G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors, with more than 800 members identified thus far in the human genome. They regulate the function of most cells in the body, and represent approximately 3% of the genes in the human genome. These receptors respond to a wide variety of structurally diverse ligands, ranging from small molecules, such as biogenic amines, nucleotides and ions, to lipids, peptides, proteins, and even light. Ligands (agonists and antagonists) acting on GPCRs are important in the treatment of numerous diseases, including cardiovascular and mental disorders, retinal degeneration, cancer, and AIDS. It is estimated that these receptors represent about one third of the actual identified targets of clinically used drugs. The determination of rhodopsin crystal structure and, more recently, of opsin, 1 and 2 adrenergic and A2A adenosine receptors provides both academia and industry with extremely valuable data for a better understanding of the molecular determinants of receptor function and a more reliable rationale for drug design. GPCR structure and function constitutes a hot topic. The book, which lies between the fields of chemical biology, molecular pharmacology and medicinal chemistry, is divided into three parts. The first part considers what receptor structures tell us about the mechanism of receptor activation. Part II focuses on receptor function. It discusses what the data from biophysical and mutational studies, and the analysis of the interactions of the receptor with ligands and regulator proteins, tell us about the process of signal transduction. The final part, on modelling and simulation, details new insights on the link between structure and mechanism and their implications in drug design.
Author(s): Jesús Giraldo, Jean-Philippe Pin
Series: RSC Drug Discovery 8
Publisher: Royal Society of Chemistry
Year: 2011
Language: English
Pages: xxxvi+512
City: Cambridge
G Protein-Coupled Receptors: From Structure to Function......Page 4
Preface......Page 6
Contents......Page 8
Early Ideas—Lingering Scepticism......Page 22
Purification and Reconstitution of Receptors......Page 24
Receptor Cloning—Convergence with Rhodopsin......Page 25
Structure–Function Studies......Page 26
Constitutively Active Mutant Receptors (CAMs) and Inverse Agonism......Page 27
A Universal Mechanism Regulates Receptor Function......Page 28
Expanding Roles of β-Arrestins and GRKs......Page 29
Biased Signalling......Page 30
Receptor Dimerization......Page 31
Conclusions......Page 32
References......Page 33
Section I: G Protein-coupled Receptors: Membrane Proteins with Privileged Structures......Page 38
1.2 Early Approaches to Analysing GPCR Structure......Page 40
1.3 The Crystal Structure of Rhodopsin......Page 44
1.4 Conformational Intermediates of Rhodopsin......Page 45
1.5 Crystal Structures of Other GPCRs......Page 49
1.6 Sequence Similarities and Conserved Motifs within GPCRs......Page 51
1.7 The D(E)RY Motif and GPCR Activation......Page 52
1.8 The NPxxYx(5,6)F Motif within GPCRs......Page 54
1.9 Ligand Binding Domains of GPCRs......Page 56
1.11 Addressing Shortcomings in our Knowledge of GPCR and G Protein Activation......Page 57
Acknowledgements......Page 58
References......Page 59
2.1 Introduction......Page 65
2.2.1 Expression and Isotopic Labelling......Page 67
2.2.2 Solid-state NMR Spectroscopy......Page 69
2.3 Retinal Conformation and Environment......Page 72
2.3.1 Retinal—the Photoreactive Trigger for Activation......Page 74
2.3.2 Retinal Conformation in the Visual Receptor Rhodopsin......Page 76
2.3.3 Location and Environment of the Retinal in Activated Rhodopsin......Page 77
2.4 Receptor Structure and Conformational Changes Associated with Activation......Page 78
2.4.1 Coupling of Retinal Isomerization to Helix Motion......Page 79
2.4.2 Disruption of the Ionic Lock and G-protein Binding......Page 81
2.5 Conclusions......Page 83
References......Page 84
3.1 Introduction......Page 91
3.2.1.1 Ground State of Rhodopsin......Page 92
3.2.1.3 Receptor States......Page 95
3.2.2.1 Conformations of Gα......Page 96
3.2.2.4 Role of Gtβγ......Page 98
3.2.3 Interaction Sites......Page 99
