGCCCCTCCTGCCAgCAACCgGAGcGgCAGTGGGTTCTGTGA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 GAAGGACGACGGACAGAAGACAAAGAGGcTACGAcGGATTGGAGAGACTcGTGGGACGTCGGGGAGGACGGTcGTTGGcCTCgCcGTCACCCAAGACACT frame 2 > S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F C E BsaXb Bbs1 Bsg1 | | | GCAGGTCTTCATCAAGCCGGAGGTCTTCCTGgCTCTgGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTG 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCCAGAAGTAGTTCGGCCTCCAGAAGGACcGAGAcCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGTCCTTACCGTTGGAC frame 2 > Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N L BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | CACTCTCCCATGTACTTCTTCCTGTGCAGcCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTGAGAGGGTACATGAAGAAGGACACGTCgGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCGGCACTAGTTGT frame 2 > H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I N S BsaXb BsaXa BsaXa PflM1 | BsaB1 | | || | GCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCgACTCTATgATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGcTGAGATAcTAAACGTAGAGGGACCACCGGAGGTAGACGTTGGAGGA frame 2 > D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N L L Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | GgCCaTTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 CcGGtAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAACTAGCCCCAGTAGACC frame 2 > A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I W Msc1 Nco1 BsaB1 BsaB1 BseR1 | | | || GTCTGCTGCGGCATCTGCGGCGTGATGGCCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCA 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CAGACGACGCCGTAGACGCCGCACTACCGGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTACCACGAGGAGT frame 2 > V C C G I C G V M A I I Y S E S K M V I V C L I T M F F A M V L L M BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | TGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 ACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGGGGTGTCGTCGTGAG frame 2 > G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Q H S Nar1 Kas1 | BseY1 BseY1 || | | || CTGCATGAAGGGGGCTGtCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGTACTTCCCCCGACaGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGAGTAGTAGTGGACGGGG frame 2 > C M K G A V T I T I L L G V F I F C W A P F F L H L V L I I T C P BspM1 BfuA1 Aar1 | ACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTTCCGCA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 TGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGATGCGGAAGGCGT frame 2 > T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A F R S SpAcc Fsp1 | | GCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+-962 CGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 2 > L E L R N T F K E I L C G C N S M N L G

PAGE 270

270 F179S FlagmMC3R/pCDNA3 AGAACTCTTCCTGCTGCCTGTCTTCtGTTTCTCCGATGCTGCCTaacctCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGgagCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGA GG TCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCT GG TGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATcTTCGACTCTATGATTTGCATCTCCCTGGT GG CCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTG CG GCATCTGCGGCGTGATGTCCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAG GC TCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCcCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTT CT TCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTT CC GCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bts1 Bbs1 BspM1 Ear1 | Pst1 BsaXa BsrB1 BfuA1 | | | | | | AGAACTCTTCCTGCTGCCTGTCTTCtGTTTCTCCGATGCTGCCTaacctCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGgagCGGCAGTGGGTT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TCTTGAGAAGGACGACGGACAGAAGaCAAAGAGGCTACGACGGAttggaGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCctcGCCGTCACCCAA frame 2 > N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F BsaXb Bbs1 | Bbs1 Bsg1 | | | | CTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGC 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGTCCTTACCG frame 2 > C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | AACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 TTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCGGCACT frame 2 > N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I BsaXb BsaXa BsaXa PflM1 | BsaB1 | | || | TCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATcTTCGACTCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 AGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAgAAGCTGAGATACTAAACGTAGAGGGACCACCGGAGGTAGACGTT frame 2 > N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTC 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAACTAGCCCCAG frame 2 > L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V Nco1 BsaB1 BsaB1 BseR1 | | | || ATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTCCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TAGACCCAGACGACGCCGTAGACGCCGCACTACAGGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTACCACG frame 2 > I W V C C G I C G V M S I I Y S E S K M V I V C L I T M F F A M V L BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | TCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCcCCACAGCA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 AGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGgGGTGTCGT frame 2 > L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Q Nar1 Kas1 | BseY1 BseY1 || | | || GCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGAGTAGTAGTGG frame 2 > H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L I I T BspM1 BfuA1 Aar1 | TGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 ACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGATGCGGA frame 2 > C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A F SpAcc Fsp1 | | TCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+-------968 AGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 2 > R S L E L R N T F K E I L C G C N S M N L G

PAGE 271

271 Y182A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCGCCTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGtGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCcACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACaCCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCaACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BtgZ1 BsaB1 BsaB1 | | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCGCCTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGCGGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I A S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGtGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCaCGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCcACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACaCCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGgTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGtGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCaACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGtTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 272

272 Y182S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTCCTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R BsaB1 SpDon BseR1| BsaB1 | || | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTCCTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGAGGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I S S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 273

273 E184A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGCGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGCGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCGCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S A S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 274

274 E184S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCTCGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R BseR1 SpDon BsaB1 | Xho1 BsaB1 | | | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCTCGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGAGCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S S S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 275

275 M195A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCGCGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCGCGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGCGCAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T A F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 276

276 M195S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCAGCTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCAGCTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTCGAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T S F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 277

277 F196A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCgGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGGCCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCgGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGcCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 | || | | | | | ATCACCATGGCCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACCGGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M A F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 278

