Rhodopsin/transducin interactions. I. 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. less than 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.
Phillips
W J
WJ
Department of Pharmacology, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853.
Cerione
R A
RA
D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D007700:Kinetics; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D011485:Protein Binding; D012243:Rhodopsin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D015293:Transducin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D000817:Anilino Naphthalenesulfonates; D008081:Liposomes; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D013439:Sulfhydryl Reagents; C021310:2-(4'-maleimidylanilino)naphthalene-6-sulfonic acid; D012243:Rhodopsin; D015293:Transducin