Conformation- and activation-based BRET sensors differentially report on GPCR-G protein coupling

G protein-coupled receptors (GPCRs) regulate cellular signaling processes by coupling to diverse combinations of heterotrimeric G proteins composed of G alpha, G beta, and G gamma subunits. Biosensors based on bioluminescence resonance energy transfer (BRET) have advanced our understanding of GPCR functional selectivity. Some BRET biosensors monitor ligand-induced conformational changes in the receptor or G proteins, whereas others monitor the recruitment of downstream effectors to sites of G protein activation. Here, we compared the ability of conformation-and activation-based BRET biosensors to assess the coupling of various class A and B GPCRs to specific G alpha proteins in cultured cells. These GPCRs included serotonin 5-HT2A and 5-HT7 receptors, the GLP-1 receptor (GLP-1R), and the M-3 muscarinic receptor. We observed different signaling profiles between the two types of sensors, highlighting how data interpretation could be affected by the nature of the biosensor. We also found that the identity of the G beta gamma subunits used in the assay could differentially influence the selectivity of a receptor toward G alpha subtypes, emphasizing the importance of the receptor-G beta gamma pairing in determining G alpha coupling specificity. Last, the addition of epitope tags to the receptor could affect stoichiometry and coupling selectivity and yield artifactual findings. These results highlight the need for careful sensor selection and experimental design when probing GPCR-G protein coupling.