278 F196S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTCCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTcTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTCCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAGGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M S F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTcTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAgACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 279

279 F197A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCGCCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCGCCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGCGGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F A A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 280

280 F197A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCGCCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1 | BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCGCCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGCGGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F A A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1 | BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 281

281 F197S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTCCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 Eci1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 | || | | | | | ATCACCATGTTCTCCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAGGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F S A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 282

282 M199A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAgcCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATcATGATCGcCgtgaTcaAcAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCGCGGTGCTCcTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCcTGCTGGCGTGGTGGCCCCACAgCAGCACTCCTGCATGAaGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAgcCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTcgGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATcATGATCGcCgtgaTcaAcAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAgTACTAGCgGcactAgtTgTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Sac2 BseR1 | BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | | | ATCACCATGTTCTTCGCCGCGGTGCTCcTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGCGCCACGAGgAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A A V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || cTGCTGGCGTGGTGGCCCCACAgCAGCACTCCTGCATGAaGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 gACGACCGCACCACCGGGGTGTcGTCGTGAGGACGTACTtCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 283

283 M199S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCAGCGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L BseR1 BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | | ATCACCATGTTCTTCGCCAGCGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTCGCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A S V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 284

284 L247A FlagmMC3R/pCDNA3 GAaCTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCtGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAG GT CTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTG GT GAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTG GC CTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGC GG CATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGG CT CCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCGCGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTC TT CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTTC CG CAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bts1 Bbs1 BspM1 Ear1 | Pst1 BsaXa BsrB1 BfuA1 | | | | | | GAaCTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCtGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTtGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGaCGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGCCGTCACCCAAG frame 1 > N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S G F BsaXb Bbs1 | Bbs1 Bsg1 | | | | TGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 ACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGTCCTTACCGT frame 1 > C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N G N BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | ACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGAT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 TGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCGGCACTA frame 1 > L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A V I BsaXb BsaXa BsaXa PflM1| BsaB1 | | || | CAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAAC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGAGGTAGACGTTG frame 1 > N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S I C N Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAACTAGCCCCAGT frame 1 > L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V I Nco1 BsaB1 BsaB1 BseR1| | | || TCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTACCACGA frame 1 > W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F A M V L BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | CCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGGGGTGTCGTC frame 1 > L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Q Nar1 Kas1| BseY1 BseY1 || | | || CACTCCTGCATGAAGGGGGCTGTCACCATCACTATCGCGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGCGCGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGAGTAGTAGTGGA frame 1 > H S C M K G A V T I T I A L G V F I F C W A P F F L H L V L I I T C BspM1 BfuA1 Aar1 | GCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTACGCCTT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 CGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGATGCGGAA frame 1 > P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A F SpAcc Fsp1 | | CCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+------967 GGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > R S L E L R N T F K E I L C G C N S M N L G

PAGE 285

285 L247S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATcCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCAGCCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATcCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAgGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 || | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCAGCCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGTCGGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I S L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 286

286 W255A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCGCGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 BseY1 Kas1| | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCGCGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGCGCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C A A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 287

287 W255S FlagmMC3R/pCDNA3 CGaCGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATC AA GCCGGAGGTCTTCCTGgCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCC GA CATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATC TC CCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACgTCACCATCTTCTATGCCCTTCGGTACCACAgCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCTGG GT CTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTC TT CGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTCG GC GCCTTTCTTCCTCCACCTGGTCCTCaTCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATC TA CGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | CGaCGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 GCtGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGCCG frame 1 > D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | AGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGgCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACcGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGT frame 1 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | GGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGAT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTA frame 1 > N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | CGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCTCC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGAGG frame 1 > A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | ATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACgTCACCATCTTCTATGCCCTTCGGTACCACAgCATCATGACAGTGAGGAAAGCCCTCACCTTGA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGcAGTGGTAGAAGATACGGGAAGCCATGGTGTcGTAGTACTGTCACTCCTTTCGGGAGTGGAACT frame 1 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I BsaB1 BsaB1 BseR1 | | | TCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 AGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCG frame 1 > G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F A Nco1 BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | | CATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGG frame 1 > M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 BseY1 Kas1| | || CCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTCGGCGCCTTTCTTCCTCCACCTGGTCCTCa 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGAGCCGCGGAAAGAAGGAGGTGGACCAGGAGt frame 1 > P Q Q H S C M K G A V T I T I L L G V F I F C S A P F F L H L V L I BspM1 BfuA1 Aar1 | TCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCAT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTA frame 1 > I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I SpAcc Fsp1 | | CTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-----976 GATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 288

288 P257A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGGCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P BseY1 BseY1 | | CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGGCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCCGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A A F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 289

289 P257S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGTCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P BseY1 BseY1 | | CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGTCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCAGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A S F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 290

290 F258A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTGCCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTGCCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGACGGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P A F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 291