3.3.2 Gear-shift Model......Page 101
3.3.4 Helix Shift Model......Page 103
3.3.5 Receptor Oligomers......Page 104
3.4 Open Questions......Page 105
References......Page 106
4.1 Introduction: Class B GPCRs......Page 112
4.2 Class B GPCR Ligands: A Family of Peptide Hormones with Helical Propensities......Page 113
4.3 Extracellular Domains of Class B GPCRs: Dedicated to Ligand Binding......Page 114
4.3.1 Structure of Class B GPCR Extracellular Domains......Page 115
4.3.2 Structural Basis of Ligand Recognition and Binding by the ECD......Page 116
4.3.3 Specificity of Ligand Binding: Contributions by the ECD......Page 118
4.4 A Model for Class B GPCR Activation......Page 119
4.5 Activity Modulation via Oligomerization......Page 121
References......Page 122
5.1 Introduction......Page 127
5.2 The Resonance Energy Transfer Principle......Page 128
5.3.1 Fluorescence-RET......Page 129
5.3.2 Bioluminescence-RET......Page 131
5.4 Protein–Fragment Complementation Assays to Visualize GPCR Oligomers......Page 133
5.5.1 Time-resolved FRET......Page 136
5.5.2 SNAP-tag Technology......Page 138
5.6 Detection of Higher-order GPCR Oligomers in Living Cells......Page 139
5.6.2 Integrating PCA Assays and RET Techniques......Page 140
5.7 Conclusions......Page 142
References......Page 143
6.1 Introduction......Page 148
6.2 Techniques: How are Interactions between Protomers Studied?......Page 152
6.3 Role of GPCR-interacting Proteins in the Regulation and Stability of Oligomers......Page 153
6.4.1.1 Adrenoceptors......Page 155
6.4.1.2 Dopamine Receptors......Page 160
6.4.1.3 Muscarinic Acetylcholine Receptors......Page 164
6.4.1.4 Melatonin Receptors......Page 165
6.4.2 Chemokine Receptors......Page 166
6.4.3.1 Lutropin Receptor......Page 169
6.4.3.3 Thyrotropin-releasing Hormone Receptor......Page 170
6.4.4.1 Somatostatin Receptors......Page 171
6.4.4.3 Cholecystokinin Receptors......Page 173
6.4.4.5 Receptors for Vasoactive Intestinal Peptide and the Secretin Receptor......Page 175
6.4.4.7 Neurotensin Receptors......Page 176
6.4.4.8 Oxytocin and Vasopressin Receptors......Page 177
6.5 Conclusions......Page 178
References......Page 179
7.1 Introduction......Page 190
7.2 Interactions between Lipid Molecules and GPCRs......Page 191
7.2.1 Lipid Modifications of GPCRs......Page 192
7.2.2 Lipid Modifications Influence GPCR Trafficking......Page 193
7.2.3 Lipid Modifications Influence GPCR Signalling......Page 194
7.3.1 Gα Subunit Lipid Modifications......Page 195
7.3.2 Lipid Modifications of Gγ Subunits......Page 196
7.3.3 Lipid Modifications of Ras: An Example of Lipidation of a Small Monomeric G Protein......Page 197
7.4.1 Membrane Structure......Page 198
7.4.3 Lipid Polymorphism......Page 200
7.5.1 Effects of Membrane Structure on G Protein Signalling......Page 203
7.6 Conclusions......Page 208
Acknowledgements......Page 209
References......Page 210
8.1 Introduction......Page 216
8.2 Reconstitution of Monomeric GPCRs into Model Lipid Bilayers......Page 217
8.2.1.1 High Density Lipoproteins in vivo......Page 218
8.2.1.2 Advantages of the rHDL Approach......Page 219
8.2.3.1 Reconstituted Receptors are Monomeric......Page 220
8.2.3.2 Functional Studies on Monomeric Receptors......Page 221
8.2.4 Thoughts on Monomeric GPCRs......Page 222
8.3 Reconstitution of Oligomeric GPCRs into Model Lipid Bilayers......Page 223
8.3.2.1 Methodology of GPCR incorporation into Vesicles......Page 224
8.3.3.1 Ligand Binding to Oligomeric GPCRs......Page 225
8.3.4 Thoughts on Oligomeric GPCRs......Page 226
8.4.1 Summary......Page 227
8.4.2 Conclusions and Future Directions......Page 228
References......Page 230
Section II: G Protein-coupled Receptors: Multifaceted Functional Machines......Page 234
9.1 Introduction......Page 236
9.2 Kinetic Analysis of Isolated GPCRs......Page 237
9.3 Kinetic Studies in Intact Cells by FRET......Page 240
9.4 Trans-conformational Switching Within GPCR Heterodimers......Page 243
9.5 Real-time Kinetics of Allosteric Modulation......Page 245
9.6 How Fast are GPCRs in Living Cells?......Page 247
References......Page 250
10.1 Introduction......Page 254
10.2.2 Hormone–PTHR Interaction......Page 255
