Agonist-induced conformational changes at the cytoplasmic side of transmembrane segment 6 in the beta(2) adrenergic receptor mapped by site-selectivefluorescent labeling

The environmentally sensitive, sulfhydryl-reactive, fluorescent probe N,N'- dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylene-dia mine (IANBD) was used as a molecular reporter of agonist-induced conformati onal changes in the beta (2) adrenergic receptor, a prototype hormone-activ ated G protein-coupled receptor. In the background of a mutant beta (2) adr energic receptor, with a minimal number of endogenous cysteine residues, ne w cysteines were introduced in positions 269(6.31), 270(6.32), 271(6.33), a nd 272(6.34) at the cytoplasmic side of transmembrane segment (TM) 6, The r esulting mutant receptors were fully functional and bound both agonists and antagonist with high affinities also upon IANBD labeling. Fluorescence spe ctroscopy analysis of the purified and site-selectively IANBD-labeled mutan ts suggested that the covalently attached fluorophore was exposed to a less polar environment at all four positions upon agonist binding. Whereas evid ence for only a minor change in the molecular environment was obtained for positions 269(6.31) and 270(6.32), the full agonist isoproterenol caused cl ear dose-dependent and reversible increases in fluorescence emission at pos itions 271(6.33) and 272(6.34). The data suggest that activation of G prote in-coupled receptors, which are activated by "diffusible" ligands, involves a structural rearrangement corresponding to the cytoplasmic part of TM 6, The preferred conformations of the IANBD moiety attached to the inserted cy steines were predicted by employing a computational method that incorporate d the complex hydrophobic/hydrophilic environment in which the cysteines re side. Based on these preferred conformations, it is suggested that the spec tral changes reflect an agonist-promoted movement of the cytoplasmic part o f TM 6 away from the receptor core and upwards toward the membrane bilayer.