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G Protein Pathways, Part B: G Proteins and Their Regulators
1st Edition, Volume 344 - December 11, 2001
Editors: Ravi Iyengar, John D. Hildebrandt
Language: English
Hardback ISBN:9780121822453
9 7 8 - 0 - 1 2 - 1 8 2 2 4 5 - 3
eBook ISBN:9780080496924
9 7 8 - 0 - 0 8 - 0 4 9 6 9 2 - 4
This volume covers topics such as the structure and identification of functional domains of G proteins, and activation of G proteins by receptors or other regulators. The text…Read more
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This volume covers topics such as the structure and identification of functional domains of G proteins, and activation of G proteins by receptors or other regulators. The text takes an integrated approach to studying common experimental questions at many different levels related to G proteins. Methods related to G proteins using molecular modeling, systems biology, protein engineering, protein biochemistry, cell biology, and physiology are all accessible in the same volume. The critically acclaimed laboratory standard for more than forty years, Methods in Enzymology is one of the most highly respected publications in the field of biochemistry. Since 1955, each volume has been eagerly awaited, frequently consulted, and praised by researchers and reviewers alike. Now with more than 300 volumes (all of them still in print), the series contains much material still relevant today—truly an essential publication for researchers in all fields of life sciences.
Section I: Activation of G Proteins by Receptors or Other Regulators
[1]: Analysis of G Protein Activation in Sf9 and Mammalian Cells by Agonist-Promoted [35S]GTPγS Binding
Introduction
Experimental Procedures
Technical Considerations
[2]: Elucidating Kinetic and Thermodynamic Constants for Interaction of G Protein Subunits and Receptors by Surface Plasmon Resonance Spectroscopy
Introduction
Investigating G Protein Subunit Interactions by SPR
Strategies for Immobilizing G Protein Subunits
Capturing Biotinylated Gi Subunits with Streptavidin-Modified SPR Chips and Verifying Specificity of Interaction
Investigating G Protein Subunit Interaction with G-Protein-Coupled Receptors by SPR
Analytical Methods for Determining Kinetic Rate Constants and Equilibrium Dissociation Constants
Conclusions
[3]: Neuroanatomical Localization of Receptor-Activated G Proteins in Brain
Introduction
[35S]GTPγS Autoradiography Assay
Limitations
Applicability
Acknowledgment
[4]: Design and Use of C-Terminal Minigene Vectors for Studying Role of Heterotrimeric G Proteins
Introduction
Construction of Gα Carboxyl-Terminal Minigenes
Cellular Effects of Minigene Peptide Expression
Acknowledgments
[5]: Dissecting Receptor–G Protein Specificity Using Gα Chimeras
Introduction
General Comments
Purification of Gα Subunits from E. coli or Sf9 Cells
Assessing Functional Status of Purified Gα Subunits
Purification of βγ Subunits from Tissue Extracts
Expression of Receptors in Sf9 Insect Cell vs Mammalian Cell Membranes
Preparation of Mammalian Membranes for [35S]GTPγS Binding Assays
Determination of Receptor Density in Membrane Preparations
Protocol for [35S]GTPγS Binding Assay using Sf9 Membranes in a 96-Well Plate
Adaptions to [35S]GTPγS Binding Protocol for Use with Mammalian Membrane Preparations
GTPγS Data Analysis
Affinity Shift Assay
G Protein Concentration in Assay
Receptor Density in Affinity Shift Assay
Reconstitution and Affinity Shift Assay
Affinity Shift Activity Data Analysis
Summary
[6]: Use of Dominant Negative Mutations in Analysis of G Protein Function in Saccharomyces cerevisiae
Introduction
Procedures
Use of Dominant Negative Mutations in Analysis of Yeast G Protein Function
Conclusions
[7]: Functional Assays for Mammalian G-Protein-Coupled Receptors in Yeast
Introduction
Selecting Expression Vector
Selecting Yeast Strain
Growth and Storage of Yeast
Transforming Plasmids into Yeast
Analysis of Receptor Protein Production in Whole Cell Extracts
Analysis of Receptor Production in Crude Membrane Fractions
Gel Shift Assays for Posttranslational Modifications
Subcellular Localization of GFP-Tagged Receptors
Ligand Binding Assay
FUS1-HIS3 Reporter Gene Assay for Receptor Signaling
