Site-specific fluorescence labeling of the beta(2) adrenergic receptor amino terminus

A modified human beta(2) receptor, designated OK-beta(2) has developed for site-specific labeling at the amino terminus with amine reactive fluorescent probes, OK-beta(2) has the following modifications: (1) all 16 lysines in the wildtype beta(2) receptor were mutated to arginines, (2) a FLAG epitope preceded by a cleaved hemagglutinin signal sequence was fused to the amino terminus, and (3) a hexahistidine tail was added to the carboxyl terminus, The FLAG epitope and hexahistidine tail were added to facilitate purification while lysine to arginine mutations eliminate potential labeling sites for amine-reactive fluorescent probes, The remaining primary amines in the OK-beta(2) receptor, the amino terminal amine and the epsilon-amine of Lys(3), both reside in the amino-terminal FLAG epitope, The OK-beta(2) receptor expressed in Sf9 insect cells exhibited ligand binding and G-protein coupling characteristics similar to the wild-type beta(2) receptor, The modified receptor was labeled with fluorescamine, an amine-reactive fluorescent probe. Proteolysis with factor Xa showed that labeling was confined to the amino terminus of the OK-beta(2) receptor, Our results demonstrate site-specific fluorescamine labeling at the amino terminus of the OK-beta(2) receptor, a lysine-depleted beta(2) receptor that retains functional characteristics of the wild-type receptor, (C) 1997 Academic Press.