G-protein dissociation was monitored by BRET experiments performed as previously reported58 (link). Briefly, a C-terminal fragment of the G-protein-coupled receptor kinase 3 (GRK3ct) fused to a luciferase serves as a BRET donor. Gβγ dimer is labeled with the fluorescent protein Venus, a BRET acceptor. Upon G-protein heterotrimer activation, free Gβγ-Venus is released and binds to membrane-associated GRK3ct-luciferase, leading to an increased signal detectable by BRET.
HEK 293T/17 cells were seeded onto a 10 μg ml−1 Matrigel-coated six-well plate (1 × 106 cells per well). After 4 h of culture, WT or mutant CCKAR (0.84 μg), Gα (Gαq, Gαs and Gαi, 2.1 μg each), Gβ (0.42 μg), Gγ (0.42 μg) and GRK (0.42 μg) were transiently transfected with Lipofectamine LTX reagent (Invitrogen). At 24 h post-transfection, cells were washed once with DMEM medium (no phenol red) and detached by EDTA. Cells were then collected by centrifugation at 1,000 r.p.m. for 5 min and resuspended in DMEM medium. Approximately 75,000 cells per well were distributed in 96-well flat-bottomed white microplates (PerkinElmer). The NanoBRET substrate (furimazine, 25 μl per well, Promega) was added, and the BRET signal (535 nm/475 nm ratio) was determined using an EnVision multilabel plate reader (PerkinElmer). The average baseline value recorded before CCK-8 stimulation was subtracted from BRET signal values.
HEK 293T/17 cells were seeded onto a 10 μg ml−1 Matrigel-coated six-well plate (1 × 106 cells per well). After 4 h of culture, WT or mutant CCKAR (0.84 μg), Gα (Gαq, Gαs and Gαi, 2.1 μg each), Gβ (0.42 μg), Gγ (0.42 μg) and GRK (0.42 μg) were transiently transfected with Lipofectamine LTX reagent (Invitrogen). At 24 h post-transfection, cells were washed once with DMEM medium (no phenol red) and detached by EDTA. Cells were then collected by centrifugation at 1,000 r.p.m. for 5 min and resuspended in DMEM medium. Approximately 75,000 cells per well were distributed in 96-well flat-bottomed white microplates (PerkinElmer). The NanoBRET substrate (furimazine, 25 μl per well, Promega) was added, and the BRET signal (535 nm/475 nm ratio) was determined using an EnVision multilabel plate reader (PerkinElmer). The average baseline value recorded before CCK-8 stimulation was subtracted from BRET signal values.