Description / Table of Contents:
Assessment of Conformational State Transitions of Class B GPCRs Using Molecular Dynamics -- Molecular Dynamic Simulations to Probe Water Permeation Pathways of GPCRs -- Expression and Purification of a Functional E. coli 13CH3-Methionine-Labeled Thermostable Neurotensin Receptor 1 Variant for Solution NMR Studies -- A Combined Cell-Free Protein Synthesis and Fluorescence-Based Approach to Investigate GPCR Binding Properties -- Furan Cross-Linking Technology for Investigating GPCR-Ligand Interactions -- Optical Regulation of Class C GPCRs by Photoswitchable Orthogonal Remotely-Tethered Ligands -- Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assay on GPCRs -- Time-Resolved FRET-Based Assays to Characterize G Protein-Coupled Receptor Hetero-Oligomer Pharmacology -- Combining Conformational Profiling of GPCRs with CRISPR/Cas9 Gene Editing Approaches -- Measuring GPCR Stoichiometry Using Types -1, -2 and -3 Bioluminescence Resonance Energy Transfer-Based Assays -- Combining SRET2 and BiFC to Study GPCR Heteromerization and Protein-Protein Interactions -- Quantification and Comparison of Signals Generated by Different FRET-Based cAMP Reporters -- Measuring GPCR-Induced Activation of Protein Tyrosine Phosphatases (PTP) Using In-Gel and Colorimetric PTP Assays -- Measurement of β-Arrestin Recruitment at GPCRs Using the Tango Assay -- Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry -- Monitoring the Aggregation of GPCRs by Fluorescence Microscopy -- Combining RNAi and Immunofluorescence Approaches to Investigate Post-Endocytic Sorting of GPCRs into Multivesicular Bodies -- Super-Resolution Imaging of G Protein-Coupled Receptors Using Ground State Depletion Microscopy -- Analysis of Spatial Assembly of GPCRs Using Photoactivatable Dyes and Localization Microscopy -- Optical Modulation of Metabotropic Glutamate Receptor Type 5 In Vivo Using a Photoactive Drug -- Imaging of Tissue-Specific and Temporal Activation of GPCR Signaling Using DREADD Knock-In Mice -- Laser Doppler Flowmetry to Study the Regulation of Cerebral Blood Flow by G Protein-Coupled Receptors in Rodents -- Preparation of Pulmonary Artery Myocytes and Rings to Study Vasoactive GPCRs
This detailed volume assembles comprehensive protocols to assist with the study of structural, molecular, cell biological, and in vivo facets of GPCRs, and to enable the development of experimental tools for screening novel GPCR drugs. Sections explore the tweaking of ligands, bioluminescence and FRET approaches, specific GPCR signaling properties, as well as visualization of subcellular compartmentalization. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and practical, G Protein-Coupled Receptor Signaling: Methods and Protocols serves as an ideal reference for life scientists working in a variety of research fields including molecular pharmacology, cell and developmental biology, brain behavior and physiology, drug development and screening. Chapter 4 is available open access under a CC BY 4.0 license via link.springer.com
XV, 406 p. 87 illus., 71 illus. in color. : online resource.