Dynamics of receptor/G protein coupling in living cells

The interaction of activated G protein-coupled receptors with G proteins is a key event in signal transduction. Here, using a fluorescence resonance energy transfer (FRET)-based assay, we measure directly and in living cells the interaction of YFP-labeled a2A-adrenergic receptors with CFP-labeled G proteins. Upon agonist stimulation, a small, concentration-dependent increase in FRET was observed. No specific basal FRET was detected in the absence of agonist. Kinetics of the onset of receptor/G protein inter-action were o100 ms and depended on expression levels of Ga. Simultaneously recorded G protein-regulated in-wardly rectifying Kþ channel currents revealed a maxi-mal current response already at agonist concentrations producing submaximal FRET amplitudes. By analyzing FRET signals in the presence of a Ga mutant, which dissociates more slowly from activated receptors, it was demonstrated that only a fraction of wild-type G proteins interacts with the activated receptor at any time. Our data suggest that a2A-adrenergic receptors and G proteins inter-act by rapid collision coupling and indicate that there is no significant precoupling between these receptors and G proteins