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.

G-Protein Coupled Receptors (GPCRs) are not only the largestprotein family in the human genome but are also the single biggesttarget for therapeutic agents. Research into GPCRs is thereforegrowing at a fast pace and the range of techniques that can beapplied to GPCRs is vast and continues to grow. This book providesan invaluable bench-side guide into the best and most up-to-datetechniques for current and future research on GPCRs.With contributions from leading internationalauthorities, this book equips readers with clear anddetailed protocols for both well-known and up-and-coming techniquesalong with hints and tips for success. All the methods have beentried and tested by leading international research labs and arepresented in easy-to-follow stages along with a useful overview ofeach technique.This book is an essential resource for all researchers inmolecular biology, biochemistry, pharmacology and for graduatestudents.

Preface.Contributors.1 Measurement of Ligand-G-Protein-coupled ReceptorInteractions (Katie Leach, Celine Valant, Patrick M. Sextonand Arthur Christopoulos).1.1 Introduction.1.2 Methods and Approaches.References.2 Second Messenger Assays for G-Protein-coupled Receptors:cAMP, Ca¯2+, Inositol Phosphates¯, ERK1/2(Karen J. Gregory, Patrick M. Sexton, Arthur Christopoulosand Caroline A. Hick).2.1 Introduction.2.2 Methods and Approaches.2.3 Troubleshooting.References.3 Use of the [¯35S]GTPgammaS Binding Assayto 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 (AndyR. 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 inYeast for Structural and Functional Analysis (Richard A.J.Darby, Mohammed Jamshad, Ljuban Grgic, William J. Holmes and RoslynM. Bill).5.1 Introduction.5.2 Methods and Approaches.5.3 Troubleshooting.References.6 Monitoring GPCR-Protein Complexes UsingBioluminescence 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 Transferto Study Receptor Conformation (Cornelius Krasel and CarstenHoffmann).7.1 Introduction.7.2 Methods and Approaches.7.3 Troubleshooting.References.8 A Disulfide Cross-linking Strategy Useful for StudyingLigand-induced Structural Changes in GPCRs (Jian Hua Li,Stuart D.C. Ward, Sung-Jun Han, Fadi F. Hamdan and JürgenWess).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 theDiffusion 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 GPCRPhosphorylation (Kok Choi Kong, Sharad C. Mistry and AndrewB. Tobin).10.1 Introduction.10.2 Methods.Acknowledgements.References.11 Measurement and Visualization of G-Protein-coupledReceptor Trafficking by Enzyme-linked Immunosorbent Assay andImmunofluorescence (Stuart J. Mundell, Shaista P. Nisar andEamonn 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 (AlexConner, Mark Wheatley and David Poyner).A.1 Introduction.A.2 Why Mutagenesis?A.3 Troubleshooting.A.4 Conclusion.References.Index.