291 F258S FlagmMC3R/pCDNA3 CGaCAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAgCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGgCAGTGGGTTCTGTGAGCAGGTCTTCATCAAG CC GGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGgCTGCAGCCGAC AT GCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCNACTCTATGATTTGCATCTCC CT GGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAgTGAGGAAAGCCCTCACCTTGaTCGGGGTCATCTGGGTC TG CTGCGGCATCTGCGGCgTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTC GC CAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCG CC TTCTTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTAC GC CTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | CGaCAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAgCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGgCAGT 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 GCtGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTcGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGCcGTCA frame 1 > D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G S Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCAGTCCT frame 1 > G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V R N BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | BsaXb | | | | | | | ATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGgCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 TACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACcGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTACTAGCG frame 1 > G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M I A BsaXb BsaXa BsaXa PflM1| BsaB1 | | || | CGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCNACTCTATGATTTGCATCTCCCTGGTGGCCTCCATC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGNTGAGATACTAAACGTAGAGGGACCACCGGAGGTAG frame 1 > V I N S D S L T L E D Q F I Q H M D N I F S M I C I S L V A S I C Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | TGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAgTGAGGAAAGCCCTCACCTTGaTCG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 ACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTcACTCCTTTCGGGAGTGGAACtAGC frame 1 > N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L I G V Nco1 BsaB1 BsaB1 BseR1| | | || GGGTCATCTGGGTCTGCTGCGGCATCTGCGGCgTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CCCAGTAGACCCAGACGACGCCGTAGACGCCGcACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAGCGGTA frame 1 > I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F A M V BspLU Ear1BtgZ1 BmgB1 Bts1 | | | | | GGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACCGGGGT frame 1 > L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A P Q Nar1 Kas1| BseY1 BseY1 || | | || CAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTCTTTCCTCCACCTGGTCCTCATCA 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAGAAAGGAGGTGGACCAGGAGTAGT frame 1 > Q H S C M K G A V T I T I L L G V F I F C W A P S F L H L V L I I T BspM1 BfuA1 Aar1 | TCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCATCTA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAGTAGAT frame 1 > C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P L I Y A SpAcc Fsp1 | | CGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+--973 GCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > F R S L E L R N T F K E I L C G C N S M N L G

PAGE 292

292 F259A FlagmMC3R/pCDNA3 ATGgacTaCaaGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCGCCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGgacTaCaaGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACctgAtGttCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGCG orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F A BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 293

293 F259S FlagmMC3R/pCDNA3 GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGT GA GCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGC AG CCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGAC TC TATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTG AT CGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATAT AT CCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTT TT CATCTTCTGCTGGGCGCCTTTCTCTCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTC AT CGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGG frame 1 > D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | AGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACT frame 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | TCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGA frame 1 > L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | GGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAG frame 1 > E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCT frame 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K SpDon BsaB1 BsaB1 | | | AAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTA frame 1 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGA frame 1 > T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || GCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTCTC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAGAG frame 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F S L BspM1 BfuA1 Aar1 | TCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCA frame 1 > H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V SpAcc Fsp1 | | CATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+991 GTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 294

294 H261A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCGCCCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCGCCCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGCGGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L A L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L

PAGE 295

295 H261S FlagmMC3R/pCDNA3 AGGacGAcGACgACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGT CT TCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGC TG CAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGAT TT GCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGT CA TCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACAT GT TCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTT CT GCTGGGCGCCTTTCTTCCTCAGCCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCC CC TCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa | | | | AGGacGAcGACgACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TCCtgCTgCTGcTGtTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGC frame 2 > D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R Bts1 BspM1 BsaXb BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | GAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGA frame 2 > S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | GTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCA 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGT frame 2 > V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | TCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 AGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCA frame 2 > M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | GGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTC 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 CCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAG frame 2 > A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L SpDon BsaB1 BsaB1 | | | ACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACA frame 2 > T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | TCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 AGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCA frame 2 > F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V Nar1 SpAcc Kas1| Bpu10 | BseY1 BseY1 || BbvC1 | | | || | | GGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCAGCCTG 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGTCGGAC frame 2 > V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L S L BspM1 BfuA1 Aar1 | GTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 CAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGG frame 2 > V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I D P SpAcc Fsp1 | | CCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+--983 GGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 2 > L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 296

296 L264A FlagmMC3R/pCDNA3 ATGgacTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCGCCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGgacTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACctgATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCGCCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGCGGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V A I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 297

297 L264S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCAGCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCAGCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGTCGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V S I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 298

298 I265A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCGCCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCGCCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGCGGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L A I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 299

299 I265S FlagmMC3R/pCDNA3 ACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGA GC AGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAG CC TGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTC TA TGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGAT CG GGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATAT CC ACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTT CA TCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCAGCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCAT CG ACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 BsaXa | | | | | ACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTC frame 2 > Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S Bts1 BspM1 BsaXb BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | CAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATC 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAG frame 2 > N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | CTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACC frame 2 > L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | AGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAG frame 2 > T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | CCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTT frame 2 > L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K SpDon BsaB1 BsaB1 | | | GCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCA 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGT frame 2 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACG frame 2 > M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A Nar1 Kas1| BseY1 BseY1 || | | || TGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 ACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAG frame 2 > G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L BspM1 Bpu10 BfuA1 BbvC1 BstAP Aar1 | | | CACCTGGTCCTCAGCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCA 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTGGACCAGGAGTCGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGT frame 2 > H L V L S I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I SpAcc Fsp1 | | TCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+--------989 AGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 2 > D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 300

300 T267A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCGCCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BtgZ1 | CCTCCACCTGGTCCTCATCATCGCCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGCGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I A C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 301

