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. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Using bimolecular BRET biosensors to monitor Gα-GTP formation of tagged Gα in live cells Alternate Protocol 1: Measuring GPCR dose-dependent Gα-GTP responses in endpoint format Basic Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity Alternate Protocol 2: Using unimolecular BRET biosensors to study endogenous G protein activity in mouse cortical neurons.

Keywords: BRET; G protein; G protein-coupled receptors; GTPase; bioluminescence energy transfer; optical biosensor.

Figure 1.. Kinetic measurements of Gαi-GTP levels in HEK293T cells with a bi-molecular BRET biosensor.

(A) Schematic of experimental protocol going from cell transfection to assay setup and luminescence recording in plate reader. (B) Schematic of bi-molecular sensor design and mode of action in cells. After addition of agonist, the GPCR will promote exchange of GDP to GTP on a YFP-tagged Gα, which will then interact with a Nanoluciferase-fused detector module. This brings the BRET donor (luciferase) and BRET acceptor (YFP) close enough to allow resonance energy transfer from the donor to the acceptor. (C) Detection of changes in Gαi3-GTP levels after stimulation of α2_A-AR with 1 μM brimonidine and inhibition with 25 μM yohimbine. The trace represents the raw BRET ratio from one single experiment. (D) Single trace of the same measurement as in C but now represented as ΔBRET. The BRET ratio of the 30 s prior to agonist injection in (C) was averaged and subtracted to all data points to yield the ΔBRET values represented here.

Figure 2.. Endpoint measurements of Gαi-GTP levels in HEK293T cells with a bi-molecular BRET biosensor.

(A) Schematic of assay setup in 96-well plate and luminescence recording in plate reader. For dose-dependence curves, the agonist can be plated in increasing concentration from left to right, and luciferase substrate (CTZ400a) can be added before measuring luminescence as endpoint measurements. (B) Representative result when stimulating the α2_A-AR with increasing doses of brimonidine (0 – 10 μM). The BRET ratio of the well with buffer only is subtracted from all data points to obtain the ΔBRET values represented here.

Figure 3.. Kinetic measurements of endogenous Gαi-GTP levels in HEK293T cells and primary neurons with a BERKY BRET biosensor.

(A) Schematic of experimental protocol in cell lines going from cell transfection to assay setup and luminescence recording in plate reader. (B) Schematic of unimolecular BERKY sensor design and mode of action in cells. Injection of an agonist will lead to formation of Gα-GTP. Binding of the detector module (KB1753) in the BERKY sensor to Gα-GTP will promote a switch of the ER/K bi-stable linker from an open to a closed conformation, which in turn will bring the BRET donor and BRET acceptor in close proximity to favor the resonance energy transfer event. (C) Detection of changes in endogenous Gαi-GTP levels in HEK293T cells after stimulation of α2_A-AR with 1 μM brimonidine and inhibition with 25 μM yohimbine. The traces represent raw BRET ratio (top) and processed data of ΔBRET over baseline (bottom) of a single representative measurement. (D) Schematic of lentiviral packaging of BERKY biosensor constructs and transduction of primary mouse neurons. (E) Detection of changes in endogenous Gαi-GTP levels in primary neurons after stimulation of α2-AR with 5 μM brimonidine and inhibition with 25 μM yohimbine. The traces represent raw BRET ratio (top) and processed data of ΔBRET over baseline (bottom) of a single representative measurement.