Contents

Preface xvii

Contributors xix

1. The Evolution of Receptors: From On-Off Switches to Microprocessors 1

Terry Kenakin

1.1. Introduction 1

1.2. The Receptor as an On-Off Switch 1

1.3. Historical Background and Classical Receptor Theory 2

1.4. The Operational Model of Drug Action 7

1.5. Receptor Antagonism 8

1.6. Specific Models of GPCRs (7TM Receptors) 11

1.7. The Receptor as Microprocessor: Ternary Complex Models 13

1.8. Receptors as Basic Drug Recognition Units 17

1.9. Receptor Structure 19

1.10. Future Considerations 19 References 22

2. The Evolving Pharmacology of GPCRs 27

Lauren T. May, Nicholas D. Holliday, and Stephen J. Hill

2.1. Agonists, Neutral Antagonists, and Inverse Agonists 27

2.1.1. Affinity and Efficacy 27

2.1.2. Pharmacological Models of Agonism,

Antagonism, and Inverse Agonism 32

2.2. LDTRS/Protean Agonism 34

2.3. Molecular Mechanisms of GPCR Ligand Binding 35

2.3.1. Rhodopsin-Like Receptor Binding Sites 35

2.3.2. Ligand Recognition in Class C Receptors 38

2.3.3. Molecular Mechanisms of Rhodopsin-Like

Receptor Activation 39

2.4. Two GPCR Ligands Binding at Once—

Concept of Allosterism 40

2.4.1. Classes of Allosteric Modulators 40

2.4.2. Pharmacological Models of Allosteric Interactions 41

2.4.3. Advantages of Allosteric Ligands 43

2.5. GPCR Dimerization 44

2.5.1. Dimerization Is Essential for Class C

Receptor Function 44

2.5.2. Is Dimerization Required for Class A

GPCR Activation? 46

2.5.3. Influence of Receptor Dimers on Binding Studies 47

2.5.4. GPCR Heterodimerization 48

2.6. Future Perspectives 49 Acknowledgments 50 References 50

3. The Emergence of Allosteric Modulators for

G Protein-Coupled Receptors 61

Karen J. Gregory, Celine Valant, John Simms, Patrick M. Sexton, and Arthur Christopoulos

3.1. Introduction 61

3.2. Foundations of Allosteric Receptor Theory 62

3.3. Models for Understanding the Effects of Allosteric Modulators 63

3.4. Types of Allosteric Modulators and Their Properties 65

3.5. Detection and Quantification of Allosteric Interactions 68

3.5.1. Radioligand Binding Assays 68

3.5.2. Functional Assays 70

3.6. Some Examples of GPCR Allosteric Modulators 73

3.6.1. Small Molecule Allosteric Modulators 73

3.6.2. Proteins as Allosteric Modulators 78

3.7. Concluding Remarks 80 References 81

4. Receptor-Mediated G Protein Activation:

How, How Many, and Where? 88

Ingrid Gsandtner, Christian W. Gruber, and Michael Freissmuth

4.1. The Mechanical Problem—Three Different Solutions 89

4.1.1. The Lever-Arm Model 91

4.1.2. The "Gear-Shift" Model 91

4.1.3. The "C-Terminal Latch" Model 93

4.1.4. Are the Three Models Mutually Exclusive? 94

4.2. Receptor Monomers-Dimers-Oligomers: One Size Fits All? 95

4.2.1. Evidence for GPCR Dimers 96

4.2.2. GPCR Dimers Are Not Universally Required as Prerequisites for G Protein Activation 96

4.2.3. Dimers May Allow for Conformational Switches Underlying Receptor Cross-Talk and

Other Forms of Allosterism 99

4.3. Corrals, Fences, Rafts—Are There Privileged Places for GPCR Activation? 100

4.3.1. The Actin Cytoskeleton Confines GPCRs by Several Mechanisms 100

4.3.2. Cholesterol-Rich Domains and Lipid Rafts 103 Acknowledgments 106 References 106

5. Molecular Pharmacology of Frizzleds—with Implications for Possible Therapy 113

Gunnar Schulte

5.1. Introduction 113

5.2. Frizzleds as WNT Receptors 113

5.2.1. Frizzleds—The Discovery 113

5.2.2. The Frizzled Family 114

5.2.3. Frizzled Ligands 116

5.2.4. WNT-Frizzled Interactions 116

5.2.5. Intracellular Posttranslational Modifications 117

5.3. Frizzled Signaling 120

5.3.1. P-Catenin-Dependent Signaling 122

5.3.2. P-Catenin-Independent Signaling 122

5.3.3. Intracellular Scaffolds (DVL and P-arrestin) 123

5.3.4. Evidence for G Protein Coupling of FZDs 125

5.3.5. Unconventional Signaling Modes 126

5.4. Frizzleds—Physiology and Possible Therapy 127

5.4.1. Frizzleds in Physiology 127

5.4.2. Therapeutic Potential 128

5.4.3. Attacking WNT-FZD Interface? 128

5.4.4. Anti-DVL Drugs 129

5.4.5. WNTs as Drugs 129

5.4.6. Future Directions 130 Acknowledgments 130 References 130

6. Secretin Receptor Dimerization: A Possible Functionally Important Paradigm for Family B G Protein-Coupled Receptors 138

