Measuring Gαβγ Heterotrimer Dissociation

To measure the dissociation of Gαβγ heterotrimer directly, we applied the BRET2 assay system as reported before^{21 (link)}. In brief, HEK293T cells were plated in a 6-well plate. After 2 h, cells were transiently co-transfected with plasmids encoding WT or mutated GRR20 together with Gi BRET probe (Gαi1-RLuc8, Gβ3, Gγ9-GFP2) using Lipofectamine 2000 reagent (Life Technologies). Adenosine A_2A receptor (A_2AR) that does not couple to Gi proteins was used as a negative control, Apelin receptor (APJ) that couples to Gi proteins was used as a positive control for the Gi BRET assay. 24 h after transfection, cells were distributed into a 96-well microplate (30,000–50,000 cells per well) and incubated for additional 24 h at 37 °C. For the constitutive activity measurement, white backings (Perkin Elmer) were applied to the plate bottoms, the transfected cells were washed once with HBSS and supplemented with 100 µL of 5 µM coelenterazine 400a (Nanolight Technologies). Plates were read in EnVision plate reader (Perkin Elmer) with 410 nm (RLuc8) and 515 nm (GFP2) emission filters with an integration time of 1 s per well. The GFP2 emission to RLuc8 emission ratio was used to compute the BRET2 ratios. ΔBRET represent the change of bioluminescence resonance energy transfer value. ΔBRET = BRET ratio (GPCR with G protein sensor) - BRET ratio (only G protein sensor).