Contents

Preface xi

Foreword xii

1 Chemical messengers and the cell membrane 1

1.1 Endocrine signalling by hormones 1

1.2 The nervous system and synaptic signalling by neurotransmitters 8

Small molecule neurotransmitters 11

Neuropeptides 13

1.3 Paracrine signalling by local chemical messengers 15

1.4 Hydrophobicity: effect on release and transport of messengers 16

1.5 Membrane proteins and membrane receptors 23

1.6 Ligand-receptor interactions 25

2 Radioliogand binding studies 29

2.1 Technical aspects of radioligand binding 29

2.2 Saturation binding 33

2.3 Competition binding 38

2.4 Kinetic experiments 46

2.5 Regional distribution of receptors 50

3 Functional studies 53

3.1 Dose-response curves and associated problems 53

3.2 From receptor occupation to stimulus and response 56

From receptor occupation to stimulus 56

From stimulus to response: linear relationship 59

From stimulus to response: non-linear relationship 61

3.3 Receptor classification and antagonist affinity 65

3.4 Pharmacological models 69

4 G protein-coupled receptors 77

4.1 From receptor to response: introduction to GPCRs 77

4.2 GPCR structure 86

4.3 Ligand interactions with family A, B and C receptors 97

Ligand interaction with family A receptors 98

Ligand interaction with family B receptors 102

Ligand interaction with family C receptors 104

4.4 Receptor activation 106

4.5 Activated GPCRs: interaction with G proteins 113

Techniques to study G protein-coupling preference 119

Divergence of intracellular signalling 123

4.6 Activated GPCRs: phosphorylation and internalization 125

Receptor phosphorylation 125

P-Arrestin binding mediated GPCR endocytosis 129

Mechanisms to terminate signalling 134

4.7 P-Arrestin-binding and MAP kinase activation 137

4.8 GPCR dimerization and association with other proteins 142

Introductory comments 142

GPCR dimerization 143

GPCR interaction with other membrane receptors 151

GPCR interaction with other membrane proteins 152

GPCR interactions with cytoplasmic proteins 156

4.9 Early models for GPCR activation 159

4.10 Restricted GPCR mobility and G protein coupling 163

Membrane compartimentalization 163

Restricted GPCR-G protein coupling: effector activity 173

4.11 Spontaneous receptor-G protein coupling 175

Models 175

Inverse agonism 179

4.12 Interaction of two G proteins with one activated receptor state 183

Fusion proteins between GPCRs and G proteins 185

4.13 Multiple receptor conformations 191

'Agonist trafficking': what do models predict? 196

Experimental 'evidence' for agonist trafficking: potential pitfalls 198 Multistate receptors: ligand-mediated sequential changes in receptor conformation 201 Multiple receptor states related to truncation, covalent modification and mutation 204

4.14 Multistate receptors and multiple ligand binding sites 207

The general allosteric ternary complex model 210

Exogenous and endogenous allosteric modulators 212

allosteric phenomena at GPCR: detection by radioligand binding 214

Detection of allosteric phenomena at GPCRs by functional assays 219

Usefulness of allosteric modulators 222

4.15 'Competitive', 'non-competitive' and 'insurmountable' antagonism 222

Co-incubation, no receptor reserve 222

Antagonist pre-incubation, no receptor reserve 224

4.16 Naturally occurring mutations of GPCRs 226

5 Concluding remarks 231

References 235

Index 244

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