G Protein-Coupled Receptors Essential Methods

G-Protein Coupled Receptors (GPCRs) are not only the largest protein family in the human genome but are also the single biggest target for therapeutic agents. Research into GPCRs is therefore growing at a fast pace and the range of techniques that can be applied to GPCRs is vast and continues to grow. This book provides an invaluable bench-side guide into the best and most up-to-date techniques for current and future research on GPCRs.With contributions from leading international authorities, this book equips readers with clear and detailed protocols for both well-known and up-and-coming techniques along with hints and tips for success. All the methods have been tried and tested by leading international research labs and are presented in easy-to-follow stages along with a useful overview of each technique.This book is an essential resource for all researchers in molecular biology, biochemistry, pharmacology and for graduate students.

Dr David R. Poyner, University of Aston, Birmingham, UK; School of Life and Health Sciences, Aston University, Birmingham, UK.

Professor?Mark Wheatley, University of Birmingham, UK.

Preface	p. ix
Contributors	p. xi
Measurement of Ligand-G Protein-coupled Receptor Interactions	p. 1
Introduction	p. 1
Methods and approaches	p. 5
References	p. 27
Second Messenger Assays for G Protein-coupled Receptors: cAMP, Ca²⁺, Inositol Phosphates, ERK1/2	p. 31
Introduction	p. 31
Methods and approaches	p. 33
Troubleshooting	p. 49
References	p. 50
Use of the [³⁵S]GTP¿S Binding Assay to Determine Ligand Efficacy at G Protein-coupled Receptors	p. 53
Introduction	p. 53
Methods and approaches	p. 54
Troubleshooting	p. 66
Acknowledgements	p. 67
References	p. 67
Quantitative Imaging of Receptor Trafficking	p. 69
Introduction	p. 69
Methods and approaches	p. 70
Troubleshooting	p. 80
References	p. 82
Production of Recombinant G Protein-coupled Receptor in Yeast for Structural and Functional Analysis	p. 85
Introduction	p. 85
Methods and approaches	p. 86
Troubleshooting	p. 106
References	p. 107
Monitoring GPCR-Protein Complexes Using Bioluminescence Resonance Energy Transfer	p. 111
Introduction	p. 111
Methods and approaches	p. 114
Troubleshooting	p. 128
References	p. 128
Using Intramolecular Fluorescence Resonance Energy Transfer to Study Receptor Conformation	p. 133
Introduction	p. 133
Methods and approaches	p. 135
Troubleshooting	p. 143
References	p. 144
A Disulfide Cross-linking Strategy Useful for Studying Ligand-induced Structural Changes in GPCRs	p. 147
Introduction	p. 147
Methods and approaches	p. 149
General considerations, caveats and troubleshooting	p. 162
Acknowledgements	p. 164
References	p. 164
Use of Fluorescence Correlation Spectroscopy to Study the Diffusion of G Protein-coupled Receptors	p. 169
Introduction	p. 169
Methods and approaches	p. 170
Troubleshooting	p. 190
References	p. 191
Identification and Analysis of GPCR Phosphorylation	p. 197
Introduction	p. 197
Methods	p. 198
Acknowledgements	p. 213
References	p. 213
Measurement and Visualization of G Protein-coupled Receptor Trafficking by Enzyme-linked Immunosorbent Assay and Immunofluorescence	p. 215
Introduction	p. 215
Methods and approaches	p. 217
Troubleshooting	p. 226
References	p. 227
Substituted Cysteine Accessibility Method (SCAM)	p. 229
Introduction	p. 229
Methods and approaches	p. 230
Troubleshooting	p. 247
References	p. 248
Homology Modelling of G Protein-coupled Receptors	p. 251
Introduction	p. 252
Methods and approaches	p. 252
Troubleshooting	p. 269
Automated methods for generating models of GPCRs	p. 269
References	p. 270
Appendix Site-directed Mutagenesis and Chimeras	p. 275
Introduction	p. 275
Why mutagenesis?	p. 275
Troubleshooting	p. 285
Conclusion	p. 285
References	p. 286
Index	p. 289
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G Protein-Coupled Receptors Essential Methods

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