Detecting GPCR Signals With Optical Biosensors of Gα‐GTP in Cell Lines and Primary Cell Cultures

Abstract

G 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.

Direct interrogation of context-dependent GPCR activity with a universal biosensor platform

“…Lentiviruses used for transduction of SH-SY5Y cells and primary cells were concentrated after large scale packaging as described previously 137,138 . Lenti-X 293T cells were plated on 150 mm diameter dishes (∼2.5 million cells / dish) and cultured at 37°C, 5% CO 2 in DMEM supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM L-glutamine.…”

Discovery of a Novel Chemo-Type for TAAR1 Agonism via Molecular Modeling

“…Bioluminescence Resonance Energy Transfer (BRET) Measurement BRET screening has been described in detail elsewhere [63,[88][89][90][91][92][93][94]. Briefly, human embryonic kidney 293 cells (HEK293T; ATCC CRL-11268) were transiently co-transfected with plasmids encoding hTAAR1 and a cAMP BRET biosensor using Lipofectamine ® 2000 reagent (ThermoFisher, Waltham, MA, USA), and then plated in 96-well white opaque microplates (Corning, Corning, NY, USA) at a density of 50 × 104 cells per well.…”