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A small fragment (SmBiT) was N-terminally fused to a C68-mutated Gγ2 subunit with a 15-amino acid flexible linker. HEK293T cells were seeded in a six-well plate and allowed to grow to 80% confluence before transfection. For measuring Gs signaling, a plasmid mixture consisting of 200 ng LgBiT-inserted Gαs subunit, 1000 ng Gβ1, 1000 ng C68S-mutant SmBiT-fused Gγ2 (C68S), 400 ng test GPCR in 200 μL of Opti-MEM (Gibco) was transiently transfected with PEI. After 1-day transfection, transfected cells were plated onto a 96-well plate treated by cell adherent reagent (Applygen).
Cell Counting Kit-F
To obtain the concentration-response curves, serially diluted agonists were added to each well to stimulate the cells. The luminance signal was measured using 0.5-s intervals after ligand addition (TECAN, 25 °C). CAMP accumulation was analyzed by a standard dose-response curve using GraphPad Prism 8.0 (GraphPad Software). EC50 and pEC50 ± SEM were calculated using nonlinear regression (curve fit). Data were means ± SEM from at least three independent experiments performed in technical triplicates. Span, sample size, and expression are provided in Supplementary Tables S2, S5, and S6.