G Protein-Coupled Receptors as Drug Targets Editedby RolandSeifert,ThomasWieland GProtein-CoupledReceptors.EditedbyR.SeifertandT.Wieland Copyrightª2005WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim ISBN:3-527-30819-9 Methods andPrinciplesin Medicinal Chemistry Editedby R. R.Mannhold,H. Kubinyi,G. Folkers EditorialBoard H.-D.Ho¨ltje, H.Timmerman, J.Vacca, H.vande Waterbeemd, T.Wieland Previous Volumes of this Series: D.Smith, D.Walker, H.van de Waterbeemd, H.Lennerna¨s, H.van de Waterbeemd P. Artursson(eds.) Pharmacokinetics and Drug Bioavailability Metabolism in Drug Design Vol. 18 2003,ISBN3-527-30438-X Vol. 13 2001,ISBN3-527-30197-6 H.-J. Bo¨hm, S.S.Abdel-Meguid (eds.) T.Lenaguer (ed.) Protein Crystallography Bioinformatics – in Drug Discovery From Genomes to Drugs Vol. 20 2004,ISBN3-527-30678-1 Vol. 14 2002,ISBN3-527-29988-2 Th.Dingermann, D.Steinhilber, G. Folkers (eds.) J.K. Seydel,M.Wiese Molecular Biology Drug-Membrane Interactions in Medicinal Chemistry Vol. 15 2002,ISBN3-527-30427-4 Vol. 21 2004,ISBN3-527-30431-2 O.Zerbe (ed.) H.Kubinyi, G.Mu¨ller(eds.) BioNMR in Drug Research Chemogenomics in Vol. 16 Drug Discovery 2002,ISBN3-527-30465-7 Vol. 22 2004,ISBN3-527-30987-X P. Arloni,F. Alber (eds.) Quantum Medicinal Chemistry T.I. Oprea(ed.) Vol. 17 Chemoinformatics in Drug 2003,ISBN3-527-30456-8 Discovery Vol. 23 2005,ISBN3-527-30753-2 G Protein-Coupled Receptors as Drug Targets Analysis of Activation and Constitutive Activity Edited by Roland Seifert and Thomas Wieland SeriesEditors: AllbookspublishedbyWiley-VCHarecarefullypro- duced.Nevertheless,authors,editors,andpublisher Prof.Dr.RaimundMannhold donotwarranttheinformationcontainedinthese BiomedicalResearchCenter books,includingthisbook,tobefreeoferrors.Rea- MolecularDrugResearchGroup dersareadvisedtokeepinmindthatstatements,data, Heinrich-Heine-Universita¨t illustrations,proceduraldetailsorotheritemsmay Universta¨tsstrasse1 inadvertentlybeinaccurate. 40225Du¨sseldorf Germany LibraryofCongressCardNo.: Appliedfor. [email protected] BritishLibraryCataloguing-in-PublicationData: Prof.Dr.HugoKubinyi Acataloguerecordforthisbookisavailable Donnersbergstrasse9 fromtheBritishLibrary. 67256WeisenheimamSand Germany Bibliographicinformationpublishedby [email protected] DieDeutscheBibliothek DieDeutscheBibliothekliststhispublicationinthe Prof.Dr.GerdFolkers DeutscheNationalbibliografie;detailed CollegiumHelveticum bibliographicdataisavailableintheInternetat STW/ETHZentrum http://dnb.ddb.de 8092Zu¨rich ª2005WILEY-VCHVerlag Switzerland GmbH&Co.KGaA,Weinheim [email protected] Allrightsreserved(includingthoseoftranslationinto otherlanguages).Nopartofthisbookmaybere- VolumeEditors: producedinanyform–byphotoprinting,microfilm, oranyothermeans–nortransmitted Prof.ThomasWieland ortranslatedintoamachinelanguagewithoutwritten Institutfu¨rPharmakologieundToxikologie permissionfromthepublishers. Fakulta¨tfu¨rKlinischeMedizinMannheim DerUniversita¨tHeidelberg Maybachstraße14-16 Typesetting Mitterweger&Partner, 68169Mannheim KommunikationsgesellschaftmbH,Plankstadt Germany Printing StraussGmbH,Mo¨rlenbach [email protected] Bookbinding J.Scha¨fferGmbH,Gru¨nstadt Prof.RolandSeifert PrintedintheFederalRepublicofGermany DepartmentofPharmacologyandToxicology