G Protein-Coupled Receptors: Structure, Function, and Ligand Screening

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ISBN: 9780849327711 | 0849327717
Cover: Hardcover
Copyright: 8/4/2005

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With contributions from international experts in the fields of pharmacology, neuroscience, and biochemistry, this text provides an in-depth exploration of GPCRs. Coverage includes the screening of ligands for GPCRs, physiological and molecular characterizations of GPCRs, and structures of GPCRs and their ligands. The book explores current research issues in the post-genome era, particularly the identification of endogenous ligands that may lead to finding new hormones or neurotransmitters. It also discusses the determination of the tertiary structure of GPCRs, useful for understanding GPCR interactions and for the theoretical modeling of drugs.

PART I Screening of Ligands for GPCRs

Screening of Endogenous Ligands for Orphan GPCRs

(34)

Masaaki Mori

Shuji Hinuma

Tetsuya Ohtaki

Masahiko Fujino

Introduction

(1)

Ligand Sources

(2)

Tissue Extract

(1)

Known Molecules

(1)

Database Search

(1)

Particular Examples of Ligand Screening for the Orphan GPCRs

(30)

Galanin-Like Peptide (GALP)

(1)

Discovery of a Novel Galanin-Family Peptide, GALP

(1)

Preparation of Tissue Extract Sample

(2)

[35S] Guanosine 5'O-(γ-Thio)Triphosphate ([35S]GTPγS) Binding Assay

(1)

Isolation of GALP

(1)

Physiological Roles of GALP

(2)

Metastin

(1)

Discovery of Metastin, the Cognate Ligand of OT7T175 (= GPR54)

(1)

FLIPR Assay

(1)

Isolation of Metastin

(1)

Structure of Metastin

(1)

Receptor Interaction

(3)

Physiological Roles of Metastin

(1)

Neuropeptide W (NPW)

(1)

GPR7 and GPR8

(1)

Isolation and Identification of NPW as the Endogenous Ligand for the GPR7 and GPR8

(2)

Neuropeptide B (NPB) as a Paralog Peptide of NPW

(1)

Biological Functions of NPW and NPB

(1)

Urotensin II (UII)

(1)

GPR14

(1)

Isolation and Identification of UII as the Endogenous Ligand for GPR14

(3)

Pathophysiological Significance of UII

(1)

Free Fatty Acids (FFAs)

(1)

FFAs Are Signaling Molecules

(1)

GPR40

(1)

Identification of FFAs as Ligands for GPR40

(2)

Role of GPR40 Expressed in βCells

(1)

Pyroglutamylated RFamide Peptide (QRFP)

(1)

RFamide Peptides in Mammals

(1)

Identification of a Novel RFamide Peptide Gene

(1)

Identification of a Receptor for QRFP

(1)

Interaction between QRFP and AQ27

(1)

Functions of QRFP and Its Receptor

(1)

Acknowledgments

(2)

Abbreviations

(1)

References

(5)

Screening of Ligands for Human GPCRs by the Use of Receptor-Gα Fusion Proteins

(30)

Shigeki Takeda

Hinako Suga

Tatsuya Haga

Introduction

(1)

List of Human GPCR Genes

(4)

Screening of GPCRs from the Human Genome

(2)

List of Useful Databases for GPCRs

(2)

List of Useful Programs for GPCR and Other Protein Research

(1)

Ligand Screening System for GPCRs Using Receptor-Gα Fusion Proteins

(25)

Gene Construction for Receptor-Gα Fusion Proteins

(5)

In vivo Cell-Based Assay

(7)

In vitro [35S]GTγS Binding Assay

(7)

Acknowledgments

(1)

Abbreviations

(1)

References

(4)

Screening and Identification of Ghrelin, an Endogenous Ligand for GHS-R

(18)

Masayasu Kojima

Kenji Kangawa

Introduction

(1)

Reverse Pharmacology

(1)

GHS (Growth-Hormone Secretagogue) and Its Receptor

(2)

History

(2)

