Agonist-induced conformational changes in the G-protein-coupling domain ofthe beta(2) adrenergic receptor

The majority of extracellular physiologic signaling molecules act by stimul ating GTP-binding protein (G-protein)-coupled receptors (GPCRs), To monitor directly the formation of the active state of a prototypical GPCR, we devi sed a method to site specifically attach fluorescein to an endogenous cyste ine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the beta ( 2) adrenergic receptor (beta (2)AR), adjacent to the G-protein-coupling dom ain. We demonstrate that this tag reports agonist-induced conformational ch anges in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-beta (2)AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction b etween the fluorescein at Cys-265 and fluorescence-quenching reagents local ized to different molecular environments of the receptor. These observation s are consistent with a rotation and/or tilting of TM6 on agonist activatio n. Our studies, when compared with studies of activation in rhodopsin, indi cate a general mechanism for GPCR activation: however, a notable difference is the relatively slow kinetics of the conformational changes in the beta (2)AR, which may reflect the different energetics of activation by diffusib le ligands.