Receptor Signal Transduction Protocols: Third Edition

Part I: Developing Models for Studying GPCRs

1. Inducible Expression of G Protein-Coupled Receptors in Transfected Cells

Beryl Koener and Emmanuel Hermans

2. Using the Flp-InTM T-RexTM System to Regulate GPCR Expression

Richard J. Ward, Elisa Alvarez-Curto, and Graeme Milligan

3. Viral Infection for GPCR Expression in Eukaryotic Cells

Antonio Porcellini, Luisa Iacovelli, and Antonio De Blasi

4. Generation of Epitope-Tagged GPCRs

Yan Huang and Gary B. Willars

5. The Use of Site-Directed Mutagenesis to Study GPCRs

Alex C. Conner, James Barwell, David R. Poyner, and Mark Wheatley

6. Approaches to Study GPCR Regulation in Native Systems

Jonathon M. Willets

7. Heterologous Expression of GPCRs in Fission Yeast

John Davey and Graham Ladds

Part II: Examining GPCR Expression and Agonist-Induced Covalent Modifications

8. Radioligand Binding Methods for Membrane Preparations and Intact Cells

David B. Bylund and Myron L. Toews

9. Quantification of GPCR mRNA Using Real-Time RT-PCR

Trond Brattelid and Finn Olav Levy

10. Determining Allosteric Modulator Mechanism of Action: Integration of Radioligand Binding and Functional Assay Data

Christopher J. Langmead

11. Design and Use of Fluorescent Ligands to Study Ligand-Receptor Interactions in Single Living Cells

Stephen J. Briddon, Barrie Kellam, and Stephen J. Hill

12. Examining Site-Specific GPCR Phosphorylation

Adrian J. Butcher, Andrew B. Tobin, and Kok Choi Kong

13. Ubiquitination of GPCRs

Adriana Caballero and Adriano Marchese

Part III: Examining Early Events in GPCR Signaling

14. [35S]GTPγS Binding as an Index of Total G-Protein and Gα-Subtype-Specific Activation by GPCRs

Rajendra Mistry, Mark R. Dowling, and R.A. John Challiss

15. Using Calcium Imaging as a Readout of GPCR Activation

Martin D. Bootman and H. Llewelyn Roderick

16. Measuring Spatio-Temporal Dynamics of Cyclic AMP Signaling in Real-Time Using FRET-Based Biosensors

Frank Gesellchen, Alessandra Stangherlin, Nicoletta Surdo, Anna Terrin, Anna Zoccarato, and Manuela Zaccolo

17. Determining the Activation of Rho as an Index of Receptor Coupling to G12/13 Proteins

Michio Nakaya, Mina Ohba, Motohiro Nishida, and Hitoshi Kurose

18. The Use of Translocating Fluorescent Biosensors for Real-Time Monitoring of GPCR-Mediated Signaling Events

Carl P. Nelson and R.A. John Challiss

Part IV: Receptor-Receptor and Receptor-Protein Interactions

19. Study of GPCR-Protein Interactions Using Gel Overlay Assays and Glutathione-S-Transferase-Fusion Protein Pull-Downs

Ashley E. Brady, Yunjia Chen, Lee E. Limbird, and Qin Wang

20. Study of GPCR-Protein Interactions by BRET

Martina Kocan and Kevin D.G. Pfleger

21. Time Resolved FRET Strategy with Fluorescent Ligands to Analyze Receptor Interactions in Native Tissues: Application to GPCR Oligomerization