UniversityofRegensburg ISBN-13:978-3-527-30819-4 Universita¨tsstraße31 ISBN-10:3-527-30819-9 D-93053Regensburg Germany [email protected] VV Table of Contents Preface XI APersonalForeword XIII ListofContributors XV AbbreviationsandTerminology XX I GeneralConcepts 1 1 HistoricalBackground andIntroduction 3 2 TheNatureofConstitutiveActivityandInverseAgonism 11 2.1 Historical Perspective 11 2.2 Theoretical Basis of Inverse Agonism: Relevance of Receptor Type 13 2.3 The Interaction of Systems with Ligands 18 2.4 Inverse Agonism as a Phenotypic Behavior 23 2.5 Conclusion 25 3 MolecularMechanismsofGPCRActivation 27 3.1 Introduction 27 3.2 GPCR Structure and Ligand Recognition 28 3.3 Conformational Changes in the GPCR Activation Process 29 3.4 Conversion to the Active Receptor State Involves Release of Stabilizing Intramolecular Interactions 35 3.5 Kinetics of Agonist Binding and Receptor Activation 37 3.6 GPCR Activation in an Oligomeric Context 38 4 MolecularandCellularDeterminantsofGPCRSpliceVariant ConstitutiveActivity 43 4.1 Introduction 43 GProtein-CoupledReceptors.EditedbyR.SeifertandT.Wieland Copyrightª2005WILEY-VCHVerlagGmbH&Co.KGaA,Weinheim ISBN:3-527-30819-9 VI TableofContents 4.2 Constitutive Activation of Second Messenger Production by C-Terminal Splice Variants of GPCRs 45 4.2.1 The Constitutive Activities of C-Terminal 5-HT Receptor 4 Splice Variants: the Shortest, the Strongest 45 4.2.2 The Constitutive Activities of mGlu R and mGlu R C-t Splice Variants: 1 5 a Case for which a Physiological Control does exist 48 4.2.3 Other Examples of GPCR C-t Splice Variants with Different Constitutive Activities 50 4.3 Differential Constitutive Internalization of C-t GPCR Splice Variants 50 4.3.1 The Thromboxane A2 Receptor TPbR, but not the TPaR Splice Variant, is Constitutively Internalized by Clathrin-dependent, GRK- and Arrestin-independent Mechanisms 51 4.3.2 The Prostaglandin F2a Receptor FPBR, but not the FPAR C-Terminal Splice Variant, is Constitutively Internalized by a Clathrin-independent, PI3-Kinase-dependent Mechanism 52 4.4 Conclusion 53 5 NaturallyOccurringConstitutivelyActiveReceptors: PhysiologicalandPharmacological Implications 55 5.1 Introduction 55 5.2 Wild-type Interspecies Homologues 56 5.3 Wild-type Receptor Subtypes within a Given Species 57 5.4 Wild-type Alternatively Spliced Receptors 57 5.5 Polymorphisms in GPCRs 57 5.6 GPCR Mutation-induced Disease 59 5.7 Future Challenges 60 6 TheImpactofGProteinsonConstitutiveGPCRActivity 63 6.1 Introduction 63 6.2 The Contribution of G proteins to Constitutive Activity 64 6.2.1 Basic Features 64 6.2.2 The Distribution of G Proteins in the Plasma Membrane 65 6.3 GPCR–G Protein Fusion Proteins 66 6.3.1 Basic Features 66 6.3.2 Modulation of the GPCR–G Protein Interface Alters Constitutive Activity 66 6.3.3 Use of G Protein Variation to Detect Ligand Efficacy 68 6.4 Conclusions 69 7 (Patho)physiologicalandTherapeuticRelevanceofConstitutive ActivityandInverseAgonismatGProtein-CoupledReceptors 71 7.1 Introduction 71 7.2 Physiological Relevance of Constitutive Activity of GPCRs 72 7.3 Constitutive Activity of GPCRs and Pathophysiology of Disease 73 7.4 Physiological Relevance of Inverse Agonists 76 TableofContents VVIIII 7.5 