301 T267S FlagmMC3R/pCDNA3 GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGT GA GCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGC AG CCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGAC TC TATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTG AT CGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATAT AT CCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTT TT CATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCAGCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTC AT CGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | GGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCC 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGG frame 1 > D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | AGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGA 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACT frame 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | TCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCT 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 AGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGA frame 1 > L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | GGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATC 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAG frame 1 > E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCT frame 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K SpDon BsaB1 BsaB1 | | | AAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCAT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTA frame 1 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | CACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCT 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 GTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGA frame 1 > T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || GCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 CGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGG frame 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L Pvu2 | TCCACCTGGTCCTCATCATCAGCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGT 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 AGGTGGACCAGGAGTAGTAGTCGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCA frame 1 > H L V L I I S C P T N P Y C I C Y T A H F N T Y L V L I M C N S V SpAcc Fsp1 | | CATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+991 GTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 1 > I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 302

302 C268A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCGCCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCNACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F CCTCCACCTGGTCCTCATCATCACCGCCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGCGGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T A P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCNACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGNTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I X P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 303

303 C268S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCaAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGcACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCAGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCaAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGtTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGcACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCgTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F CCTCCACCTGGTCCTCATCATCACCAGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGTCGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T S P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 304

304 T270A FlagmMC3R/pCDNA3 ATGgacTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCGCCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCaACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGgacTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACctgATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCGCCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGCGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P A N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCaACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGtTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 305

305 T270S FlagmMC3R/pCDNA3 GACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTG AG CAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCA GC CTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACT CT ATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGA TC GGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATA TC CACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTcCTGCATGAAgGGGGCTGTCACCATCACTATCCTGCTGGGTGtTT TC ATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCTCCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCA TC GACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 BsaXa | | | | | GACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGT frame 0 > D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S Bts1 BspM1 BsaXb BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | GCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGAT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTA frame 0 > N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I BspM1 BstX1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | | CCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGAC frame 0 > L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | GAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGA frame 0 > E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | CCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTT frame 0 > L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K SpDon BsaB1 BsaB1 | | | AGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 TCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAG frame 0 > A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGAC frame 0 > T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A Nar1 Kas1| BseY1 BseY1 || | | || CTGGCGTGGTGGCCCCACAGCAGCACTcCTGCATGAAgGGGGCTGTCACCATCACTATCCTGCTGGGTGtTTTCATCTTCTGCTGGGCGCCTTTCTTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACCGCACCACCGGGGTGTCGTCGTGAgGACGTACTTcCCCCGACAGTGGTAGTGATAGGACGACCCACaAAAGTAGAAGACGACCCGCGGAAAGAAGGA frame 0 > G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L BspM1 BfuA1 Aar1 | CCACCTGGTCCTCATCATCACCTGCCCCTCCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGTGGACCAGGAGTAGTAGTGGACGGGGAGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAG frame 0 > H L V L I I T C P S N P Y C I C Y T A H F N T Y L V L I M C N S V SpAcc Fsp1 | | ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+ 990 TAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 306

306 T278A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACGCGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACgTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC frame 0 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA frame 0 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG frame 0 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT frame 0 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC frame 0 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG frame 0 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG frame 0 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA frame 0 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACGCGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGCGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG frame 0 > L H L V L I I T C P T N P Y C I C Y A A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACgTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGcAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 307

307 T278S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAgGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGgcTGtGGTCagGAATGGCAACCTGCACTCtCCCaTGTACTTCtTCCTGT GC AGCCTGGCTGCAGCCGACATgctggtgAGcCTGTCCAACTCCCTGGAGACCATCATGATCgcCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACTCGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC frame 0 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAgGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTcCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA frame 0 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGgcTGtGGTCagGAATGGCAACCTGCACTCtCCCaTGTACTTCtTCCTGTGCAGCCTGGCTGCAGCCGACATgctggtgAGcCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACcgACaCCAGtcCTTACCGTTGGACGTGAGaGGGtACATGAAGaAGGACACGTCGGACCGACGTCGGCTGTAcgaccacTCgGACAGGTTGAGG frame 0 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCgcCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGcgGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT frame 0 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC frame 0 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG frame 0 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG frame 0 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA frame 0 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACTCGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGAGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG frame 0 > L H L V L I I T C P T N P Y C I C Y S A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 308

308 F281A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATGCCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC frame 0 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA frame 0 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG frame 0 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT frame 0 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC frame 0 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG frame 0 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG frame 0 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA frame 0 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 Xcm1 | | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATGCCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTACGGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG frame 0 > L H L V L I I T C P T N P Y C I C Y T A H A N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 309

