Direct optical recording of intrinsic efficacy at a G protein-coupled receptor

Full and partial agonists activate receptors to varying degrees, presumably by inducing full or partial conformational changes in the receptor protein. Varying degrees of partial agonism corresponding to varying intrinsic efficacies have been demonstrated for many compounds acting at G-protein-coupled receptors, but a method to determine intrinsic efficacies directly at the receptor level has so far been lacking. Here we describe a method that allows the direct monitoring of agonist-induced conformational changes in G-protein-coupled receptors. The cyan (CFP) and yellow (YFP) variants of the green fluorescent protein were fused to the receptors. This resulted in fluorescence resonance energy transfer (FRET) between the CFP- and YFP-moieties. The extent of FRET was reduced in the presence of an agonist. The FRET signal strictly followed agonist occupancy of the receptor. Using the alpha(2)-adrenergic receptor as a model system, the full agonist noradrenaline produced a full signal, the partial agonist clonidine produced only a partial signal, and the antagonist phentolamine had no effect. Thus, optical recording of the agonist-induced conformational change in a G-protein-coupled receptor allows the direct analysis of the intrinsic efficacies of agonists.