G Protein-Coupled Receptors as Drug Targets

Transcription

1 G Protein-Coupled Receptors as Drug Targets Analysis of Activation and Constitutive Activity Edited by Roland Seifert and Thomas Wieland WILEY- VCH WILEY-VCH Verlag GmbH & Co. KGaA

2 Table of Contents Preface XI A Personal Foreword XIII List of Contributors XV Abbreviations and Terminology XX I General Concepts 1 1 Historical Background and Introduction 3 2 The Nature of Constitutive Activity and Inverse Agonism Historical Perspective Theoretical Basis of Inverse Agonism: Relevance of Receptor Type The Interaction of Systems with Ligands Inverse Agonism as a Phenotypic Behavior Conclusion 25 3 Molecular Mechanisms of CPCR Activation Introduction GPCR Structure and Ligand Recognition Conformational Changes in the GPCR Activation Process Conversion to the Active Receptor State Involves Release of Stabilizing Intramolecular Interactions Kinetics of Agonist Binding and Receptor Activation GPCR Activation in an Oligomeric Context 38 4 Molecular and Cellular Determinants of CPCR Splice Variant Constitutive Activity Introduction 43 G Protein-Coupled Receptors. Edited by R. Seifert and T. Wieland Copyright 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN:

3 VI Table of Contents 4.2 Constitutive Activation of Second Messenger Production by C-Terminal Splice Variants of GPCRs The Constitutive Activities of C-Terminal 5-HT 4 Receptor Splice Variants: the Shortest, the Strongest The Constitutive Activities of mglujr and mglu 5 R C-t Splice Variants: a Case for which a Physiological Control does exist Other Examples of GPCR C-t Splice Variants with Different Constitutive Activities Differential Constitutive Intemalization of C-t GPCR Splice Variants The Thromboxane A2 Receptor TP p R, but not the TP a R Splice Variant, is Constitutively Internalized by Clathrin-dependent, GRK- and Arrestin-independent Mechanisms The Prostaglandin F 2a Receptor FP B R, but not the FP A R C-Terminal Splice Variant, is Constitutively Internalized by a Clathrin-independent, PI3-Kinase-dependent Mechanism Conclusion 53 5 Naturally Occurring Constitutively Active Receptors: Physiological and Pharmacological Implications Introduction Wild-type Interspecies Homologues Wild-type Receptor Subtypes within a Given Species Wild-type Alternatively Spliced Receptors Polymorphisms in GPCRs GPCR Mutation-induced Disease Future Challenges 60 6 The Impact of C Proteins on Constitutive CPCR Activity Introduction The Contribution of G proteins to Constitutive Activity Basic Features The Distribution of G Proteins in the Plasma Membrane GPCR-G Protein Fusion Proteins Basic Features Modulation of the GPCR-G Protein Interface Alters Constitutive Activity Use of G Protein Variation to Detect ligand Efficacy Conclusions 69 7 (Patho)physiological and Therapeutic Relevance of Constitutive Activity and Inverse Agonism at C Protein-Coupled Receptors Introduction Physiological Relevance of Constitutive Activity of GPCRs Constitutive Activity of GPCRs and Pathophysiology of Disease Physiological Relevance of Inverse Agonists 76

4 Table of Contents VII 7.5 Inverse Agonists as Drugs Conclusions 79 8 Methodological Approaches Introduction Analysis of Constitutive GPCR Activity in Membranes and Intact Cells Procedure for Sf9 Cell Culture and Membrane Preparation GPCR Radioligand Binding Studies GTPase Assay [ 35 S]GTPyS Binding Assay Adenylyl Cyclase Assay Measurement of Constitutive Activity of GPCRs in Intact Cells Quantitative Determination of camp Concentrations in Cell Culture Lysates Determination of Inositol Phosphate Formation in Living Cells Determination of G Protein Activation by SRF-mediated Gene Transcription Deorphanization and Constitutive Activity of GPCRs by Aequorin-based Ca 2+ Determinations 2 25 II Constitutive Activity of Selected CPCR Systems Constitutive Activity of P-Adrenoceptors: Analysis in Membrane Systems Introduction Analysis of (3AR/G S Protein Coupling in Membranes Development of the Concept that (3ARs are Constitutively Active Probing Models of GPCR Activation with P 2 ARwt and P 2 AR CAM with Inverse Agonists Probing Models of GPCR Activation with P 2 AR wt and P 2 ARc AM and with Partial and Full Agonists Probing Models of GPCR Activation with P 2 AR wt and Purine Nucleotides Constitutive Activity of the P 2 AR Coupled to Various Ga s Proteins Probing Models of GPCR Activation with P 2 AR Coupled to Various Classes of G proteins Comparison of the Constitutive Activities of the P X AR and the P 2 AR Conclusions Constitutive Activity of p-adrenoceptors: Analysis by Physiological Methods Introduction Constitutive Activity and Inverse Agonism: Definition and Detection P r Adrenoceptors Constitutive Activity of Overexpressed PJARS 243

