Regulators of G Protein Signaling, Part A

Regulators of G Protein Signaling, Part A

1st Edition - August 19, 2004

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  • Editor: David Siderovski
  • Hardcover ISBN: 9780121827946
  • eBook ISBN: 9780080497266

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Description

Regulators of G Protein Signaling, Part A is an in-depth treatment of G-Protein Signaling, and will cover general methods of analysis of RGS protein analysis, including Expression and post-translational modification, Assays of GAP activity and allosteric control, Electrophysiological methods and RGS-insensitive Ga subunits, Mouse models of RGS protein action, Methods of RGS protein inhibition, and G-protein regulators of model organisms.

Key Features

  • Table of Contents
    • Expression and post-translational modification
    • Assays of GAP activity and allosteric control
    • Electrophysiological methods and RGS-insenstitive Ga subunits
    • Mouse Models of RGS protein action
    • Methods of RGS protein inhibition
    • G-protein regulators of model organisms

Readership

Biochemists, biophysicists, microbiologists, cell biologists, molecular biologists, geneticists, biomedical researchers

Table of Contents

  • Editors-In-Chief

    Contributors to Volume 389

    Preface

    Methods In Enzymology

    Quantitative Real-Time Polymerase Chain Reaction Measurement of Regulators of G-Protein Signaling mRNA Levels in Mouse Tissues

    Introduction

    Methods

    Application of qRT-PCR to the Measurement of RGS mRNA Levels

    Future Applications

    Regulation of Chemokine-Induced Lymphocyte Migration by RGS Proteins

    Introduction

    RGS Proteins in Lymphocytes

    Assessment of Chemokine Signaling in Lymphocytes and Alterations by RGS Proteins

    Role of Palmitoylation in RGS Protein Function

    Introduction

    Metabolic Incorporation of Palmitate into RGS Proteins

    In Vitro Incorporation of [3H]Palmitate into RGS Proteins

    Functional Assays with Palmitoylated RGS Proteins

    Fluorescence-Based Assays for RGS Box Function

    Introduction

    Materials

    Fluorescence Resonance Energy Transfer Assay Design Considerations

    Cloning and Purification of Gαi1-CFP and YFP-RGS4

    FRET-Based Assay of RGS4/Gαi1 Interaction

    Development of a High-Throughput FRET Assay for Gαi1/RGS4 Interaction

    Use of RGS Box Proteins as Biosensors for G-Protein Activation

    Concluding Remarks

    Application of RGS Box Proteins to Evaluate G-Protein Selectivity in Receptor-Promoted Signaling

    Abstract

    Introduction

    Methodology

    Materials and Methods

    Background

    Application

    Concluding Remarks

    Allosteric Regulation of GAP Activity by Phospholipids in Regulators of G-Protein Signaling

    Introduction

    Reagents and Methods

    Regulation of RGS Domain GAP Activity

    Conclusions

    Assays of RGS Protein Modulation by Phosphatidylinositides and Calmodulin

    Introduction

    Background

    Assays

    Summary

    Analyses of RGS Protein Control of Agonist-Evoked Ca2+ Signaling

    Introduction

    Selection of the Experimental System

    Assay of RGS Protein Function in Permeabilized Pancreatic Acinar Cells

    Assay of RGS Protein Effects on GPCR-Evoked Ca2+ Signaling in Single Cells

    Conclusions

    Measuring the Modulatory Effects of RGS Proteins on GIRK Channels

    Introduction

    Measuring RGS Modulation of GIRK Channels in CHO-K1 Cells

    Measuring Native GIRK Channel-Gating Properties in Cardiomyocytes and Neurons

    Single Cell RT-PCR Analysis of Endogenous RGS Expression

    Measuring RGS Modulation of GIRK Channels in Xenopus Oocytes

    Future Applications

    Assays for G-Protein-Coupled Receptor Signaling Using RGS-Insensitive Gα Subunits

    Introduction

    Overview of Methods and Rationale

    Generation and Characterization of Stable PTX-i and RGS/PTX-i Gα Expressing Cells

    Assays for Gα Protein Activity

    Second Messenger Assays

    Example of Experimental Findings

    Concluding Remarks

    Use of RGS-Insensitive Gα Subunits to Study Endogenous RGS Protein Action on G-Protein Modulation of N-Type Calcium Channels in Sympathetic Neurons

    Introduction

    Gα Mutations Conferring Pertussis Toxin and RGS Insensitivity

    Expression of G–Protein Subunits in Rat Sympathetic Neurons

    Reconstituting G-Protein Modulation of N-Type Ca2+ Channel with PTX/RGS–i Gα Mutants