309 F281S FlagmMC3R/pCDNA3 GACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCA TC AAGCCGGAGGTCTTCCTGgCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAG CC GACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA TC TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCgACAGGTACgTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCT GG GTCTGCTGCGGCATCTgCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCC TC TTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCT GG GCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATAGCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCA TC TACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | GACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGC frame 0 > D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGgCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACcGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCA frame 0 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | CAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTAC frame 0 > R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | ATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGA frame 0 > I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCATCTGCAACCTCCTGGCCATTGCCATCgACAGGTACgTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGTAGACGTTGGAGGACCGGTAACGGTAGcTGTCCATGcAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAA frame 0 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L BsaB1 BsaB1 | | GATCGGGGTCATCTGGGTCTGCTGCGGCATCTgCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTAGCCCCAGTAGACCCAGACGACGCCGTAGAcGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAG frame 0 > I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACC frame 0 > A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 Kas1| BseY1 BseY1 || | | || CCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGA frame 0 > P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L BspM1 BfuA1 Aar1 | CATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATAGCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTATCGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAG frame 0 > I I T C P T N P Y C I C Y T A H S N T Y L V L I M C N S V I D P L SpAcc Fsp1 | | ATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-------978 TAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 310

310 N282A FlagmMC3R/pCDNA3 GAcGACGACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCtGCcTaACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCA TC AAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAG CC GACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA TC TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCT GG GTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCC TC TTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCT GG GCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCGCCACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTCA TC TACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | GAcGACGACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCtGCcTaACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTgCTGCTGtTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGaCGgAtTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGC frame 0 > D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCA frame 0 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | CAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTAC frame 0 > R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | ATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGA frame 0 > I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAA frame 0 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L BsaB1 BsaB1 | | GATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAG frame 0 > I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACC frame 0 > A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 Kas1| BseY1 BseY1 || | | || CCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGA frame 0 > P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L BspM1 BfuA1 Aar1 BstX1 | | CATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCGCCACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGACCCCCTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGCGGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCTGGGGGAG frame 0 > I I T C P T N P Y C I C Y T A H F A T Y L V L I M C N S V I D P L SpAcc Fsp1 | | ATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-------978 TAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 311

311 N282S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTcCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAGCACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC frame 0 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA frame 0 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG frame 0 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT frame 0 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTcCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAgGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC frame 0 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG frame 0 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG frame 0 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA frame 0 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAGCACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTCGTGGATGGACCAAGAGTAGTACACGTTGAGG frame 0 > L H L V L I I T C P T N P Y C I C Y T A H F S T Y L V L I M C N S SpAcc Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > V I D P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 312

312 D295A FlagmMC3R/pCDNA3 GAcGACgACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCA TC AAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAG CC GACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA TC TCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTTGATCGGGGTCATCT GG GTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCC TC TTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCT GG GCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGCCCCCCTCA TC TACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 Ear1 | Pst1 BsaXa BsrB1 | | | | | GAcGACgACaAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 CTgCTGcTGtTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGACGGTCGTTGGCCTCGC frame 0 > D D D K N S S C C L S S V S P M L P N L S E H P A A P P A S N R S G Bts1 BspM1 BsaXb BfuA1 Bbs1 | Bbs1 Bsg1 | | | | | GCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 CGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCACTAGGACCGACACCA frame 0 > S G F C E Q V F I K P E V F L A L G I V S L M E N I L V I L A V V BspM1 BstX1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | | CAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 GTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGGGACCTCTGGTAGTAC frame 0 > R N G N L H S P M Y F F L C S L A A A D M L V S L S N S L E T I M BsaXb BsaXb BsaXa BsaXa PflM1| BsaB1 | | | || | ATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCATCTCCCTGGTGGCCT 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 TAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGTAGAGGGACCACCGGA frame 0 > I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I S L V A S Kpn1 BsrD1 Drd1 Acc65 | BspH1 SpDon | | | | | | CCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCTT 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 GGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTCCTTTCGGGAGTGGAA frame 0 > I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R K A L T L BsaB1 BsaB1 | | GATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAGTAGTGGTACAAGAAG frame 0 > I G V I W V C C G I C G V M F I I Y S E S K M V I V C L I T M F F Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | GCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGG 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGGGACGACCGCACCACC frame 0 > A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P A G V V A Nar1 Kas1| BseY1 BseY1 || | | || CCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAAGGAGGTGGACCAGGA frame 0 > P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F L H L V L BspM1 BfuA1 Aar1 BtgZ1 | | CATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCGTCATCGCCCCCCTC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGGCAGTAGCGGGGGGAG frame 0 > I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S V I A P L SpAcc Fsp1 | | ATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+-------978 TAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC frame 0 > I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 313

313 D295S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCAGCCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S SpAcc Fsp1 | | GTCATCAGCCCCCTCATCTACGCCTTCCGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGTCGGGGGAGTAGATGCGGAAGGCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I S P L I Y A F R S L E L R N T F K E I L C G C N S M N L G

PAGE 314

314 R302A FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAGCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCGCCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCATCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGTAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAGCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTCGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S Fsp1 | GTCATCGACCCCCTCATCTACGCCTTCGCCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGCGGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F A S L E L R N T F K E I L C G C N S M N L G

