Mechanisms and Pathways of Heterotrimeric G Protein Signaling (eBook)

Cover 1
Contents 4
Preface 6
Chapter 1: Structural Basis of Effector Regulation and Signal Termination in Heterotrimeric Galpha Proteins 10
I. Introduction and Scope 12
II. A Selective Survey of Galpha Protein Structure and Function 14
III. Mechanisms of Effector Recognition and Regulation by GalphabullGTP 18
A. A Common Galpha:Effector Interface 20
B. Functional Consequences of Galpha:Effector Binding 24
IV. Signal Termination: The Mechanism of GTP Hydrolysis and Conformational Deactivation 32
A. Structure of the Ground State for GTP Hydrolysis 34
B. Reaction Trajectory for GTP Hydrolysis 37
V. Signal Termination Through GAPs and Effector GAP Domains 47
A. Deactivation of Galphaq by PLCbeta 48
B. RGS GAPs 50
C. Synergy Between RGS and Effector Domains 55
D. Deactivation of Galpha 13 by the alphaGAP Element of p115RhoGEF 56
VI. Conclusions 60
Acknowledgments 61
References 61
Chapter 2: How do Receptors Activate G Proteins? 76
I. Introduction 76
A. Structure of Heptahelical Receptors 77
B. Heterotrimeric G Protein Structure 79
II. Toward a Model of the Receptor-G Protein Complex 81
A. Structural Determinants of Receptor-G Protein Specificity 81
B. Point to Point Interactions Between Receptors and G Proteins 86
C. Current Approaches to Modeling the Receptor-G Protein Complex 86
III. Molecular Basis for G Protein Activation 89
IV. Summary and Conclusions 93
References 95
Chapter 3: Some Mechanistic Insights into GPCR Activation from Detergent-Solubilized Ternary Complexes on Beads 104
I. Perspectives 105
II. Survey of Experimental Approaches and Representative Data 108
A. Flow Cytometric Approaches to Assess GPCR Function In Vivo 108
B. Rapid Mix Flow Cytometry 109
C. Modular Assembly of Molecular Complexes on Beads 109
III. Analysis of Soluble Receptor Ternary Complex Assemblies 113
A. General Considerations 113
B. Simple Ternary Complex Model: General Considerations 114
C. Application of Ternary Complex Model 117
D. Landscapes of G-Bead Assemblies Based on Application of Experimentally Derived Binding Constants to the Ternary Complex Model 118
E. Ternary Complex Analysis of Soluble Receptor Assemblies 120
F. Are Unique Conformational Changes in Receptors Elicited by Interactions with Ligands Resulting in Varied G Protein Interactions by the Ligand-Bound Receptor? 124
IV. Guanine Nucleotide Activation of Ternary Complex: Some Dynamic Aspects of Structure and Reactivity 126
A. General Considerations 126
B. Structural Studies 127
C. Some Dynamic Aspects of GPCR Activation 128
D. Modular Disassembly of the Ternary Complexes: Guanine Nucleotide Activation Causes the Rapid Separation of the GPCR and Ligand from the G Protein 130
E. GDP Activity? 134
F. Galpha Subunit Dissociation? 135
G. Outlook 136
Acknowledgments 137
References 137
Chapter 4: Activation of G Protein-Coupled Receptors 146
I. Introduction 147
II. Structural and Mechanistic Homology Among GPCRs 149
A. Rhodopsin as a Structural Model for GPCRs 149
B. GPCRs Activated by Diffusible Agonists 150
C. GPCR Oligomers 151
III. Conformational States 152
A. Basal Activity and Ligand Efficacy 153
B. Multiple Agonist-Specific States 154
C. Defining the "Active State" 155
IV. Activation by Agonists 157
A. Insights from Constitutively Active Mutants 157
B. Molecular Switches 159
C. Activation of Molecular Switches by Ligands 162
D. Agonist Binding and Activation Is a Multistep Process 164
E. The beta2AR as a Model System for Ligand Binding and Activation: Biophysical Analysis of Agonist-Induced Conformational Changes 164
V. Concluding Remarks 168
References 168
Chapter 5: Kinetic Analysis of G Protein-Coupled Receptor Signaling Using Fluorescence Resonance Energy Transfer in Living Cells 176
I. Introduction 176
II. Assays and Methods 179
A. Principle of the Assays 179
B. Construction and Expression of Fluorescent Receptor and G Protein Constructs 185
C. Microscopic FRET Measurements and Imaging 186
III. Results and Discussion 188
A. Agonist Binding 188
B. Receptor Activation 189
C. Receptor-G Protein Interaction 191
D. G Protein Activation 192
IV. Conclusions 193
Acknowledgments 194
References 194
Chapter 6: Regulation of Rho Guanine Nucleotide Exchange Factors by G Proteins 198
I. Introduction 199
A. Intrinsic Mechanisms of G Proteins 199
B. Regulation of Rho Proteins by Heterotrimeric G Proteins 202
II. RGS-RhoGEFs 202
A. Discovery and Relationships 202
B. The RGS Domain and GTPase Activity 205
C. The DH and PH Domains: Structure and Relative Activities 209
III. Mechanisms of Regulation 210
A. Direct Regulation of GEF Activity by G12 and G13 210
B. Indirect Regulation of RGS-RhoGEF Activity 212
C. Role of the C-Terminus in Oligomerization and Regulation of In Vivo Activity 214
D. Specificity of Regulation by G Proteins 215
E. Regulation of RGS-RhoGEFs by Phosphorylation 217
F. Function of the PDZ Domain 218
G. Expression of PDZ-RhoGEFs 222
IV. Physiological Function of the RGS-RhoGEFs 222
A. Pathway Specificity 223
B. Regulation in Hematopoietic Cells 223
C. Interactions with Other Proteins 225
Acknowledgments 230
References 230
Author Index 238
Subject Index 256