Bioluminescence resonance energy transfer (BRET)

BRET¹ was utilised to determine ligand-mediated receptor-Gα protein interactions, as described previously¹⁹. Briefly, cells were rinsed and harvested into PBS, and transferred into 96-well plates at a density of ∼200,000 cells/well. Following addition of Coelenterazine h substrate, the baseline BRET ratio (475 nm/535 nm) was recorded for 1 min, before addition of ligand and further recording for an additional 1 min. To calculate BRET signals, the values at 535 nm were divided by that omitted at 475 nm. For assessing constitutive receptor-G protein or receptor-receptor associations, net BRET values were calculated by subtracting the basal BRET ratio of receptor-tagged Rluc8 alone from all readings. The effect of ligand addition were assessed by subtracting basal net BRET readings from ligand-stimulated values, followed by subtracting any changes observed with control, PBS-treated wells. Constitutive receptor-G protein association readings were carried out in duplicate and ligand-induced BRET readings in triplicate.