G Protein Signaling: Methods and Protocols

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

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