Receptor Signal Transduction Protocols

As our understanding of G protein-coupled receptor (GPCR) signal transduction continues to grow, we cannot help but be struck by the emerging complexity and the ability of this receptor superfamily to continually surprise us as new facets are discovered. In this third edition of Receptor Signal Transduction Protocols, the expert contributors have taken into account the constant evolution of the GPCR field and dealt with methods that allow researchers to address many of the important rising issues. Serving as a companion to the previous editions, this volume contains thorough revisions of some core chapters, the complete rewriting of others to encompass new technological developments since the publication of the prior editions, and the commissioning of brand new, cutting-edge chapters to expand on previous coverage. Written in the highly successful Methods in Molecular Biology(TM) series format, chapters include introductions to the respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Inducible Expression of G Protein-Coupled Receptors in Transfected Cells. - Using the Flp-In
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T-Rex
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System to Regulate GPCR Expression. - Viral Infection for GPCR Expression in Eukaryotic Cells. - Generation of Epitope-Tagged GPCRs. - The Use of Site-Directed Mutagenesis to Study GPCRs. - Approaches to Study GPCR Regulation in Native Systems. - Heterologous Expression of GPCRs in Fission Yeast. - Radioligand Binding Methods for Membrane Preparations and Intact Cells. - Quantification of GPCR mRNA Using Real-Time RT-PCR. - Determining Allosteric Modulator Mechanism of Action: Integration of Radioligand Binding and Functional Assay Data. - Design and Use of Fluorescent Ligands to Study Ligand-Receptor Interactions in Single Living Cells. - Examining Site-Specific GPCR Phosphorylation. - Ubiquitination of GPCRs. - [
³⁵
S]GTP S Binding as an Index of Total G-Protein and G -Subtype-Specific Activation by GPCRs. - Using Calcium Imaging as a Readout of GPCR Activation. - Measuring Spatio-Temporal Dynamics of Cyclic AMP Signaling in Real-Time Using FRET-Based Biosensors. - Determining the Activation of Rho as an Index of Receptor Coupling to G
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Proteins. - The Use of Translocating Fluorescent Biosensors for Real-Time Monitoring of GPCR-Mediated Signaling Events. - Study of GPCR-Protein Interactions Using Gel Overlay Assays and Glutathione-
S
-Transferase-Fusion Protein Pull-Downs. - Study of GPCR-Protein Interactions by BRET. - Time Resolved FRET Strategy with Fluorescent Ligands to Analyze Receptor Interactions in Native Tissues: Application to GPCR Oligomerization. - Peptide Affinity Purification for the Isolation and Identification of GPCR-AssociatedProtein Complexes. - Tandem Affinity Purification and Identification of GPCR-Associated Protein Complexes. - Identification of GPCR Localization in Detergent Resistant Membranes. - Analysis of GPCR Localization and Trafficking. - Statistical Methods in Research.