Receptor Signal Transduction Protocols

This new edition combines updates of key chapters from the first edition with a large number of new key methodologies that have emerged more recently for studying G protein-coupled receptors (GPCRs) and events immediately downstream of their activation. The methods are focused primarily on events at the receptor level, including ligand binding, the genetic manipulation of receptors, the generation of model cell lines in which to study them, and the interaction and activation of G-proteins. Additional methods concentrate on receptor expression and localization, receptor internalization and post-translational modification, GPCR-protein interactions, and the use of knock-out and knock-in strategies for determining the physiological roles of receptors.

Table of Contents:
Radioligand-Binding Methods for Membrane Preparations and Intact Cells David B. Bylund, Jean D. Deupree, and Myron L. Toews Determination of Allosteric Interactions Using Radioligand-Binding Techniques Sebastian Lazareno Generation, Use, and Validation of Receptor-Selective Antibodies John J. Mackrill Immunocytochemical Identification of G-Protein-Coupled Receptor Expression and Localization Laura C. Mongan and Blair D. Grubb Generation and Use of Epitope-Tagged Receptors R. A. Jeffrey McIlhinney Identification of G-Protein-Coupled Receptor mRNA Expression by Northern Blotting and In Situ Hybridization Lucy F. Donaldson Electron Microscopic Studies of Receptor Localization Rafael Lujan Generation of Model Cell Lines Expressing Recombinant G-Protein-Coupled Receptors Emmanuel Hermans Viral Infection Protocols Antonio Porcellini and Antonio De Blasi Expression of G-Protein Signaling Components in Adult Mammalian Neurons by Microinjection Stephen R. Ikeda Covalent Modification of G-Proteins by Affinity Labeling Martin Hohenegger, Michael Freissmuth, and Christian Nanoff Measurement of Agonist-Stimulated [35S]GTPgS Binding to Assess Total G-Protein and Ga-Subtype-Specific Activation by G-Protein-Coupled Receptors Mark R. Dowling, Stefan R. Nahorski, and R. A. John Challiss Identification and Quantitation of G-Protein a-Subunits Ian Mullaney and Graeme Milligan Analysis of Function of Receptor-G-Protein and Receptor-RGS Fusion Proteins Richard J. Ward and Graeme Milligan Assessment of Receptor Internalization and Recycling Jennifer A. Koenig G-Protein-Coupled Receptor Phosphorylation and Palmitoylation Andrew B. Tobin and Mark Wheatley Identification of G-Protein-Coupled Receptor Phosphorylation Sites by 2D Phosphopeptide Mapping Andree Blaukat Ubiquitination of G-Protein-Coupled Receptors Adriano Marchese and Jeffrey L. Benovic Receptor Mutagenesis Strategies for Examination of Structure-Function Relationships Marion Blomenrohr, Henry F. Vischer, and Jan Bogerd Study of G-Protein-Coupled Receptor-Protein Interactions by Bioluminescence Resonance Energy Transfer Karen M. Kroeger and Karin A. Eidne Fluorescence Resonance Energy Transfer to Study Receptor Dimerization in Living Cells Jurgen E. Bader and Annette G. Beck-Sickinger Identification of Protein Interactions by Yeast Two-Hybrid Screening and Coimmunoprecipitation Michael Tanowitz and Mark von Zastrow Study of G-Protein-Coupled Receptor-Protein Interactions Using Gel Overlay Assays and Glutathione-S-Transferase-Fusion Protein Pull-Downs Ashley E. Brady, Qin Wang, and Lee E. Limbird Receptor Knock-Out and Knock-In Strategies Ikuo Matsuda and Atsu Aiba Statistical Methods in G-Protein-Coupled Receptor Research Pat Freeman and Domenico Spina Index