RHODOPSIN TRANSDUCIN INTERACTIONS .1. CHARACTERIZATION OF THE BINDING OF THE TRANSDUCIN-BETA-GAMMA SUBUNIT COMPLEX TO RHODOPSIN USING FLUORESCENCE SPECTROSCOPY

In this work we have used fluorescence spectroscopic approaches to examine the binding of the beta-gamma(T) subunit complex of transducin to the photoreceptor, rhodopsin. To do this, we have covalently labeled the beta-gamma(T) subunit complex with the environmentally sensitive fluorescent cysteine reagent 2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid (MIANS). By using the MIANS moiety as a fluorescent reporter group, we were able to monitor directly the binding of the MIANS-beta-gamma(T) complex to light-activated rhodopsin, which was reconstituted into phosphatidylcholine vesicles, through an enhancement (30-50%) in the MIANS fluorescence. Phosphatidylcholine vesicles, alone, elicited only minor changes in the MIANS-beta-gamma(T) fluorescence (i.e. < 10% enhancement), whereas the addition of rhodopsin in the absence of lipid vesicles and in minimal detergent fully mimicked the effects of reconstituted rhodopsin and caused a significant enhancement of the MIANS fluorescence. The interactions between the MIANS-beta-gamma(T) complex and rhodopsin also resulted in a quenching of the rhodopsin tryptophan fluorescence (approximately 30%), which most likely reflected resonance energy transfer between the tryptophan residues and the MIANS moieties. The binding of the MIANS-beta-gamma(T) species to the alpha(T) subunit was accompanied by an enhancement of the MIANS fluorescence (30-50%) and a slight blue shift of the emission maximum, as described previously (Phillips, W. J., and Cerione, R. A. (1991) J. Biol. Chem. 266, 11017-11024). However, the alpha(T)-induced enhancement of the MIANS-beta-gamma(T) fluorescence was not additive with the enhancement elicited by rhodopsin. Conditions which resulted in the activation of the alpha(T) subunit reversed the alpha(T)-induced enhancement of the MIANS emission, whereas the rhodopsin-induced enhancement persisted, thereby suggesting that the rhodopsin-beta-gamma(T) complex can remain intact throughout the G protein activation event. Studies with synthetic peptides representing different regions of the cytoplasmic domain of rhodopsin demonstrated that a portion of the putative carboxyl-terminal tail (amino acid residues 310-324) was capable of eliciting changes in the MIANS-beta-gamma(T) fluorescence as well as inhibiting the MIANS-beta-gamma(T)-induced quenching of the rhodopsin tryptophan fluorescence. These results suggest that this region of the rhodopsin molecule may constitute a portion of the binding domain for the beta-gamma(T) complex.