Inverse Agonists as Drugs 77 7.6 Conclusions 79 8 MethodologicalApproaches 81 8.1 Introduction 81 8.2 AnalysisofConstitutiveGPCRActivityinMembranesandIntactCells 82 8.2.1 Procedure for Sf9 Cell Culture and Membrane Preparation 84 8.2.2 GPCR Radioligand Binding Studies 86 8.2.3 GTPase Assay 90 8.2.4 [35S]GTPcS Binding Assay 96 8.2.5 Adenylyl Cyclase Assay 101 8.3 Measurement of Constitutive Activity of GPCRs in Intact Cells 106 8.3.1 Quantitative Determination of cAMP Concentrations in Cell Culture Lysates 109 8.3.2 Determination of Inositol Phosphate Formation in Living Cells 110 8.3.3 Determination of G Protein Activation by SRF-mediated Gene Transcription 113 8.3.4 Deorphanization and Constitutive Activity of GPCRs by Aequorin-based Ca2+ Determinations 115 II ConstitutiveActivityofSelectedGPCRSystems 121 9 ConstitutiveActivityofb-Adrenoceptors:AnalysisinMembraneSystems 123 9.1 Introduction 123 9.2 Analysis of bAR/G Protein Coupling in Membranes 124 s 9.3 Development of the Concept that bARs are Constitutively Active 127 9.4 Probing Models of GPCR Activation with b AR and b AR 2 wt 2 CAM with Inverse Agonists 128 9.5 Probing Models of GPCR Activation with b AR and b AR 2 wt 2 CAM and with Partial and Full Agonists 130 9.6 Probing Models of GPCR Activation with b AR and Purine 2 wt Nucleotides 131 9.7 Constitutive Activity of the b AR Coupled to Various Ga Proteins 133 2 s 9.8 Probing Models of GPCR Activation with b AR Coupled to 2 Various Classes of G proteins 135 9.9 ComparisonoftheConstitutiveActivitiesoftheb ARandtheb AR 135 1 2 9.10 Conclusions 136 10 ConstitutiveActivityofb-Adrenoceptors:AnalysisbyPhysiologicalMethods 141 10.1 Introduction 141 10.2 ConstitutiveActivityandInverseAgonism:DefinitionandDetection 142 10.3 b -Adrenoceptors 143 1 10.3.1 Constitutive Activity of Overexpressed b ARs 143 1 VIII TableofContents 10.3.2 Is there any Evidence for a Physiological Effect of Constitutively Active Receptors in Normal Cardiomyocytes? 145 10.3.3 Substates of the b AR: the Putative b AR 147 1 4 10.4 b -Adrenoceptors 148 2 10.4.1 Constitutive Activity of Overexpressed b ARs 148 2 10.4.2 Inverse Agonism at the b AR 150 2 10.4.3 bAR Antagonists: Inverse Agonists at b AR-G or Full 2 s Agonists at b AR-G? 152 2 i 10.4.4 Involvement of the b AR in the “Putative b AR“ Effect 153 2 4 10.5 Homo- and Heterodimerization of b - and b ARs 154 1 2 10.6 Conclusions 154 11 ConstitutiveActivityatthea-Adrenoceptors:PastandFutureImplications 159 1 11.1 Introduction 159 11.1.1 The a -Adrenoceptors: Main Structure–Functional Features 159 1 11.1.2 The Discovery of Constitutively Activating Mutations and its Implications 161 11.2 Theoretical and Experimental Approaches for Study of Constitutive GPCR Activity 162 11.2.1 Theoretical Analysis of CAM GPCR Pharmacology 162 11.2.2 Computational Modeling of the a AR 163 1B 11.2.3 Measuring Constitutive Activity of the a AR Subtypes 165 1 11.3 Constitutively Activating Mutations of the a AR Subtypes 166 1 11.3.1 Where the Mutations have been Found 166 11.3.2 Constitutive Activation of Multiple Signaling Pathways 169 11.4 A Putative Model of Receptor Activation for the a AR 169 1B 11.5 Constitutive Activity of Wild-type a ARs and Inverse Agonism 171 1 11.5.1 Constitutive