PAGE 315

315 R302S FlagmMC3R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTGCCAGCAACCGGAGCGGCAGTGGGTTCT GT GAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGTGATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGT GC AGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCCCTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCG AC TCTATGATTTGCATCTCCCTGGTGGCCTCCaTCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAGGAAAgCCCTCACCT TG ATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTCATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTAT AT ATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCCCTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTG TT TTCATCTTCTGCTGGGCGCCTTTCTTCCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCCG TC ATCGACCCCCTCATCTACGCCTTCAGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG Bbs1 SpAcc Ear1 | Pst1 | | | | ATGGACTACAAGGACGACGACGACAAGAACTCTTCCTGCTGCCTGTCTTCTGTTTCTCCGATGCTGCCTAACCTCTCTGAGCACCCTGCAGCCCCTCCTG 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCTTGAGAAGGACGACGGACAGAAGACAAAGAGGCTACGACGGATTGGAGAGACTCGTGGGACGTCGGGGAGGAC orf 1 > M D Y K D D D D K N S S C C L S S V S P M L P N L S E H P A A P P A Bts1 BspM1 BsaXb BsaXa BsrB1 BfuA1 Bbs1 | Bbs1 | | | | | | CCAGCAACCGGAGCGGCAGTGGGTTCTGTGAGCAGGTCTTCATCAAGCCGGAGGTCTTCCTGGCTCTGGGCATCGTCAGTCTGATGGAAAACATCCTGGT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 GGTCGTTGGCCTCGCCGTCACCCAAGACACTCGTCCAGAAGTAGTTCGGCCTCCAGAAGGACCGAGACCCGTAGCAGTCAGACTACCTTTTGTAGGACCA orf 1 > S N R S G S G F C E Q V F I K P E V F L A L G I V S L M E N I L V BspM1 Bsg1 BfuA1 Pst1 Bsg1 SpDon Bsa1 | | | | | | GATCCTGGCTGTGGTCAGGAATGGCAACCTGCACTCTCCCATGTACTTCTTCCTGTGCAGCCTGGCTGCAGCCGACATGCTGGTGAGCCTGTCCAACTCC 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CTAGGACCGACACCAGTCCTTACCGTTGGACGTGAGAGGGTACATGAAGAAGGACACGTCGGACCGACGTCGGCTGTACGACCACTCGGACAGGTTGAGG orf 1 > I L A V V R N G N L H S P M Y F F L C S L A A A D M L V S L S N S BsaXb BstX1 BsaXb BsaXa BsaXa PflM1| BsaB1 | | | | || | CTGGAGACCATCATGATCGCCGTGATCAACAGCGACTCCCTGACCTTGGAGGACCAGTTTATCCAGCACATGGATAATATCTTCGACTCTATGATTTGCA 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 GACCTCTGGTAGTACTAGCGGCACTAGTTGTCGCTGAGGGACTGGAACCTCCTGGTCAAATAGGTCGTGTACCTATTATAGAAGCTGAGATACTAAACGT orf 1 > L E T I M I A V I N S D S L T L E D Q F I Q H M D N I F D S M I C I Kpn1 BsrD1 Drd1 Acc65 | BspH1 | | | | | TCTCCCTGGTGGCCTCCaTCTGCAACCTCCTGGCCATTGCCATCGACAGGTACGTCACCATCTTCTATGCCCTTCGGTACCACAGCATCATGACAGTGAG 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 AGAGGGACCACCGGAGGtAGACGTTGGAGGACCGGTAACGGTAGCTGTCCATGCAGTGGTAGAAGATACGGGAAGCCATGGTGTCGTAGTACTGTCACTC orf 1 > S L V A S I C N L L A I A I D R Y V T I F Y A L R Y H S I M T V R SpDon BsaB1 BsaB1 | | | GAAAgCCCTCACCTTGATCGGGGTCATCTGGGTCTGCTGCGGCATCTGCGGCGTGATGTTCATCATCTACTCCGAGAGCAAGATGGTCATCGTGTGTCTC 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 CTTTcGGGAGTGGAACTAGCCCCAGTAGACCCAGACGACGCCGTAGACGCCGCACTACAAGTAGTAGATGAGGCTCTCGTTCTACCAGTAGCACACAGAG orf 1 > K A L T L I G V I W V C C G I C G V M F I I Y S E S K M V I V C L Nco1 BseR1| BspLU Ear1BtgZ1 BmgB1 Bts1 || | | | | | ATCACCATGTTCTTCGCCATGGTGCTCCTCATGGGCACCCTATATATCCACATGTTCCTCTTCGCCAGGCTCCACGTCCAGCGCATCGCAGTGCTGCCCC 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 TAGTGGTACAAGAAGCGGTACCACGAGGAGTACCCGTGGGATATATAGGTGTACAAGGAGAAGCGGTCCGAGGTGCAGGTCGCGTAGCGTCACGACGGGG orf 1 > I T M F F A M V L L M G T L Y I H M F L F A R L H V Q R I A V L P P Nar1 Kas1| BseY1 BseY1 || | | || CTGCTGGCGTGGTGGCCCCACAGCAGCACTCCTGCATGAAGGGGGCTGTCACCATCACTATCCTGCTGGGTGTTTTCATCTTCTGCTGGGCGCCTTTCTT 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 GACGACCGCACCACCGGGGTGTCGTCGTGAGGACGTACTTCCCCCGACAGTGGTAGTGATAGGACGACCCACAAAAGTAGAAGACGACCCGCGGAAAGAA orf 1 > A G V V A P Q Q H S C M K G A V T I T I L L G V F I F C W A P F F BspM1 BfuA1 Aar1 | CCTCCACCTGGTCCTCATCATCACCTGCCCCACCAATCCCTACTGCATCTGCTACACGGCCCATTTCAACACCTACCTGGTTCTCATCATGTGCAACTCC 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGAGGTGGACCAGGAGTAGTAGTGGACGGGGTGGTTAGGGATGACGTAGACGATGTGCCGGGTAAAGTTGTGGATGGACCAAGAGTAGTACACGTTGAGG orf 1 > L H L V L I I T C P T N P Y C I C Y T A H F N T Y L V L I M C N S Eco57 Fsp1 | | GTCATCGACCCCCTCATCTACGCCTTCAGCAGCCTGGAGCTGCGCAACACGTTCAAGGAGATTCTCTGCGGCTGCAACAGCATGAACTTGGGCTAG 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+--993 CAGTAGCTGGGGGAGTAGATGCGGAAGTCGTCGGACCTCGACGCGTTGTGCAAGTTCCTCTAAGAGACGCCGACGTTGTCGTACTTGAACCCGATC orf 1 > V I D P L I Y A F S S L E L R N T F K E I L C G C N S M N L G