GHS Receptor (GHS-R) Superfamily

(1)

Purification of Ghrelin

(8)

Construction of GHS-R Expressing Cells

(2)

Intracellular Calcium Measurement Assay by FLIPR

(1)

Purification of Rat Ghrelin

(3)

Structural Determination of Rat Ghrelin

(1)

Minor Molecular Forms of Human Ghrelin

(1)

Ghrelin Precursor and Des-Gln14-Ghrelin

(2)

Nonmammalian Ghrelin

(6)

Bullfrog Ghrelin

(1)

Chicken Ghrelin

(1)

Fish Ghrelins

(1)

References

(4)

Ligand Screening of Olfactory Receptors

(28)

Kazushige Touhara

Sayako Katada

Takao Nakagawa

Yuki Oka

Introduction

(2)

The G Protein-Coupled Olfactory Receptor Superfamily

(1)

Odorant Signaling Pathways

(2)

Functional Expression of Olfactory Receptors in Olfactory Neurons

(4)

Adenovirus-Mediated Gene Transfer

(1)

Generation of GFP-Tagged Gene-Targeted Mice

(1)

Ca2+ Imaging and EOG Recording

(1)

Ca2+ Imaging Assay in Dissociated Olfactory Neurons

(2)

EOG Recording

(1)

Methods for Functional Receptor Expression in Heterologous Cells

(3)

General Considerations

(1)

N-Terminal Tagging and Glycosylation

(1)

Chaperones for Membrane Trafficking

(1)

Methods for Odorant Response Assays of Olfactory Receptors

(9)

Ca2+ Imaging Assay

(2)

cAMP Assay

(2)

Luciferase Reporter Gene Assay

(1)

Xenopus Oocyte Assay

100

(1)

Assays for Multiple G Protein-Mediated Signals

100

(2)

Measurement of Ion Currents with a Voltage Clamp Method

102

(1)

Measurement with Depolarization Step Pulses

103

(1)

Other Functional Assay Systems

104

(1)

Structure-Activity Matrices of Olfactory Receptors

105

(1)

Conclusions

106

(7)

Acknowledgments

106

(1)

References

107

(6)

PART II Functions and Regulations of GPCRs

Generation and Phenotypical Analysis of Muscarinic Acetylcholine Receptor Knockout Mice

113

(24)

Jurgen Wess

Dinesh Gautam

Sung-Jun Han

Jongrye Jeon

Cuiling Li

Chuxia Deng

Introduction

113

(1)

General Strategy Used to Generate Mice Lacking Specific mAChRs

114

(7)

Phenotypical Analysis of mAChR Knockout (KO) Mice

121

(8)

Summary of Mutant Phenotypes

121

(5)

Potential Compensatory Changes of mAChR Expression Levels in mAChR KO Mice

126

(1)

Issues Related to the Mouse Genetic Background of the Analyzed mAChR KO Mice

127

(1)

Generation and Analysis of mAChR Double KO Mice

128

(1)

Relevance for Drug Therapy

129

(2)

M1 Receptors

129

(1)

M2 Receptors

129

(1)

M3 Receptors

130

(1)

M4 Receptors

130

(1)

M5 Receptors

130

(1)

Conclusions and Future Outlook

131

(6)

Acknowledgments

131

(1)

References

131

(6)

Systematic Mutagenesis of M1 Muscarinic Acetylcholine Receptors

137

(42)

Edward C. Hulme

Introduction

138

(2)

G Protein-Coupled Receptors

138

(1)

The Muscarinic Acetylcholine Receptors

139

(1)

Mutagenesis Studies

140

(1)

The Mechanistic Framework for the Interpretation of Mutagenesis Studies on GPCRs

141

(8)

The Ternary Complex Model of Receptor-G Protein Interaction

141

(6)

The Extended Ternary Complex Model

147

(1)

The Dimeric Receptor Ternary Complex Model

148

(1)

Experimental Techniques

149

(8)

Choice of Target Sequences

149

(1)

Expression Vector

150

(1)