Activity of Wild-type a AR Subtypes 171 1 11.5.2 Inverse Agonism at the a ARs 172 1 11.6 Receptor Regulation and Constitutive Activity of the a ARs 173 1 11.7 Conclusions 174 12 ConstitutiveActivityofMuscarinicAcetylcholineReceptors: ImplicationsforReceptorActivationandPhysiologicalRelevance 177 12.1 Introduction 177 12.2 Constitutive Activity – Native Systems 178 12.3 Constitutive Activity – Recombinant Systems 178 12.4 Constitutive Activation by G Proteins 179 12.5 Structure–Function Analysis of Receptor Activation 180 12.5.1 Transmembrane Domain 3 182 12.5.2 Transmembrane Domain 6 183 12.5.3 Transmembrane Domain 7 185 12.5.4 Other Transmembrane Domains and Extracellular Domains 186 12.5.5 Cytoplasmic Domains 186 12.5.6 i3 Loop 187 TableofContents IIXX 12.5.7 i2 Loop 188 12.6 Structure–Function Model for Activation 189 12.7 Conclusions 189 13 ConstitutivelyActiveHistamineReceptors 195 13.1 Introduction 195 13.2 The Histamine Receptors 196 13.2.1 The H R 197 1 13.2.2 The H R 201 2 13.2.3 The H R 202 3 13.2.4 The H R 206 4 13.3 Assay Systems for Detection of Constitutive Activity of Histamine Receptors 209 13.3.1 Histamine Receptor Expression and the Detection of Constitutive Activity 209 13.3.2 Changes in Intracellular Ca2+ 210 13.3.3 [35S]GTPcS Binding Assays (see also Chapter 8) 211 13.3.4 IP Formation (see also Chapter 8) 212 3 13.3.5 cAMP Assays (see also Chapter 8) 212 13.3.6 Measurements of Arachidonic Acid (AA) Release 213 13.3.7 Reporter Gene Assays (see also Chapter 8) 213 13.3.8 Activation of Kinases 214 13.3.9 Effects oftheCellular Environment onHistamine Receptor Activity 214 13.3.10 Construction and Expression of Constitutively Active Mutant Receptors 215 13.3.11 Contamination with Endogenous Histamine 216 13.4 Conclusions 216 14 ConstitutivelyActiveSerotoninReceptors 223 14.1 Introduction 223 14.2 5-HT Receptor (5-HT R) 224 1A 1A 14.3 5-HT and 5-HT Receptors (5-HT R and 5-HT R) 226 1B 1D 1B 1D 14.4 5-HT Receptor (5-HT R) 228 2A 2A 14.5 5-HT Receptor (5-HT R) 231 2C 2C 14.6 Conclusion 237 15 VirallyEncodedConstitutivelyActiveChemokineReceptors 243 15.1 Introduction 243 15.1.1 Viral Strategies to Evade the Host Immune System 243 15.1.2 Chemokines and Chemokine Receptors 243 15.1.3 Viral Homologues of Chemokines and Chemokine Receptors and Viral Chemokine-binding Proteins 246 15.2 The Human Cytomegalovirus-encoded Chemokine Receptor Homologue pUS28 248 15.2.1 Characteristics of Human Cytomegalovirus Infection 248 X TableofContents 15.2.2 Functional Characteristics of pUS28 249 15.2.3 Signaling Pathways Regulated by pUS28 249 15.2.4 Regulation of Transcriptional Activity by pUS28 250 15.2.5 Regulation of Constitutively Active pUS28 252 15.2.6 Cellular Functions of pUS28 253 15.3 The Human Kaposi’s Sarcoma Virus-encoded Chemokine Receptor KSHV-GPCR 255 15.3.1 Characteristics of Human Kaposi’s Sarcoma Virus Infection 255 15.3.2 Functional Characteristics of KSHV-GPCR 255 15.3.3 Signaling Pathways Regulated by KSHV-GPCR 256 15.3.4 Regulation of Transcriptional Activity by KHSV-GPCR 256 15.3.5 Regulation of KSHV Activity by Chemokines 258 15.3.6 Structure–Function Relationships of KHSV-GPCR 258 15.3.7 Cellular Functions of KHSV-GPCR in vivo 259 15.4 Conclusions 260 Index 265