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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 Expres… Read more
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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 “Trapâ€: 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
DS