Site-Directed Mutagenesis

150

(1)

Preparation of Plasmid DNA

151

(1)

Transfection of Cells

151

(1)

Membrane Preparations for Binding Assays

152

(1)

Binding Assays on Wild-Type and Mutant M1 mAChRs

153

(2)

Functional Assays on Mutant Receptors

155

(1)

Immunocytochemistry and Immunofluorescence

156

(1)

Data Analysis

157

(1)

Key Considerations in Performing Mutagenesis Experiments

157

(12)

Measuring the Expression of Functional Receptors

157

(1)

How Should Functional Data Be Expressed? The Contribution of Basal Signaling Activity

158

(3)

The Effect of Variable Receptor Expression on the Functional Dose-Response Curve: The Effective Receptor:G Protein Ratio

161

(3)

Mutants that Give No Measurable Binding of [3H]NMS

164

(1)

Reduced Expression

164

(2)

Reduced Ligand Affinity

166

(1)

Should We Be Using a Dimeric Receptor Ternary Complex Model to Analyze Binding and Dose-Response Data?

166

(3)

The Interpretation of Ala-Scanning Mutagenesis Studies

169

(4)

Determining the Functional Phenotype of Mutant Receptors

169

(1)

Phenotypes Predicted by the Extended Ternary Complex Model of Agonist-Receptor-G Protein Interaction

170

(1)

A Case Study: Transmembrane Helix 7

171

(2)

Conclusions

173

(6)

Acknowledgments

175

(1)

References

175

(4)

Analysis of the Regulation of Muscarinic Acetylcholine Receptors

179

(18)

Chris J. van Koppen

Introduction

179

(1)

Radioligand Binding Assays to Measure mAChR Internalization and Downregulation

180

(3)

Analysis of mAChR Internalization in Intact Cells Using [3H]NMS Binding Assay

180

(2)

Analysis of mAChR Downregulation Using [3H]QNB Binding Assay to Cell Membranes

182

(1)

[35S]GTPγS Assay to Measure Coupling of mAChR to Heterotrimeric G Proteins

183

(3)

Analysis of mAChR Signal Transduction Pathways

186

(11)

Phospholipase C Stimulation

186

(2)

Phospholipase D Activation

188

(3)

Elevation of [Ca2+]i

191

(1)

Inhibition of cAMP Accumulation

192

(3)

Acknowledgments

195

(1)

References

195

(2)

Single-Molecule Analysis of Chemotactic Signaling Mediated by cAMP Receptor on Living Cells

197

(22)

Masahiro Ueda

Yukihiro Miyanaga

Toshio Yanagida

Introduction

198

(3)

Single-Molecule Detection Techniques

198

(1)

Chemotactic Signaling and GPCRs

199

(2)

Total Internal Reflection Fluorescence Microscopy (TIRFM)

201

(4)

Theory: Evanescent Field

202

(1)

Configuration of Objective-Type TIRFM

203

(1)

Measurement of Angle of Incident Laser Beam

204

(1)

Ligand-Binding Analysis

205

(8)

Preparation of Fluorescent-Labeled cAMP Analogue

205

(1)

Single-Molecule Imaging on Living Cells and Crude Membranes

206

(1)

Coverslip Preparation

206

(1)

Cell Preparation and Observation

207

(1)

Crude Membrane Preparation

207

(1)

Verification of Single-Molecule Imaging

208

(1)

Dissociation Rate Analysis

208

(4)

GTP Sensitivity

212

(1)

Receptor States on Living Cells

212

(1)

Green Fluorescent Protein (GFP) Imaging at Single-Molecule Level

213

(6)

Acknowledgments

216

(1)

Abbreviations

216

(1)

References

216

(3)

Oligomerization of G Protein-Coupled Purinergic Receptors

219

(24)

Hiroyasu Nakata

Kazuaki Yoshioka

Toshio Kamiya

Introduction

220

(1)

Oligomerization of G Protein-Coupled Receptors

220

(1)

Purinergic Receptors

220

(1)