FUS1-lacZ Reporter Gene Assay for Receptor Signaling
Genetic Strategies for Identifying Mutant Receptors
Future Directions
Acknowledgments
[8]: Role of G Protein βγ Complex in Receptor–G Protein Interaction
Introduction
Preparation of M2 Receptor-Containing Membranes
Purification and Reconstitution of M2
Purification of G Protein
Assays to Measure G Protein Coupling to Receptor
Testing Effect of Peptides Specific to G Protein βγ Complex on Receptor-G Protein Interaction
Acknowledgments
[9]: Phosducin Down-Regulation of G-Protein Coupling: Reconstitution of Phosducin Transducin of cGMP Cascade in Bovine Rod Photoreceptor Cells
Introduction
Preparation of Rod Outer Segment Membrane and Purification of Proteins
Phosducin Inhibition of Retinal cGMP Cascade
Dissociation of Tα and Tβγ Subunits by Phosducin
Role of ROS Disk Membranes in Phosducin/Transducin Interaction
Concluding Remarks
[10]: Analysis of Signal Transfer from Receptor to Go/Gi in Different Membrane Environments and Receptor-Independent Activators of Brain G Protein
Introduction
Signal Restoration Assay
Receptor-Independent Regulators of G-Protein Activation State
Materials
Acknowledgments
[11]: Identification of Modulators of Mammalian G-Protein Signaling by Functional Screens in the Yeast Saccharomyces cerevisiae
Introduction
General Considerations in Working with Yeast
Creation of Screening Strains and Libraries
Yeast Screens for Pheromone Pathway Activators
Using Yeast to Evaluate Site of Action and Mechanism of Isolated G-Protein Activators
Establishing Novel Readouts for Negative Regulators of G-Protein Signaling
Concluding Remarks
Acknowledgments
Section II: Isolation or Production of Native or Modified
[12]: Expression of α Subunit of Gs in Escherichia coli
Introduction
Conclusion
Acknowledgments
[13]: Purification of G Protein Isoforms GOA GOC from Bovine Brain
Introduction
General Methods and Materials
Preparation of G Protein from Bovine Brain
Purification of G Protein Isoforms
Notes and Discussion
Acknowledgment
[14]: Coexpression of Proteins with Methionine Aminopeptidase/or N-Myristoyltransferase in Escherichia coli to Increase Acylation Homogeneity of Protein Preparations
Introduction
Myristoylation of Proteins in Bacteria Expressing Full-Length hNMT1 or hNMT2
Myristoylation of hArf1 in Bacteria Expressing hNMT1 and Met-AP
Summary
Acknowledgments
[15]: Purification of G Protein βγ from Bovine Brain
Introduction
Solutions and Assay Methods
Membrane Preparation and Detergent Extraction
DEAE Column Chromatography
AcA 34 Gel Filtration Chromatography
Procedure
Octyl-Agarose Hydrophobic Chromatography
FPLC Mono Q Ion Exchange Chromatography
Summary Gel Analysis and Discussion
Acknowledgment
[16]: Separation and Analysis of G Protein γ Subunits
Introduction
Materials and Reagents
Isolation and Analysis of γ Subunits of G Protein Heterotrimers
Analysis of Isolated γ Subunits
Discussion
Acknowledgments
[17]: Activity of Gγ Prenylcysteine Carboxyl Methyltransferase
Introduction
Methylation of CAAX Proteins in Intact Cells
Acknowledgment
[18]: Preparation and Application of G Protein γ Subunit-Derived Peptides Incorporating a Photoactive Isoprenoid
Introduction
Materials and Methods
Attachment of a Photoactive Isoprenoid to Peptides
Photolysis of a Prenylated Peptide with RhoGDI
Section III: Functional Analysis of G Protein Subunits
[19]: Expression and Functional Analysis of G Protein α Subunits in S49 Lymphoma Cells
Introduction
Cell Lines
Transient Expression of αs Constructs in cyc− S49 Lymphoma Cells
Stable Expression
Characterization of αs Constructs Expressed in Stable Cell Lines
Discussion
Acknowledgments
[20]: Mouse Gene Knockout Knockin Strategies in Application to α Subunits of Gi/Go Family of G Proteins
Introduction
Inactivation of Nonsensory PTX-Sensitive Gi/Go Class of G Proteins
Handling of ES Cells
Acknowledgment
[21]: Determining Cellular Role of Gα12
Introduction
Strategies
Establishing Model System in Which Gα12-Mediated Cellular Transformation Can Be Reversibly Induced
Interpretation of Results
Summary
Acknowledgments
[22]: Targeted, Regulatable Expression of Activated Heterotrimeric G Protein α Subunits in Transgenic Mice
Introduction
Methods
[23]: Inducible, Tissue-Specific Suppression of Heterotrimeric G Protein α Subunits in Vivo
