Zusammenfassung
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.
Nutzer