Purification of G Protein Subunits and G Protein Effectors -- Purification of Recombinant G Protein ? Subunits from Escherichia coli -- Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged ? and ?? Subunits -- Expression and Purification of Soluble Adenylyl Cyclase from Escherichia coli -- Purification of Phospholipase C ? and Phospholipase C ? from Sf9 Cells -- Assays for G Protein-Dependent Effector Activation In Vitro -- Assay for G Protein-Dependent Activation of Phospholipase C ? Using Purified Protein Components -- Assays of Recombinant Adenylyl Cyclases Expressed in Sf9 Cells -- Assays For G Protein-Coupled Receptor-Mediated and G Protein Subunit-Mediated Effector Activation in Intact Cells -- Measurement of G Protein-Coupled Receptor-Stimulated Phospholipase D Activity in Intact Cells -- Analysis of G Protein-Mediated Activation of Phospholipase C in Cultured Cells -- Receptor Mechanisms, Activity, and Localization -- Intensive Mutational Analysis of G Protein-Coupled Receptors in Yeast -- Green Fluorescent Protein-Tagged ?-Arrestin Translocation as a Measure of G Protein-Coupled Receptor Activation -- Detection of G Protein-Coupled Receptors by Immunofluorescence Microscopy -- Assay of G Protein-Coupled Receptor Activation of G Proteins in Native Cell Membranes Using [35S]GTP?S Binding -- Analysis of the Coupling of G12/13 to G Protein-Coupled Receptors Using a Luciferase Reporter Assay -- Disruption of Endogenous G Protein-Signaling Systems to Understand the Role of G Protein Subunits in Intact Systems -- Selective Inhibition of G Protein-Mediated Pathways Using RGS Domains -- Ribozymes as Tools for Suppression of G Protein ? Subunits -- In Vivo Adenoviral-Mediated Gene Transfer of the ?ARKct to Study the Role of G?? in Arterial Restenosis -- Posttranslational Modifications of Regulators of G Protein Signaling -- Analysis of RGS Protein Palmitoylation -- Methods for Measuring RGS Protein Phosphorylation by G Protein-Regulated Kinases -- Use of Gfp-Tagged G Proteins and Effectors in Intact Cells -- The Use of Green Fluorescent Proteins to View Association Between Phospholipase C? and G Protein Subunits in Cells -- Cellular Localization of GFP-Tagged ? Subunits.

G proteins and G protein-coupled receptors are ubiquitously expressed proteins that regulate a wide range of physiological processes and are consequently the targets of many pharmaceuticals. In G Protein Signaling: Methods and Protocols, leading researchers describe in detail key methods for investigating G protein signaling from a variety of perspectives ranging from in vitro biochemistry to whole animal studies. The authors focus on the mechanisms of G protein and G protein-coupled receptor function and the roles of G protein subunits in cell biology and disease. Among the readily reproducible techniques presented are those for the purification of G proteins and effector enzymes, assays of these purified G proteins and effector enzymes, and for the study of G protein interactions with effectors in intact cells. Additional methods are provided for assaying G protein-coupled receptor structure, function, and localization, for studying the physiological roles for endogenous G proteins, and for examining lipid and phosphate modifications of RGS proteins. Each fully tested protocol includes a background introduction explaining the principle behind the technique, equipment and reagent lists, tips on troubleshooting and avoiding known pitfalls, and, where needed, a discussion of the interpretation and use of results. Comprehensive and highly practical, G Protein Signaling: Methods and Protocols offers novice and expert researchers alike an array of cutting-edge tools to illuminate the roles of G protein and G protein-coupled receptors, as well as a better understanding of their action with drugs, neurotransmitters, and sensory stimuli.