5 VIII Table of Contents Is there any Evidence for a Physiological Effect of Constitutively Active Receptors in Normal Cardiomyocytes? Substates of the P X AR: the Putative P 4 AR , P 2 -Adrenoceptors Constitutive Activity of Overexpressed P 2 ARs Inverse Agonism at the P 2 AR PAR Antagonists: Inverse Agonists at P 2 AR-G S or Full Agonists at PJAR-G,? Involvement of the P 2 AR in the "Putative P 4 AR" Effect Homo- and Heterodimerization of p r and p 2 ARs Conclusions Constitutive Activity at the a,-adrenoceptors: Past and Future Implications Introduction The a r Adrenoceptors: Main Structure-Functional Features The Discovery of Constitutively Activating Mutations and its Implications Theoretical and Experimental Approaches for Study of Constitutive GPCR Activity Theoretical Analysis of CAM GPCR Pharmacology Computational Modeling of the a 1B AR Measuring Constitutive Activity of the ajar Subtypes Constitutively Activating Mutations of the ajar Subtypes Where the Mutations have been Found Constitutive Activation of Multiple Signaling Pathways A Putative Model of Receptor Activation for the a 1B AR Constitutive Activity of Wild-type ajars and Inverse Agonism Constitutive Activity of Wild-type a t AR Subtypes Inverse Agonism at the o^ars Receptor Regulation and Constitutive Activity of the ajars Conclusions Constitutive Activity of Muscarinic Acetylcholine Receptors: Implications for Receptor Activation and Physiological Relevance Introduction Constitutive Activity - Native Systems Constitutive Activity - Recombinant Systems Constitutive Activation by G Proteins Structure-Function Analysis of Receptor Activation Transmembrane Domain Transmembrane Domain Transmembrane Domain Other Transmembrane Domains and Extracellular Domains Cytoplasmic Domains i3 Loop 287

6 Table of Contents \ IX i2 Loop Structure-Function Model for Activation Conclusions Constitutively Active Histamine Receptors Introduction The Histamine Receptors The HiR The H 2 R The H 3 R The H 4 R Assay Systems for Detection of Constitutive Activity of Histamine Receptors Histamine Receptor Expression and the Detection of Constitutive Activity Changes in Intracellular Ca [ 35 S]GTPyS Binding Assays (see also Chapter 8) IP 3 Formation (see also Chapter 8) camp Assays (see also Chapter 8) Measurements of Arachidonic Acid (AA) Release Reporter Gene Assays (see also Chapter 8) Activation of Kinases Effects of the Cellular Environment on Histamine Receptor Activity Construction and Expression of Constitutively Active Mutant Receptors Contamination with Endogenous Histamine Conclusions Constitutively Active Serotonin Receptors Introduction HT 1A Receptor (5-HT 1A R) HT 1B and 5-HT m Receptors (5-HT 1B R and 5-HT 1D R) HT 2A Receptor (5-HT 2A R) HT 2C Receptor (5-HT 2C R) Conclusion Virally Encoded Constitutively Active Chemokine Receptors Introduction Viral Strategies to Evade the Host Immune System Chemokines and Chemokine Receptors Viral Homologues of Chemokines and Chemokine Receptors and Viral Chemokine-binding Proteins The Human Cytomegalovirus-encoded Chemokine Receptor Homologue pus Characteristics of Human Cytomegalovirus Infection 248

7 X Table of Contents Functional Characteristics of pus Signaling Pathways Regulated by pus Regulation of Transcriptional Activity by pus Regulation of Constitutively Active pus Cellular Functions of pus The Human Kaposi's Sarcoma Virus-encoded Chemokine Receptor KSHV-GPCR Characteristics of Human Kaposi's Sarcoma Virus Infection Functional Characteristics of KSHV-GPCR Signaling Pathways Regulated by KSHV-GPCR Regulation of Transcriptional Activity by KHSV-GPCR Regulation of KSHV Activity by Chemokines Structure-Function Relationships of KHSV-GPCR Cellular Functions of KHSV-GPCR in vivo Conclusions 260 Index 265