Introduction
Methods
[24]: Construction of Replication Defective Adenovirus That Expresses Mutant Gαs Q227L
Introduction
Materials
[25]: Expression of Adenovirus-Directed Expression of Activated Gαs in Rat Hippocampal Slices
Introduction
Hippocampal Transduction of Adv-Gα*s Resulting in Increase in Basal PKA Activity
Expression of Gα*s Resulting in Increase in Long-Term Spatial Memory
Immunocytochemical Localization
Conclusions
[26]: Quench-Flow Kinetic Measurement of Individual Reactions of G-Protein-Catalyzed GTPase Cycle
Introduction
Equipment and Reagents
Dissociation of GDP
Receptor-Catalyzed Nucleotide Exchange
Hydrolysis of Gα-Bound GTP
[27]: Analysis of Genomic Imprinting of Gsα Gene
Introduction
Maternal vs Paternal GNAS1/Gnas Mutations Leading to Distinct Phenotypes
Parental Allele-Specific Expression of GNAS1/Gnas Gene Products
Parental Allele-Specific Methylation of GNAS1/Gnas Gene
Conclusions
[28]: Subcellular Localization of G Protein Subunits
Introduction
Subcellular Fractionation
Immunofluorescence
Acknowledgments
[29]: Fluorescence Approaches to Study G Protein Mechanisms
Introduction
Intrinsic Fluorescence of G Proteins
MANT Fluorophore
Use of Fluorescent Nucleotides in G Protein Purification
BODIPY Fluorophore
Spectroscopic Analysis of BODIPY Nucleotide Binding to G Proteins
Affinity and Specificity of BODIPY GTP Analogs for Different G Protein α Subunits
Mastoparan-Induced Guanine Nucleotide Exchange
Future Perspectives
[30]: Defining G Protein βγ Specificity for Effector Recognition
Introduction
Construction of Recombinant Baculovirus Vectors
Expression and Purification of Recombinant Gβγ and Mutants
Analysis of Gβγ Mutants with Various Effectors
Discussion
Acknowledgment
[31]: Ribozyme-Mediated Suppression of G Protein γ Subunits
Introduction
Ribozymes
G Protein γ Subunits as Targets of Ribozymes
In Vitro Analysis of Ribozyme Activity
Ribozyme Delivery into Cells
In Vivo Analyis of Ribozyme Activity
Summary
Acknowledgment
Section IV: G Protein Structure and Identification
[32]: Use of Scanning Mutagenesis to Delineate Structure–Function Relationships in G Protein α Subunits
Introduction
Mutagenesis Approaches
Functional Analysis of Mutant α Subunits
Determining Role(s) of Functionally Important Residues
General Principles of α Subunit Function Derived from Scanning Mutagenesis
Acknowledgments
[33]: Development of Gs-Selective Inhibitory Compounds
Introduction
General Considerations
Assays for Inhibitors of Gαs
Conclusions
Acknowledgments
[34]: Characterization of Deamidated G Protein Subunits
Introduction
Resolution of Goα Subunits by Urea/SDS–PAGE
Limited Tryptic Digestion of G Protein α Subunits: Functional Conditions
Discussion
Proteolytic Digestion of G Protein α Subunits: Denaturing Conditions
Method
Chemical Derivatization of Peptides
Method
Sequencing of Deamidated Peptides
Acknowledgments
[35]: Determining G Protein Heterotrimer Formation
[35S]GTPγS Binding Assay of Purified Recombinant α Subunits
Expression and Purification of Recombinant G Protein βγ Complex from Baculovirus–Insect Cell System
Acknowledgment
[36]: Use of Peptide Probes to Determine Function of Interaction Sites in G Protein Interactions with Effectors
Introduction
Using Peptides to Address Function
Selecting Peptide Regions
Truncating Peptide Regions
Designing Control Peptides: Scrambling of Peptide Regions vs Substitutions within Peptide Regions
Substitutions within Peptide Regions: Identifying Critical Amino Acids
Substitutions within Peptide Regions: Identifying Critical Features of Amino Acids
Conclusions
Acknowledgments
[37]: Protein Interaction Assays with G Proteins
Introduction
General Considerations
Materials
Method of Assay
Applications
Acknowledgments
[38]: Evolutionary Traces of Functional Surfaces along G Protein Signaling Pathway
Introduction
Principles of Evolutionary Trace Method
Evolutionary Traces along G Protein Signaling Pathway
Limitations and Future Direction
Acknowledgments
[39]: Discovery of Ligands for βγ Subunits from Phage-Displayed Peptide Libraries
General Phage and Bacterial Methods
Preparation of βγ Subunits for Screening or ELISA
Libraries
Screening of Phage-Displayed Peptide Libraries against G Protein βγ Subunits
Construction of Phage Displaying Selected Peptides
