G Protein Signaling: Methods and Protocols

ISBN-13: 9781617373817 / Angielski / Miękka / 2010 / 250 str.

Alan V. Smrcka

Opis

Since the initial discovery of the G protein-coupled receptor system that regulates cyclicAMP production, the G protein field has rapidly expanded. Cell surface receptors that couple to heterotrimeric G proteins, the G prote- coupled receptors (GPCRs), number in the hundreds and bind to a wide div- sity of ligands including, biogenic amines (e. g., adrenaline), lipid derivatives (e. g., lysophosphatidic acid), peptides (e. g., opioid peptides), proteins (e. g., thyroid-stimulating hormone), and odorants to name a few. The GPCR system is found throughout biology in such simple organisms as yeast and in such more complex organisms as Dictyostelium discoideum (slime mold), Caen- habditis elegans (nematode worm), and of course in humans. GPCRs and their associated G protein systems are the subject of intense academic research and because of their involvement in a human biology and disease, the pharmac- tical industry has large research initiatives dedicated to the study of GPCRs. By some estimates, more than 50% of the pharmaceuticals on the market are targeted at GPCRs.The G protein/G protein-coupled receptor system consists of a receptor (GPCR), a heterotrimeric G protein consisting of ?, ?, and ? subunits, and an effector. G protein effector molecules, such as enzymes or ion channels, respond to acti- tion by the G protein to generate second messengers or changes in membrane potential that lead to alterations in cell physiology.

Recenzje

"...a timely and useful set of protocols...a significant contribution." -Doody's Health Sciences Book Review Journal

Spis treści

Part I. Purification of G Protein Subunits and G Protein Effectors Purification of Recombinant G Protein a Subunits from Escherichia coli Wendy K. Greentree and Maurine E. Linder Purification of G Protein Subunits from Sf9 Insect Cells Using Hexahistidine-Tagged a and bg Subunits Tohru Kozasa Expression and Purification of Soluble Adenylyl Cyclase from Eschericia coli Jeff A. Beeler and Wei-Jen Tang Purification of Phospholipase C b and Phospholipase C e from Sf9 Cells Mousumi Ghosh, Huan Wang, Grant G. Kelley, and Alan V. Smrcka Part II. Assays for G Protein-Dependent Effector Activation In Vitro Assay for G Protein-Dependent Activation of Phospholipase C b Using Purified Protein Components Mousumi Ghosh and Alan V. Smrcka Assays of Recombinant Adenylyl Cyclases Expressed in Sf9 Cells Ronald Taussig Part III. 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 Stephanie J. Walker and H. Alex Brown Analysis of G Protein-Mediated Activation of Phospholipase C in Cultured Cells Bo Liu and Dianqing Wu Part IV. Receptor Mechanisms, Activity, and Localization Intensive Mutational Analysis of G Protein-Coupled Receptors in Yeast Andjelka ´Celi´c, Sara M. Connelly, Negin P. Martin, and Mark E. Dumont Green Fluorescent Protein-Tagged b-Arrestin Translocation as a Measure of G Protein-Coupled Receptor Activation Stephen S. G. Ferguson and Marc G. Caron Detection of G Protein-Coupled Receptors by Immunofluorescence Microscopy Patricia M. Hinkle and John A. Puskas Assay of G Protein-Coupled Receptor Activation of G Proteins in Native Cell Membranes Using [35S]GTPgS Binding Jean M. Bidlack and Amy L.Parkhill Analysis of the Coupling of G12/13 to G Protein-Coupled Receptors Using a Luciferase Reporter Assay Bo Liu and Dianqing Wu Part V. 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 Tohru Kozasa and Richard D. Ye Ribozymes as Tools for Suppression of G Protein g Subunits Janet D. Robishaw, Zheng-ping Guo, and Qin Wang In Vivo Adenoviral-Mediated Gene Transfer of the bARKct to Study the Role of Gbg in Arterial Restenosis Guido Iaccarino and Walter J. Koch Part VI. Posttranslational Modifications of Regulators of G Protein Signaling Analysis of RGS Protein Palmitoylation Leah S. Bernstein, Maurine E. Linder, and John R. Hepler Methods for Measuring RGS Protein Phosphorylation by G Protein-Regulated Kinases Susanne Hollinger and John R. Hepler Part VII. Use of GFP-Tagged G Proteins and Effectors in Intact Cells The Use of Green Fluorescent Proteins to View Association Between Phospholipase Cb and G Protein Subunits in Cells Suzanne Scarlata and Louisa Dowal Cellular Localization of GFP-Tagged a Subunits Thomas R. Hynes, Thomas E. Hughes, and Catherine H. Berlot Index

Blurb

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.