2.8. Bioluminescence Resonance Energy Transfer (BRET)

Cellular measurements of BRET between Venus-Gβ1γ2 and masGRK3ct-Nluc-HA were performed to examine GAP activity of RGS9-2/Gβ5s complex in living cells [40,48,49]. Then, at 16 to 24 h post-transfection, HEK293T/17 cells were washed once with BRET buffer (Dulbecco’s phosphate-buffered saline (PBS) containing 0.5 mM MgCl₂ and 0.1% glucose) and detached by gentle pipetting over the monolayer. Cells were harvested by centrifugation at 500× g for 5 min and resuspended in BRET buffer. Approximately 50,000 to 100,000 cells per well were distributed in 96-well flatbottomed white microplates (Greiner Bio-One, Kremsmunster, Austria). The NanoLuc (Nluc) substrate, furimazine [50], was purchased from Promega and used according to the manufacturer’s instructions. BRET measurements were made using a microplate reader (POLARstar Omega; BMG Labtech, Ortenberg, Germany) equipped with two emission photomultiplier tubes, allowing us to detect two emissions simultaneously with the highest possible resolution of 20 ms per data point. All measurements were performed at room temperature. To activate and then deactivate, we applied the final concentration of 100 μM dopamine and 100 μM haloperidol on the transfected cells to control the activity of those GPCRs. The BRET signal was determined by calculating the ratio of the light emitted by the Venus- Gβ1γ2 (535 nm with a 30 nm band path width) over the light emitted by the masGRK3ct-Nluc-HA (475 nm with a 30 nm band path width). The average lowest value (basal BRET ratio) recorded after haloperidol application was subtracted from the experimental BRET signal values, and the resulting difference (DBRET ratio) was normalized against the maximal DBRET value recorded upon agonist stimulation. The rate constants (1/μ) of the deactivation phases were obtained by fitting a single exponential curve to the traces with Clampfit 10.3. k_GAP rate constants were determined by subtracting the basal deactivation rate (k_app) from the deactivation rate measured in the presence of exogenous RGS protein. Obtained k_GAP rate constants were used to quantify GAP activity. Bioluminescence resonance energy transfer (BRET)-based signaling assay and measuring the GAP of Regulator G-protein signaling (RGS) complexes in expression assays were used to investigate the impact of the variant on the function of Gβ5S.