G Protein–Coupled Receptors – Essential Methods

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. Contributors. 1 Measurement of Ligand-G-Protein-coupled Receptor Interactions (Katie Leach, Celine Valant, Patrick M. Sexton and Arthur Christopoulos). 1.1 Introduction. 1.2 Methods and Approaches. References. 2 Second Messenger Assays for G-Protein-coupled Receptors: cAMP, Ca2+, Inositol Phosphates, ERK1/2 (Karen J. Gregory, Patrick M. Sexton, Arthur Christopoulos and Caroline A. Hick). 2.1 Introduction. 2.2 Methods and Approaches. 2.3 Troubleshooting. References. 3 Use of the [35S]GTPgammaS Binding Assay to Determine Ligand Efficacy at G-protein-coupled Receptors (Elodie Kara and Philip G. Strange). 3.1 Introduction. 3.2 Methods and Approaches. 3.3 Troubleshooting. Acknowledgements. References. 4 Quantitative Imaging of Receptor Trafficking (Andy R. James, Takeo Awaji, F. Anne Stephenson and Nicholas A. Hartell). 4.1 Introduction. 4.2 Methods and Approaches. 4.3 Troubleshooting. References. 5 Production of Recombinant G-Protein-coupled Receptor in Yeast for Structural and Functional Analysis (Richard A.J. Darby, Mohammed Jamshad, Ljuban Grgic, William J. Holmes and Roslyn M. Bill). 5.1 Introduction. 5.2 Methods and Approaches. 5.3 Troubleshooting. References. 6 Monitoring GPCR-Protein Complexes Using Bioluminescence Resonance Energy Transfer (Werner C. Jaeger, Kevin D.G. Pfleger and Karin A. Eidne). 6.1 Introduction. 6.2 Methods and Approaches. 6.3 Troubleshooting. References. 7 Using Intramolecular Fluorescence Resonance Energy Transfer to Study Receptor Conformation (Cornelius Krasel and Carsten Hoffmann). 7.1 Introduction. 7.2 Methods and Approaches. 7.3 Troubleshooting. References. 8 A Disulfide Cross-linking Strategy Useful for Studying Ligand-induced Structural Changes in GPCRs (Jian Hua Li, Stuart D.C. Ward, Sung-Jun Han, Fadi F. Hamdan and Jurgen Wess). 8.1 Introduction. 8.2 Methods and Approaches. 8.3 General Considerations, Caveats and Troubleshooting. Acknowledgements. References. 9 Use of Fluorescence Correlation Spectroscopy to Study the Diffusion of G-protein-coupled Receptors (Stephen J. Briddon, Jonathan A. Hern and Stephen J. Hill). 9.1 Introduction. 9.2 Methods and Approaches. 9.3 Troubleshooting. References. 10 Identification and Proteomic Analysis of GPCR Phosphorylation (Kok Choi Kong, Sharad C. Mistry and Andrew B. Tobin). 10.1 Introduction. 10.2 Methods. Acknowledgements. References. 11 Measurement and Visualization of G-Protein-coupled Receptor Trafficking by Enzyme-linked Immunosorbent Assay and Immunofluorescence (Stuart J. Mundell, Shaista P. Nisar and Eamonn Kelly). 11.1 Introduction. 11.2 Methods and Approaches. 11.3 Troubleshooting. References. 12 Substituted Cysteine Accessibility Method (SCAM) (George Liapakis and Jonathan A. Javitch). 12.1 Introduction. 12.2 Methods and Approaches. 12.3 Troubleshooting. References. 13 Homology Modelling of G-Protein-coupled Receptors (John Simms). 13.1 Introduction. 13.2 Methods and Approaches. 13.3 Troubleshooting. 13.4 Automated Methods for Generating Models of GPCRs. References. Appendix: Site-directed Mutagenesis and Chimeras (Alex Conner, Mark Wheatley and David Poyner). A.1 Introduction. A.2 Why Mutagenesis? A.3 Troubleshooting. A.4 Conclusion. References. Index.