    Conclusions

    Endogenous RGS Proteins Regulate Presynaptic and Postsynaptic Function: Functional Expression of RGS-Insensitive Gα Subunits in Central Nervous System Neurons

    Introduction

    Presynaptic Inhibition: Replacing Endogenous Gαi/o Subunits with RGSi Variants

    Postsynaptic Excitation: Bypassing Endogenous Gαq/11 Subunits with RGSi Chimeras

    Conclusions and Caveats

    In Situ Hybridization Analysis of RGS mRNA Regulation and Behavioral Phenotyping of RGS Mutant Mice

    Introduction

    Background to In Situ Hybridization Studies

    In Situ Hybridization Protocols

    Background to Behavioral Analyses of RGS Protein Mutant Mouse Strains

    Behavioral Analysis Protocols

    Summary

    RGS-Insensitive G-Protein Mutations to Study the Role of Endogenous RGS Proteins

    Introduction

    Characterization of an RGS-Insensitive Mutation

    Use of RGSi Gα to Unmask the Role of Endogenous RGS Proteins

    Ribozyme- and siRNA-Mediated Suppression of RGS-Containing RhoGEF Proteins

    Introduction

    Ribozymes

    RNA Interference

    RGS Proteins as Targets of Ribozyme and siRNA Knockdown Methods

    Comparison of RGS-RhoGEF siRNA vs Ribozyme Activity in Cells

    Efficacy and Gene Specificity of RGS-RhoGEF siRNAs

    Potential Problems

    Structure-Based Design, Synthesis, and Activity of Peptide Inhibitors of RGS4 GAP Activity

    Introduction

    Results and Discussion

    Materials and Methods

    Yeast-Based Screening for Inhibitors of RGS Proteins

    Introduction

    RGS Proteins as Small Molecule Targets

    RGS4 Yeast Pheromone Response Assay

    RGS4 HTS Results

    RGSZ1 Yeast Two-Hybrid Screen

    RGSZ1 Screen Results

    Summary

    Genetic Analysis of RGS Protein Function in Caenorhabditis elegans

    Introduction

    RGS and G Proteins Present in Caenorhabditis elegans

    Function of RGS and G Proteins in C. elegans

    Transgenic Expression of RGS Proteins in C. elegans as an Experimental System for Studying RGS Function

    Quantitation of Locomotion Rate by Counting Body Bends per Minute

    Quantitative Assessment of Egg-Laying Behavior by Counting Unlaid Eggs

    Quantitative Assessment of Egg-Laying Behavior by Measuring the Stage of Freshly Laid Eggs

    Purification and In Vitro Functional Analysis of the Arabidopsis thaliana Regulator of G-Protein Signaling-1

    Introduction

    Materials

    Bioinformatic Identification and Analysis of AtRGS1

    Purification of AtGPA1 and AtRGS1

    Protein Interaction Assays

    GAP Assays

    AtRGS1 Function in Arabidopsis thaliana

    Introduction

    Materials and Methods

    Experimental Results

    Identification and Functional Analysis of the Drosophila Gene loco

    Introduction

    RGS Proteins in Drosophila

    Enhancer Trap Technique to Identify Genes

    Identification of loco

    loco Expression during Development

    The loco Mutant Phenotype

    “GAP” or “Trap”: Molecular Function(s) of loco?

    Open Questions

    Analysis of the Roles of Pins and Heterotrimeric G Proteins in Asymmetric Division of Drosophila Neuroblasts

    Introduction

    Yeast Two-Hybrid Screen with the Functional Domain of Insc

    In Vitro Protein Interaction Assay

    Protein Complex Purification, Coimmunoprecipitation, and Western Blotting

    RNA In Situ Hybridization Assay

    Immunohistochemistry

    Generation of pins and Gαi Mutants

    RNA Interference

    Germline Transformation and Rescue Experiment

    Mathematical Modeling of RGS and G-Protein Regulation in Yeast

    Introduction

    The Yeast Pheromone Response Pathway

    Model Development

    Results

    Conclusions

    Identification of Yeast Pheromone Pathway Modulators by High-Throughput Agonist Response Profiling of a Yeast Gene Knockout Strain Collection

    Background

    Library Maintenance

    96-Well Format Pheromone Response Assay

    Data Analysis

    High-Throughput Considerations

    Author Index

    Subject Index

Product details

  • No. of pages: 480
  • Language: English
  • Copyright: © Academic Press 2004
  • Published: August 19, 2004
  • Imprint: Academic Press
  • Hardcover ISBN: 9780121827946
  • eBook ISBN: 9780080497266

About the Serial Volume Editor

David Siderovski

Affiliations and Expertise

University of North Carolina, Chapel Hill, North Carolina, USA

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