G Protein Pathways, Part B: G Proteins and Their Regulators

1st Edition, Volume 344 - December 11, 2001
Imprint: Academic Press
Editors: Ravi Iyengar, John D. Hildebrandt
Language: English
Hardback ISBN:
9 7 8 - 0 - 1 2 - 1 8 2 2 4 5 - 3
eBook ISBN:
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

G Protein Pathways, Part B: G Proteins and Their Regulators

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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.

Contributors to Volume 344
Preface
Methods in Enzymology
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 G_i 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 T_α and T_βγ Subunits by Phosducin
  - Role of ROS Disk Membranes in Phosducin/Transducin Interaction
  - Concluding Remarks
- [10]: Analysis of Signal Transfer from Receptor to G_o/G_i 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 G_s in Escherichia coli
  - Introduction
  - Conclusion
  - Acknowledgments
- [13]: Purification of G Protein Isoforms G_OA G_OC 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 α_s Constructs in cyc⁻ S49 Lymphoma Cells
  - Stable Expression
  - Characterization of α_s Constructs Expressed in Stable Cell Lines
  - Discussion
  - Acknowledgments
- [20]: Mouse Gene Knockout Knockin Strategies in Application to α Subunits of G_i/G_o Family of G Proteins
  - Introduction
  - Inactivation of Nonsensory PTX-Sensitive G_i/G_o 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 Gα_s Q227L
  - Introduction
  - Materials
- [25]: Expression of Adenovirus-Directed Expression of Activated Gα_s 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 G_sα 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 G_s-Selective Inhibitory Compounds
  - Introduction
  - General Considerations
  - Assays for Inhibitors of Gα_s
  - Conclusions
  - Acknowledgments
- [34]: Characterization of Deamidated G Protein Subunits
  - Introduction
  - Resolution of G_oα 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 Gα_i − 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 Gα_i3 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 G_iα, G_oα, G_sα, G₁₂α, G₁₃α, and G_zα
  - [γ-³²P] GTP Binding to G_qα and G_tα
  - Removal of [³²P]P_i Released during GTP Binding
  - Single-Turnover GTPase Reaction
  - RGS and Gα Subunit Interactions
  - Determining Fractionally Active RGS Protein Pool
  - Determining K_I Values for Gα/RGS Interactions
  - Steady-State GTPase Activity in Reconstituted Proteoliposomes
  - Reconstitution of M2-Muscarinic Receptors, G_oα, and Gβ₅/RGS9 in Phospholipid Vesicles
  - Steady-State GTPase Assays
  - Summary
- [48]: Measuring RGS Protein Interactions with G_qα
  - Introduction
  - Special Materials
  - RGS Stimulation of G_qα GTPase Activity
  - RGS Inhibition of G_qα 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

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G Protein Pathways, Part B: G Proteins and Their Regulators

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Ravi Iyengar

John D. Hildebrandt

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