Analysis of Oligomerization of Purinergic Receptors

221

(22)

Receptor Pharmacology

221

(1)

Transfection and Expression in HEK293T Cells

222

(1)

Ligand Binding

223

(2)

Adenylyl Cyclase Coupling in Co-Transfected Cells

225

(2)

Immunoprecipitation

227

(1)

Co-Immunoprecipitation of Epitope-Tagged A1R and P2Y1R in Co-Expressed HEK293T Cells

227

(2)

Co-Immunoprecipitation of A1R and P2Y1R from Rat Brain

229

(3)

Immunocytochemical Analysis

232

(1)

Double Immunofluorescence Microscopy of Co-Expressed Purinergic Receptors

232

(1)

BRET Analysis

233

(1)

BRET of Co-Expressed Purinergic Receptors

234

(1)

Plasmid Constructs, Transfection, and BRET2 Assay

235

(3)

Acknowledgments

238

(1)

Abbreviations

238

(1)

References

238

(5)

PART III Tertiary Structure of GPCRs and Their Ligands

Methods and Results in X-Ray Crystallography of Bovine Rhodopsin

243

(18)

Tetsuji Okada

Rumi Tsujimoto

Miho Muraoka

Chihiro Funamoto

Introduction

243

(1)

Purification and Crystallization

244

(6)

Isolation of Membranes from Retina

244

(2)

Purification of Membranes

246

(1)

Selective Solubilization

247

(1)

Crystallization

247

(2)

Application to Rhodopsin Analogue

249

(1)

Structure Determination and Refinement

250

(1)

Characterization of Crystals

250

(1)

Summary of Structure Determination

251

(1)

Structure Refinements at Higher Resolution

251

(1)

Crystal Structure

251

(6)

Crystal Lattice

251

(1)

Overall Structure

252

(1)

Transmembrane Region

252

(3)

Constitutive Activity

255

(2)

Remarks

257

(4)

Acknowledgments

258

(1)

References

258

(3)

Determination of Steric Structure of Muscarinic Ligands Bound to Muscarinic Acetylcholine Receptors: Approaches by TRNOE (Transferred Nuclear Overhauser Effect)

261

(22)

Hiroyasu Furukawa

Toshiyuki Hamada

Hiroshi Hirota

Masaji Ishiguro

Tatsuya Haga

Introduction

262

(1)

Expression and Purification of the M2 Receptors

263

(6)

Methods for Expressing the M2 Mutant Using Sf9/Baculovirus

264

(2)

Methods for Membrane Preparation and Solubilization

266

(2)

Methods for Purification of the M2 Mutant

268

(1)

Synthesis and Characterization of (S)-Methacholine

269

(5)

Methods for Synthesis of (S)-Methacholine

271

(1)

Methods for Activity Assays

271

(3)

Determination of the (S)-Methacholine Conformation by NOESY and TRNOESY

274

(4)

Methods for Determination of the Free (S)-Methacholine Conformation

275

(2)

Methods for Determination of the Receptor-Bound (S)-Methacholine Conformation

277

(1)

Assessment of (S)-Methacholine Conformation by Docking Studies

278

(5)

Acknowledgment

280

(1)

References

280

(3)

Modeling of G Protein-Coupled Receptors for Drug Design

283

(20)

Masaji Ishiguro

Introduction

283

(2)

Structural Models of the Photointermediates, Batho, Lumi, and Meta I

285

(4)

Structural Models of Photointermediates, Meta Ib and I380

289

(1)

Structural Model of Fully Activated Photointermediate, Meta II

290

(1)

Functional Structures of GPCRs

291

(2)

Receptor-Ligand Complex Models for Muscarinic Acetylcholine Receptor

293

(3)

The Ligand-Binding Modes of Adrenergic b2 Receptor

296

(2)

Summary

298

(5)

Abbreviations

299

(1)

References

299

(4)

Index

303

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G Protein-Coupled Receptors: Structure, Function, and Ligand Screening

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Table of Contents

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