Kaleeckal G. Harikumar, Maoqing Dong, and Laurence J. Miller

6.1. Methodological Approaches to GPCR Oligomerization 139

6.2. Structural Themes for GPCR Oligomerization 141

6.3. Functional Effects of GPCR Oligomerization 150

6.4. Secretin Receptor Oligomerization 151 References 153

7. Past and Future Strategies for GPCR Deorphanization 165

Angélique Levoye, Nathalie Clement, Elodie Tenconi and Ralf Jockers

7.1. Introduction 165

7.2. Current Strategies to Identify the Ligand and Function of Orphan 7TM Proteins 168

7.2.1. Reverse Pharmacology 168

7.2.2. Orphan Receptor Strategy 168

7.2.3. Use of Sequence Homology, Cross Genome Phylogenetic Analysis, and Chemogenomics to Predict Candidate Ligands 168

7.2.4. Determination of the Expression Pattern and the Phenotype of Knockout Mice of Orphan

7TM Proteins 170

7.3. Functional Assays for Deorphanization 170

7.3.1. Classical Assays of GPCR Deorphanization 173

7.3.2. Recent Assays in GPCR Deorphanization 174

7.4. Future Directions and New Concepts 176

7.5. Controversial Issues 179 Acknowledgments 181 References 181

8. High-Throughput GPCR Screening Technologies and the Emerging Importance of the Cell Phenotype 191

Terry Reisine and Richard M. Eglen

8.1. Introduction 191

8.2. How Are GPCR Drugs Discovered? 192

8.3. GPCR Dependence on G Proteins 193

8.4. Technologies for GPCR Compound Screening and Drug Discovery 195

8.4.1. Cell-Free Assays 195

8.4.2. Cell-Based Assays 195

8.4.3. Ca++ Transients for GPCR HTS 196

8.4.4. Reporter Assays for GPCR HTS 198

8.4.5. Universal HTS Assays for GPCRs? 198

8.5. Importance of Target Cells in GPCR HTS Assays 199

8.6. Summary 203 References 204

9. Are "Traditional" Biochemical Techniques Out of Fashion in the New Era of GPCR Pharmacology? 209

Maria Teresa Dell'anno and Maria Rosa Mazzoni

9.1. Overview 209

9.2. Receptor Binding Assays 210

9.3. Methods for Measurement of cAMP 216

9.3.1. Assessments of Adenylyl Cyclase Activity:

Methods Using Labeled ATP 216

9.3.2. Methods Using Nonlabeled ATP 218

9.4. Conclusions 223 References 223

10. Fluorescence and Resonance Energy Transfer Shine New Light on GPCR Function 226

Carsten Hoffmann and Moritz Bünemann

10.1. Overview 226

10.2. Introduction 226

10.3. Labeling GPCRs with Fluorescent Tags 227

10.3.1. Tagging GPCRs with Fluorescent Proteins 227

10.3.2. Labeling of GPCRs with Fluorescent Dyes 228

10.4. Detection of Fluorescence and Bioluminescence 231

10.5. Fluorescence-Based Assays to Study Receptor Localization, Trafficking and Receptor Function 232 10.5.1. How to Monitor Receptor Function by Means of Fluorescence Microscopy 233

10.6. Resonance Energy Transfer, a Tool to Get New Insights into GPCR Function 234

10.6.3. Comparison of BRET and FRET 235

10.7. Analysis of Steady-State Protein-Protein Interaction by Means of RET 236

10.8. Kinetic Analysis of Protein-Protein Interactions by Means of FRET 237

10.8.1. G Protein Activity Measured by FRET 238

10.8.2. Receptor-G Protein Interaction Studied by RET 239

10.8.3. Kinetics of Receptor-G Protein Interactions 240

10.8.4. Receptor-ß-arrestin Interaction Detected by RET 242

10.9. Detection of Receptor Function by Fluorescence Resonance Energy 243

10.9.1. Partial Agonism Detected on the Level of the Receptor 245

10.9.2. Inverse Agonism Detected at the Level of the Receptor 246

References 247

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