Analysis of Binding to βγ Subunits using Phage ELISA
Analysis of Isolated Peptides in Functional Assays
Summary
Appendix Solutions for Bacterial and Phage Manipulation
[40]: Exploring Protein–Protein Interactions by Peptide Docking Protocols
Introduction
Calculation of Residue Solvent Accessibility
Secondary Structure Prediction
Protein Surface Visualization
Protein Docking
Predictions by the Docking Model and Experimental Testing
Utility of Molecular Modeling in Analyzing Protein–Protein Interactions
[41]: Structural Characterization of Intact G Protein γ Subunits by Mass Spectrometry
Introduction
Methods
Results
Discussion
Conclusions
Acknowledgments
Section V: RGS Proteins and Signal Termination
[42]: Quantitative Assays for GTPase-Activating Proteins
Single-Turnover GAP Assays
Steady-State GAP Assays
Acknowledgments
[43]: Analysis of RGS Proteins in Saccharomyces cerevisiae
Introduction
I Expression of RGS Proteins in Yeast
II Assay of RGS Function in Vivo
III Screening for RGS Activators/Inhibitors in Yeast
Conclusions
[44]: Purification of RGS Protein, Sst2, from Saccharomyces cerevisiae and Escherichia coli
Introduction
Construction of Expression Vectors
Denaturing Purification of Sst2 from Yeast
Nondenaturing Purification of Sst2 from Escherichia coli
[45]: RGS Domain: Production and Uses of Recombinant Protein
Introduction
Preparation of Box Recombinant Protein
RGS-Catalyzed Gα–GTP Hydrolysis
Preparation of Gα–GTP Substrate
GAP Reaction
Protocol for Performing GAP Assay Using Gαi − 1 Subunits
Retention of Full GAP Activity by 4Box in Single-Turnover Assay
Inhibition by 4Box of Agonist-Bound Receptor Complexes in Cells
Evaluation of Kinetic Data
Conclusion
[46]: Screening for Interacting Partners for Gαi3 and RGS–GAIP Using the Two-Hybrid System
Introduction
Materials
General Procedures for Two-Hybrid Library Screening
Confirmation of Interactions between Bait and Prey Proteins
Acknowledgments
[47]: Assay of RGS Protein Activity in Vitro Using Purified Components
Overview of Single-Turnover Gα Protein GTPase Activity
Binding of [35S]GTPγS to Purified Gα Subunits
HPLC Purification of [γ32P]GTP
[γ-32P]GTP Binding to Giα, Goα, Gsα, G12α, G13α, and Gzα
[γ-32P] GTP Binding to Gqα and Gtα
Removal of [32P]Pi Released during GTP Binding
Single-Turnover GTPase Reaction
RGS and Gα Subunit Interactions
Determining Fractionally Active RGS Protein Pool
Determining KI Values for Gα/RGS Interactions
Steady-State GTPase Activity in Reconstituted Proteoliposomes
Reconstitution of M2-Muscarinic Receptors, Goα, and Gβ5/RGS9 in Phospholipid Vesicles
Steady-State GTPase Assays
Summary
[48]: Measuring RGS Protein Interactions with Gqα
Introduction
Special Materials
RGS Stimulation of Gqα GTPase Activity
RGS Inhibition of Gqα Signaling Functions
Acknowledgments
[49]: Assays of Complex Formation between RGS Protein Gγ Subunit-like Domains and Gβ Subunits
Introduction
In Vitro Analysis of Gβ/GGL Domain Association
Cell Lysate Coimmunoprecipitation Analysis of Gβ/GGL Domain Association
Purification of Gβ5/RGS11 Heterodimers from Insect Cell Expression
Acknowledgments
[50]: RGS Function in Visual Signal Transduction
GTP Hydrolysis and Recovery of Light Responses
Proteins Implicated in Regulating GTP Hydrolysis in Photoreceptors
Biochemical Assays of GTPase Acceleration
Candidate Gene Approach
mRNA Expression Analysis
Sterile culture tubes and RNase-free microfuge tubes Centrifugal vacuum evaporator
Protein Localization by Antibodies
Immunodepletion
Gene Inactivation
Heterologous Expression Systems
[51]: Molecular Cloning of Regulators of G-Protein Signaling Family Members and Characterization of Binding Specificity of RGS 12 PDZ Domain
Introduction
Molecular Cloning of Novel RGS Family Members
Characterization of RGS12 PDZ Domain Binding
Concluding Remarks
Acknowledgments
Author index
Subject Index
No. of pages: 813
Language: English
Edition: 1
Volume: 344
Published: December 11, 2001
Imprint: Academic Press
Hardback ISBN: 9780121822453
eBook ISBN: 9780080496924
RI
Ravi Iyengar
Affiliations and expertise
Mount Sinai School of Medicine, New York, U.S.A.
JH
John D. Hildebrandt
Affiliations and expertise
Medical University of South Carolina, Charleston, U.S.A.
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