Bioluminescence Resonance Energy Transfer (BRET) Assay

The Gα-γ protein activation assay uses RLuc8-fused Gα protein subunit and GFP10-fused Gγ protein for a resonance energy transfer pair. Flag-tagged receptor and untagged Gβ constructs were co-transfected. The BRET assays were performed as described previously.⁵⁸ Briefly, human embryonic kidney cells (HEK-293T) were transfected, using a constant amount of plasmid DNA but various ratios of plasmids encoding the fusion protein partners. Expression of GFP10 fusion proteins was estimated by measuring fluorescence at 515 nm following excitation at 405 nm. Expression of RLuc8 fusion proteins was estimated by measuring the luminescence of the cells after incubation with 5 µM coelenterazine 400a. In parallel, BRET was measured as the fluorescence of the cells at 535 nm at the same time points using a Mithras LB940 reader (Berthold). In order to measure the antagonist activity, 10 min preincubation of 1 µM quinpirole precedes the 10 min incubation of tested compounds before the sample reading.