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

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 Gα subunit undergoes GDP-GTP exchange, dissociating from the Gβγ 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 Gs and Gi3 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.