10.2.3 PTHR Activation/Deactivation......Page 257
10.2.5 G Protein Activation/Deactivation......Page 259
10.3.1 PTHR Conformations......Page 260
10.3.2 Sustained PTHR Signalling......Page 261
10.4 Perspectives and Conclusions......Page 263
References......Page 265
11.1 Introduction......Page 269
11.2 Structural Organization of mGlu Receptors......Page 270
11.2.1 The Agonist Binding Domain: A Venus Flytrap Domain......Page 271
11.2.2 The Cysteine-Rich Domain......Page 272
11.2.3 The 7TM Domain......Page 273
11.2.4 The C-terminal Tail......Page 274
11.3.2 Can mGlu Subunits Form Heterodimeric Receptors?......Page 275
11.4.2 Dimeric Functioning of MGlu VFTs......Page 276
11.4.3 Symmetric versus Asymmetric Functioning of the VFT Dimer......Page 278
11.5 Cysteine-Rich Domain and Intramolecular Transduction in mGluRs......Page 279
11.6.1 Active and Inactive States of the 7TM Domain......Page 281
11.6.2 Asymmetric Functioning of mGlu 7TM Dimer......Page 282
11.7 Implications for Other GPCR Dimers......Page 283
References......Page 286
12.1 Introduction......Page 292
12.2 Signalling Complexities......Page 293
12.2.1 Potential Crosstalk due to Dimerization......Page 294
12.2.2 Potential Crosstalk due to Signal Integration......Page 297
12.3.1 Individual Components Involved in a Signalling Unit......Page 298
12.3.2 Differentiating Signalling of Heteromers from Homomers......Page 300
References......Page 302
13.1 Introduction......Page 306
13.2.1 Potentiation of NMDA Receptor-mediated Responses by group I mGlu receptors......Page 310
13.2.2 Inhibition of NMDA Receptor-mediated Responses by Group I mGlu Receptors......Page 311
13.2.3 Role of Group I mGlu Receptors in the Plasticity of NMDA Receptor-mediated Synaptic Responses......Page 312
13.2.5 Endocannabinoid-dependent Modulation of Synaptic Strength by Synergistic Action of NMDA and Group I mGlu Receptors......Page 313
13.3 Group I mGlu Receptor-mediated Long-term Depression......Page 314
Acknowledgements......Page 316
References......Page 317
14.1 Introduction......Page 321
14.3 Pharmacology......Page 322
14.4 Distribution......Page 323
14.5 Structure of RAMPs......Page 324
14.6 RAMP Interactions with Receptors......Page 325
14.6.2 Signalling......Page 326
14.6.3 Mechanisms of Receptor Interaction......Page 327
14.6.4 RAMP Domains/Residues Involved in Ligand Interactions......Page 328
References......Page 330
15.1 Introduction......Page 334
15.2 Biochemical Evidence for a Receptor Docking Site......Page 337
15.3.1 Studies using Receptor-derived Peptides......Page 338
15.3.2 Studies using Modified Receptors......Page 339
15.4 Structural Elements of GRKs Involved in Binding GPCRs......Page 340
15.5.1 Structure of GRK6 in a Closed Conformation......Page 342
15.5.2 An Alternative role for the N-terminal Region?......Page 343
15.5.3 A Receptor Docking Model and a Proposed Mechanism of Activation......Page 344
15.6 Conclusions......Page 347
References......Page 348
16.1 Introduction......Page 353
16.2 GRK2 is a Multidomain Protein......Page 354
16.4 GRK2 Phosphorylates non-GPCR Substrates and Displays a Complex Network of Functional Interactions......Page 355
16.5 GRK2 in Cardiovascular Cells: Implications in Heart Failure and Hypertension......Page 358
16.6 GRK2 Interactome in Immune Cell Migration:
Physiopathological Implications in Inflammation
and Sepsis......Page 360
16.7 GRK2 Interactome in Epithelial Cell Migration......Page 362
16.8 GRK2 and Cell Cycle Progression......Page 363
16.10 GRK2 and Pain Modulation......Page 365
16.11 Conclusions......Page 367
References......Page 368
17.1 Arrestins: A Small Family of Proteins
with Many Functions......Page 372
17.2 Receptor Elements Engaged by Arrestins......Page 373
17.3 Receptor-binding Arrestin Elements and GPCR
Specificity......Page 374
17.4 Stoichiometry of the Arrestin–Receptor Complex......Page 375
17.5 Arrestin Effects on the Receptor......Page 377
17.6 Receptor Binding-induced Conformational
Changes in Arrestin......Page 378
17.7 What Does the Receptor–Arrestin Complex
Do that the Components Don’t?......Page 380
17.8 What Do We Need to Know About Arrestins?......Page 383
References......Page 385
