Regulators of G Protein Signaling, Part A

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