BRET-based G Protein Biosensors for Measuring G Protein-Coupled Receptor Activity in Live Cells

Summary

This protocol describes the use of BRET-based biosensors for real-time measurement of G protein activation in living HEK293 cells upon G protein-coupled receptor ligand stimulation. Here, the β2-adrenergic receptor and cannabinoid type 1 receptor serve as examples to demonstrate the efficiency of sensors for several G protein subtypes and across various GPCRs.

Abstract

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane receptors, playing a crucial role in cellular signaling by transducing extracellular stimuli into intracellular responses. GPCR activation leads to conformational changes that enable interactions with heterotrimeric G proteins. Upon activation, the G_α subunit undergoes GDP-GTP exchange, dissociating from the G_βγ dimer and triggering downstream signaling cascades. To study GPCR-mediated G protein activation, bioluminescence resonance energy transfer (BRET)-based biosensors is a highly sensitive and non-invasive approach. The G protein-based tricistronic activity sensors (G-CASE biosensors), developed by Schihada et al., detect heterotrimer dissociation as a proxy for activation and enable the real-time monitoring of GPCR activity using a single plasmid transfection, overcoming the limitations of co-transfection approaches. BRET assays offer several advantages over fluorescence-based techniques, such as lower background noise, reduced photobleaching, and improved sensitivity. In this protocol, the BRET-based G-CASE biosensors were used in live-cells and 96-well plates. Specifically, we assess the activity of the β2-adrenergic receptor (β2-AR) and the cannabinoid type 1 receptor (CB1R) in terms of G protein activation. Using HEK 293T wild-type cells transiently transfected with β2-AR and HEK293T cells stably expressing CB1R, the robustness of G_s and G_i3 G-CASE biosensors is confirmed. This protocol supports the utility of this approach for pharmacological studies and high-throughput GPCR screening to better understand the ability of ligands to activate specific pathways, for example, via a biased signaling and thus, facilitating the discovery of novel therapeutic compounds.

Introduction

G protein-coupled receptors (GPCRs) represent the largest superfamily of transmembrane receptors, responsible for transducing extracellular stimuli into intracellular signaling cascades resulting in specific biological responses. They are pivotal pharmacological targets, with approximately 30% of marketed drugs acting on GPCRs¹. GPCR ligand binding induces conformational changes in the receptor, facilitating interactions with heterotrimeric G proteins and/or GPCR kinases (GRKs) and β-arrestins, thereby initiating downstream signaling or receptor internalization².

Heterotrimeric G proteins serv....

Protocol

The reagents and the equipment used in this study are listed in the Table of Materials.

1. Well plate seeding (Day 1)

NOTE: Follow all cell culture protocols in a sterile laminar flow hood to maintain sterile conditions. White well plates with transparent flat bottoms are used to follow cell growth and viability. Use the full white well plate to avoid using a sticker in step 3.2.1.

1. Well plate coating
   NOTE: Pre-coated plates may be used to bypass this step.
   1. Pour into a multichannel reservoir around 6 mL of sterile-filtered Poly-L-lysine (PLL) solution ....

Results

A generalized scheme of the experimental setup and execution is detailed in Figure 2. The activation of G_s or G_i/o family proteins following ligand activation of two GPCRs was evaluated using the G-CASE biosensors. First, a prototypical family A GPCR was studied, the β2-AR transiently transfected in HEK 293T cells. When an agonist (i.e., isoproterenol) was applied to HEK wt cells expressing the β2-AR along with the G_s(short)-CASE protein sensor, a concentrati.......

Discussion

The present protocol describes the use of BRET biosensors for real-time measurement of G protein activation in living cells in response to GPCR stimulation with specific ligands. By using the bioluminescent donor-acceptor energy transfer principle, this approach allows for the detection of conformational changes and dissociation of heterotrimeric G proteins upon receptor activation, providing a robust and sensitive platform for studying GPCR signaling. This protocol can be adapted and applied to various GPCR families, in.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Advanced 96-well culture plate, white with transparent flat bottom, with lid, Greiner bio-one	Dutscher	655983
BrightMax ClearLine White Film	Dutscher	760245
ClarioSTAR LVF	BMG Labtech
Dimethyl sulfoxide (DMSO)	Sigma	D4540
DPBS Modified, without calcium chloride and magnesium chloride, liquid, sterile-filtered, suitable for cell	Sigma	D8537
Dulbecco’s modified Eagle’s medium (DMEM) GlutaMAX	Sigma	D0822	Warm in 37 °C water bath before use
Fetal bovine serum (FBS)	Sigma	F9665
Galphai3-Nluc-Gbeta1-Ggamma2-Venus	Addgene	168122	https://www.addgene.org/Gunnar_Schulte/ Gift from Gunnar Schulte
Galphas(short isoform)-Nluc-Gbeta3-Ggamma1-Venus	Addgene	168124	https://www.addgene.org/Gunnar_Schulte/ Gift from Gunnar Schulte
HEK-293T stably expressing CB1R (HEK CB1)			Kind gift from M. Guzman (Complutense University, Madrid, Spain)
HEK-293T wild-type (HEK wt)
Isoproterenol	Sigma	I6504	Solubilised in H20 and stored at – 20 °C
Lipofectamine 2000	Invitrogen	11668019
Opti-MEM I Reduced Serum Medium GlutaMAX Supplement	Fischer	11564506
pcDNA3 Flag beta-2-adrenergic-receptor tag FLAG C-ter			Kind gift of D. Perrais (IINS, Bordeaux)
pcDNA3.1-(empty)-TAG	Addgene	138209
Penicillin/streptomycin	Sigma	P4333
Poly-L-lysine solution (PLL)	Sigma	RNBL7086
Trypsin-EDTA	Sigma	T3924
WIN-55,212-2	Cayman Chemicals	10009023	Solubilised in DMSO and stored at – 20 °C