Affiliation:
1. Department of Biochemistry & Cell Biology, Chobanian & Avedisian School of Medicine Boston University Boston Massachusetts
2. Department of Biology Boston University College of Arts & Sciences Boston Massachusetts
Abstract
AbstractG protein–coupled receptors (GPCRs) are the largest class of transmembrane receptors and mediate a wide variety of physiological processes. GPCRs respond to a plethora of extracellular ligands and initiate signaling pathways inside cells via heterotrimeric G proteins (Gαβγ). Because of the critical role GPCRs play in regulating biological processes and as pharmacological targets, the availability of tools to measure their signaling activity are of high interest. Live‐cell biosensors that detect the activity of G proteins in response to GPCR stimulation have emerged as a powerful approach to investigate GPCR/G protein signaling. Here, we detail methods to monitor G protein activity through direct measurement of GTP‐bound Gα subunits using optical biosensors based on bioluminescence resonance energy transfer (BRET). More specifically, this article describes the use of two types of complementary biosensors. The first protocol explains how to use a multicomponent BRET biosensor that relies on expression of exogenous G proteins in cell lines. This protocol yields robust responses that are compatible with endpoint measurements of dose‐dependent ligand effects or with kinetic measurements of subsecond resolution. The second protocol describes the implementation of unimolecular biosensors that detect the activation of endogenous G proteins in cell lines expressing exogenous GPCRs or in primary cells upon stimulation of endogenous GPCRs. Overall, using the biosensors as described in this article will help users characterize the mechanisms of action of many pharmacological agents and natural ligands that modulate GPCR and G protein signaling with high precision. © 2023 Wiley Periodicals LLC.Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα‐GTP formation of tagged Gα in live cellsAlternate Protocol 1: Measuring GPCR dose‐dependent Gα‐GTP responses in endpoint formatBasic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activityAlternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons
Funder
National Institutes of Health
American Heart Association
Subject
Medical Laboratory Technology,Health Informatics,General Pharmacology, Toxicology and Pharmaceutics,General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Neuroscience
Cited by
1 articles.
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