G Protein-Coupled Receptors : Essential Methods / Edition 1

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More About This Textbook


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, GPCR Essential Methods 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 will be of interest to researchers in molecular biology, biochemistry, pharmacology and will be a useful resource for graduate students.

Highlights the best and most up-to-date techniques for working on GPCRs from the vast range currently in use

Explains clearly and precisely how to carry out selected techniques in addition to background information on the various approaches

Chapters are written by leading international authorities in the field and cover both new and well-known, tried and tested methods for working with GPCRs

An essential laboratory manual for researchers at all levels

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Product Details

  • ISBN-13: 9780470749142
  • Publisher: Wiley
  • Publication date: 2/9/2010
  • Edition number: 1
  • Pages: 310
  • Product dimensions: 6.80 (w) x 9.80 (h) x 0.90 (d)

Table of Contents

Preface ix

Contributors xi

1 Measurement of Ligand-G Protein-coupled Receptor Interactions Katie Leach Celine Valant Patrick M. Sexton Arthur Christopoulos 1

1.1 Introduction 1

1.2 Methods and approaches 5

References 27

2 Second Messenger Assays for G Protein-coupled Receptors: cAMP, Ca2+, Inositol Phosphates, ERK1/2 Karen J. Gregory Patrick M. Sexton Arthur Christopoulos Caroline A. Hick 31

2.1 Introduction 31

2.2 Methods and approaches 33

2.3 Troubleshooting 49

References 50

3 Use of the [35S]GTPγS Binding Assay to Determine Ligand Efficacy at G Protein-coupled Receptors Elodie Kara Philip G. Strange 53

3.1 Introduction 53

3.2 Methods and approaches 54

3.3 Troubleshooting 66

Acknowledgements 67

References 67

4 Quantitative Imaging of Receptor Trafficking Andy R. James Takeo Awaji F. Anne Stephenson Nicholas A. Hartell 69

4.1 Introduction 69

4.2 Methods and approaches 70

4.3 Troubleshooting 80

References 82

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 Roslyn M. Bill 85

5.1 Introduction 85

5.2 Methods and approaches 86

5.3 Troubleshooting 106

References 107

6 Monitoring GPCR-Protein Complexes Using Bioluminescence Resonance Energy Transfer Werner C. Jaeger Kevin D.G. Pfleger Karin A. Eidne 111

6.1 Introduction 111

6.2 Methods and approaches 114

6.3 Troubleshooting 128

References 128

7 Using Intramolecular Fluorescence Resonance Energy Transfer to Study Receptor Conformation Cornelius Krasel Carsten Hoffmann 133

7.1 Introduction 133

7.2 Methods and approaches 135

7.3 Troubleshooting 143

References 144

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 Jürgen Wess 147

8.1 Introduction 147

8.2 Methods and approaches 149

8.3 General considerations, caveats and troubleshooting 162

Acknowledgements 164

References 164

9 Use of Fluorescence Correlation Spectroscopy to Study the Diffusion of G Protein-coupled Receptors Stephen J. Briddon Jonathan A. Hern Stephen J. Hill 169

9.1 Introduction 169

9.2 Methods and approaches 170

9.3 Troubleshooting 190

References 191

10 Identification and Analysis of GPCR Phosphorylation Kok Choi Kong Sharad C. Mistry Andrew B. Tobin 197

10.1 Introduction 197

10.2 Methods 198

Acknowledgements 213

References 213

11 Measurement and Visualization of G Protein-coupled Receptor Trafficking by Enzyme-linked Immunosorbent Assay and Immunofluorescence Stuart J. Mundell Shaista P. Nisar Eamonn Kelly 215

11.1 Introduction 215

11.2 Methods and approaches 217

11.3 Troubleshooting 226

References 227

12 Substituted Cysteine Accessibility Method (SCAM) George Liapakis Jonathan A. Javitch 229

12.1 Introduction 229

12.2 Methods and approaches 230

12.3 Troubleshooting 247

References 248

13 Homology Modelling of G Protein-coupled Receptors John Simms 251

13.1 Introduction 252

13.2 Methods and approaches 252

13.3 Troubleshooting 269

13.4 Automated methods for generating models of GPCRs 269

References 270

Appendix Site-directed Mutagenesis and Chimeras Alex Conner Mark Wheatley David R. Poyner 275

A.1 Introduction 275

A.2 Why mutagenesis? 275

A.3 Troubleshooting 285

A.4 Conclusion 285

References 286

Index 289

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