INTERACTION OF TRANSDUCIN WITH LIGHT-ACTIVATED RHODOPSIN PROTECTS IT FROM PROTEOLYTIC DIGESTION BY TRYPSIN

The tryptic cleavage pattern of transducin (G(t)) in solution was comp ared with that in the presence of phospholipid vesicles, rod outer seg ment (ROS) membranes kept in the dark, or ROS membranes containing lig ht-activated rhodopsin, metarhodopsin II (Rh). When G(t) was in the h igh affinity complex with Rh, the alpha(t) subunit was almost complet ely protected from proteolysis. The protection of at at Arg(310) was c omplete, while Arg(204) was substantially protected. The cleavage of a lpha(t) at Lys's was protected in the presence of phospholipid vesicle s, ROS membranes kept in the dark, or ROS membranes containing Rh. Th e cleavage of beta(t) was slower in the presence of ROS membranes or p hospholipid vesicles. When the Rh. G(t) complex was incubated with gu anyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between G(t) and Rh, the protection at Arg(310) and Arg(304) was diminished. From our results, we propose th at Rh either physically blocks access of trypsin to Arg(204) and Arg( 310) or maintains the heterotrimer in such a conformation that these c leavage sites are not available. Since Arg(204) is involved in the swi tch interface with beta gamma(t) (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 31 1-319), it may be that beta gamma(t) is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg(310) is not near the beta gamma(t) subunit, thus we believe that the high affinity binding of G(t) to Rh physically or sterically blocks access of trypsin to this site. Thus, Arg(310), only a few angstroms away fr om the carboxyl terminus of a,, which is known to directly bind to Rh , is likely to also be a part of the Rh binding site. This is in agre ement with other studies and has implications for the mechanism by whi ch receptors catalyze GDP release from G proteins. The protection of L ys(18) in the presence of phospholipid vesicles suggests that the amin o-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B.(1996) Nature 37 9, 311-319).