Section III: Modelling G protein-coupled Receptor Structure and Function......Page 394
18.1 Introduction......Page 396
18.2 The Interhelical Binding Cavity as a Target
for Virtual Screening......Page 398
18.3.1 Virtual Screening Campaigns......Page 399
18.3.2 Controlled Virtual Screening Experiments......Page 400
18.3.3 Identification of GPCR Agonists and Blockers
Through Virtual Screening......Page 402
18.4.1 Useful Homology Models can be Constructed......Page 404
18.4.2 Controlled Virtual Screening Experiments......Page 406
18.4.3 Examples of Virtual Screening Campaigns based
on GPCR Homology Models......Page 408
Acknowledgements......Page 409
References......Page 410
19.1 Introduction......Page 412
19.2.1 General Strategies......Page 413
19.2.2 Computational Approaches......Page 414
19.2.3 Probing Receptor Activation Using Genetically
Encoded Non-Natural Amino Acids......Page 417
19.2.4 Reconstitution of Expressed Receptors in Membrane
Nanoparticles......Page 418
19.3 Conclusions......Page 419
References......Page 420
20.1 Introduction......Page 421
20.2 Molecular Dynamics Simulation: Training a
Computational Microscope on GPCR Function......Page 422
20.3 A Brief History of Molecular Dynamics
Simulation of GPCRs......Page 426
20.4 Future Prospects......Page 430
Acknowledgements......Page 432
References......Page 433
21.1 Introduction......Page 438
21.2.1 Insights from Random Acceleration Molecular Dynamics......Page 439
21.2.2 Binding Pathways as Assessed by Well-Tempered
Metadynamics......Page 442
21.3.1 Exploration of Global Conformational Motions
of GPCRs with Elastic Network Models and
Normal Mode Analysis......Page 445
21.3.2 Signal Transmission Mechanisms Assessed By MD
Simulations Restrained by Normal Modes......Page 447
21.3.3 Insights from Biased MD Using Mass-Weighted RMSD
Restraints and an Implicit Membrane Mimetic......Page 449
21.3.4 Activation Pathways from Adiabatic Biased Molecular
Dynamics Combined with Metadynamics......Page 451
21.4.1 Coarse-Grained Representations of GPCR Complexes......Page 454
21.4.2 Estimates of Dimerization Constants from Umbrella
Sampling Methods......Page 456
21.4.3 Normal Mode Analysis of Oligomeric Assemblies......Page 460
References......Page 461
22.1 Receptors as Allosteric Proteins......Page 466
22.2 Functional Selectivity: Historical Perspective......Page 469
22.3 Biased Agonism......Page 470
22.4 Biased Antagonism......Page 473
22.5 The Impact of Functional Selectivity on
New Drug Discovery......Page 474
References......Page 476
23.1 Introduction......Page 483
23.2 Originally Signal Transduction was Simple:
A Monomeric Receptor and a Single
Signalling Pathway......Page 484
23.3 Later Signal Transduction was Multiple......Page 487
23.4 Currently Signal Transduction is Complex:
Oligomeric Receptors and Multiple
Signalling Pathways......Page 488
23.4.1 The Protomers within Activated Receptor Dimers
can be Arranged in Either Symmetric or Asymmetric
Conformations......Page 490
23.4.2.1 Dosage Dependent Switch from G Protein-dependent
Pathway to G Protein-independent Pathway......Page 492
23.4.2.2 Inverse Agonists Displaying Agonist Behaviour......Page 494
23.5 Conclusions......Page 495
A23.1 Two-state Receptor Model (Figure 23.1A)......Page 496
A23.2 Three-state Receptor Model (Figure 23.1B)......Page 497
A23.3 Two-state Dimer Receptor Model (Figure 23.2A)......Page 498
A23.4 Three-state Dimer Receptor Model (Figure 23.2B)......Page 499
A23.5 The Asymmetric/Symmetric Three-state Dimer Receptor
Model (Figure 23.2C)......Page 500
References......Page 502
24.1 Introduction......Page 506
24.2 Models of GPCR-induced Calcium Signalling......Page 507
24.3 Phase-locking and Sub-threshold Calcium Responses......Page 510
24.4 Phase-locking Analysis of GPCR-induced Calcium Signalling in Two Models......Page 512
24.5 Microfluidics to Enable Pulsatile Stimulation of Cells......Page 515
24.6 Imaging of Signalling Dynamics in a Microfluidic Device......Page 517
24.7 Experimental Observations of Phase-locking
in GPCR-induced Calcium Signalling......Page 518
24.9 Model Revision......Page 520
24.10 Future Directions......Page 522
References......Page 523
Subject Index......Page 526