PAGE 316

316 FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTgTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCtGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCC TG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTgTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAAcAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCtGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGaCGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BseY1 | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCTGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGGACTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I L I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 317

317 L250A FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGNGGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCG CG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GNGGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CNCCCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > X E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BtgZ1 Nru1 BseY1 | | | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCGCGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGCGCTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I A I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 318

318 L250E FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCG AG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BseY1 | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCGAGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGCTCTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I E I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 319

319 L250F FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCT TT ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 polyA BseY1 | | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCTTTATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGAAATAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I F I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 320

320 L250K FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGtGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCA AG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GtGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CaCTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BseY1 | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCAAGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGTTCTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I K I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 321

321 L250N FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCgtgGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCA AC ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAgAACTGAGGAAAACCTTCaAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCgtgGGATGCACACTTCTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGcacCCTACGTGTGAAGAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BseY1 | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCAACATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGTTGTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I N I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAgAACTGAGGAAAACCTTCaAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTcTTGACTCCTTTTGGAAGtTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

PAGE 322

322 L250Q FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTcTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAgAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAgCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCC AG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAgGAAAAcCTTCAAAGAGATCATCTGTTGCTATCCCCTGgGAgGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTcTCTGCACCTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAgAGACGTGGAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAgAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAgCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTcTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTcGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BseY1 | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCAGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGGTCTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I Q I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAgGAAAAcCTTCAAAGAGATCATCTGTTGCTATCCCCTGgGAgGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTcCTTTTgGAAGTTTCTCTAGTAGACAACGATAGGGGACcCTcCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

