FLUORESCENT LABELING OF PURIFIED BETA(2) ADRENERGIC-RECEPTOR - EVIDENCE FOR LIGAND-SPECIFIC CONFORMATIONAL-CHANGES

The purpose of the present study was to develop an approach to directl y monitor structural changes in a G protein coupled receptor in respon se to drug binding, Purified human beta(2) adrenergic receptor was cov alently labeled with the cysteine-reactive, fluorescent probe N,N '-di methyl-N-(iodoacetyl)-N (7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylenedia mine (IANBD), IANBD is characterized by a fluorescence which is highly sensitive to the polarity of its environment, We found that the full agonist, isoproterenol, elicited a stereoselective and dose-dependent decrease in fluorescence from IANBD-labeled beta(2) receptor. The chan ge in fluorescence could be plotted against the concentration of isopr oterenol as a simple hyperbolic binding isotherm demonstrating interac tion with a single binding site in the receptor, The ability of severa l adrenergic antagonists to reverse the response confirmed that this b inding site is identical to the well described binding site in the bet a(2) receptor, Comparison of the response to isoproterenol with a seri es of adrenergic agonists, having different biological efficacies, rev ealed a linear correlation between biological efficacy and the change in fluorescence, This suggests that the agonist-mediated decrease in f luorescence from IANBD-labeled beta(2) receptor is due to the same con formational change as involved in receptor activation and G protein co upling, In contrast to agonists, negative antagonists induced a small but significant increase in base line fluorescence. Despite the small amplitude of this response, it supports the notion that antagonists by themselves may alter receptor structure, In conclusion, our data prov ide the first direct evidence for ligand-specific conformational chang es occurring in a G protein coupled receptor, Furthermore, the data de monstrate the potential of fluorescence spectroscopy as a tool for fur ther delineating the molecular mechanisms of drug action at G protein- coupled receptors.