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323 L250R FlaghMC4R/pCDNA3 ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTcTCTGCACcTCTGGAACCGCAGCAGTTACAGACTGCACAGCAATGCCAGTGAGTCCCTTGGAAAAGGCT AC TCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTTGTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCAC CC ATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTGGTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATA TT GATAATGTCATTGACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAACATTATGACAGTTA AG CGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCTGTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTC TC ATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGATTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCC GG ATTGGCGTCTTTGTTGTCTGCTGGGCCCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATGT GT AATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTTGTGACTTGTCTAGCAGATATTAG SpAcc SpDon Bsg1 Bsg1 | | | | ATGGACTACAAGGACGACGACGACAAGGTGAACTCCACCCACCGTGGGATGCACACTTcTCTGCACcTCTGGAACCGCAGCAGTTACAGACTGCACAGCA 1 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 100 TACCTGATGTTCCTGCTGCTGCTGTTCCACTTGAGGTGGGTGGCACCCTACGTGTGAAgAGACGTGgAGACCTTGGCGTCGTCAATGTCTGACGTGTCGT orf 1 > M D Y K D D D D K V N S T H R G M H T S L H L W N R S S Y R L H S N BsrD1 Bsu36 | | ATGCCAGTGAGTCCCTTGGAAAAGGCTACTCTGATGGAGGGTGCTACGAGCAACTTTTTGTCTCTCCTGAGGTGTTTGTGACTCTGGGTGTCATCAGCTT 101 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 200 TACGGTCACTCAGGGAACCTTTTCCGATGAGACTACCTCCCACGATGCTCGTTGAAAAACAGAGAGGACTCCACAAACACTGAGACCCACAGTAGTCGAA orf 1 > A S E S L G K G Y S D G G C Y E Q L F V S P E V F V T L G V I S L Bsm1 Pst1 | | GTTGGAGAATATCTTAGTGATTGTGGCAATAGCCAAGAACAAGAATCTGCATTCACCCATGTACTTTTTCATCTGCAGCTTGGCTGTGGCTGATATGCTG 201 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 300 CAACCTCTTATAGAATCACTAACACCGTTATCGGTTCTTGTTCTTAGACGTAAGTGGGTACATGAAAAAGTAGACGTCGAACCGACACCGACTATACGAC orf 1 > L E N I L V I V A I A K N K N L H S P M Y F F I C S L A V A D M L SpDon T7Ter Ssp1 | | | GTGAGCGTTTCAAATGGATCAGAAACCATTGTCATCACCCTATTAAACAGTACAGATACGGATGCACAGAGTTTCACAGTGAATATTGATAATGTCATTG 301 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 400 CACTCGCAAAGTTTACCTAGTCTTTGGTAACAGTAGTGGGATAATTTGTCATGTCTATGCCTACGTGTCTCAAAGTGTCACTTATAACTATTACAGTAAC orf 1 > V S V S N G S E T I V I T L L N S T D T D A Q S F T V N I D N V I D SpDon Mfe1 Bts1 Bpm1| | | || ACTCGGTGATCTGTAGCTCCTTGCTTGCATCCATTTGCAGCCTGCTTTCAATTGCAGTGGACAGGTACTTTACTATCTTCTATGCTCTCCAGTACCATAA 401 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 500 TGAGCCACTAGACATCGAGGAACGAACGTAGGTAAACGTCGGACGAAAGTTAACGTCACCTGTCCATGAAATGATAGAAGATACGAGAGGTCATGGTATT orf 1 > S V I C S S L L A S I C S L L S I A V D R Y F T I F Y A L Q Y H N CATTATGACAGTTAAGCGGGTTGGGATCATCATAAGTTGTATCTGGGCAGCTTGCACGGTTTCAGGCATTTTGTTCATCATTTACTCAGATAGTAGTGCT 501 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 600 GTAATACTGTCAATTCGCCCAACCCTAGTAGTATTCAACATAGACCCGTCGAACGTGCCAAAGTCCGTAAAACAAGTAGTAAATGAGTCTATCATCACGA orf 1 > I M T V K R V G I I I S C I W A A C T V S G I L F I I Y S D S S A SpDon BspLU SpDon | | | GTCATCATCTGCCTCATCACCATGTTCTTCACCATGCTGGCTCTCATGGCTTCTCTCTATGTCCACATGTTCCTGATGGCCAGGCTTCACATTAAGAGGA 601 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 700 CAGTAGTAGACGGAGTAGTGGTACAAGAAGTGGTACGACCGAGAGTACCGAAGAGAGATACAGGTGTACAAGGACTACCGGTCCGAAGTGTAATTCTCCT orf 1 > V I I C L I T M F F T M L A L M A S L Y V H M F L M A R L H I K R I PspOM Eci1 BspE1 BseY1 | | | | | TTGCTGTCCTCCCCGGCACTGGTGCCATCCGCCAAGGTGCCAATATGAAGGGAGCGATTACCTTGACCATCCGGATTGGCGTCTTTGTTGTCTGCTGGGC 701 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 800 AACGACAGGAGGGGCCGTGACCACGGTAGGCGGTTCCACGGTTATACTTCCCTCGCTAATGGAACTGGTAGGCCTAACCGCAGAAACAACAGACGACCCG orf 1 > A V L P G T G A I R Q G A N M K G A I T L T I R I G V F V V C W A Apa1 Ssp1 SpDon | | | CCCATTCTTCCTCCACTTAATATTCTACATCTCTTGTCCTCAGAATCCATATTGTGTGTGCTTCATGTCTCACTTTAACTTGTATCTCATACTGATCATG 801 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 900 GGGTAAGAAGGAGGTGAATTATAAGATGTAGAGAACAGGAGTCTTAGGTATAACACACACGAAGTACAGAGTGAAATTGAACATAGAGTATGACTAGTAC orf 1 > P F F L H L I F Y I S C P Q N P Y C V C F M S H F N L Y L I L I M BspE1 SpAcc Xmn1 T7Ter Stu1 | | | | | TGTAATTCAATCATCGATCCTCTGATTTATGCACTCCGGAGTCAAGAACTGAGGAAAACCTTCAAAGAGATCATCTGTTGCTATCCCCTGGGAGGCCTTT 901 ---------+---------+---------+---------+---------+---------+---------+---------+---------+---------+ 1000 ACATTAAGTTAGTAGCTAGGAGACTAAATACGTGAGGCCTCAGTTCTTGACTCCTTTTGGAAGTTTCTCTAGTAGACAACGATAGGGGACCCTCCGGAAA orf 1 > C N S I I D P L I Y A L R S Q E L R K T F K E I I C C Y P L G G L C GTGACTTGTCTAGCAGATATTAG 1001 ---------+---------+ 1020 CACTGAACAGATCGTCTATAATC orf 1 > D L S S R Y

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324 APPENDIX B AGONIST AND ANTAGONIST PHARMACOLOGY CURVES AT THE WILDTYPE AND MUTANT MOUSE MELANOCORTIN-3 RECEPTORS The agonist and antagonist pharmacology curves illustrated in this appendix are either representative examples of one experiment, done in duplicate or are averag es from at least two experiments, done in duplicate.

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363 BIOGRAPHICAL SKETCH Bettina Proneth was born in Regensburg, Germany on September 3rd 1978. After graduating from high school in 1997 she starte d Pharmacy School at the University of Regensburg in Germany. She received her bach elors degree in 2002 and started her pharmacy internships in the Hospital Pharmacy and Retail Pharmacy in Regensburg. In fall 2002 she came to the University of Florida as exchange student. She worked with Dr. Paul Doering in the Center of Drug Information and did research with Dr. Sean Sullivan in the field of Cancer Gene Therapy. In fall 2003 she passed her pharmacy board exam to practice as pharmacist in the European Union. In spring 2004 she started the graduate program in the Department of Medicinal Chemistry at the University of Fl orida in Gainesville. She worked under the supervision of Dr. Carrie Haskell-Luevano on ra tional drug design approaches towards the brain melanocortin receptors potentially leadi ng to new treatment options for obesity.