Interaction of transducin with light-activated rhodopsin protects it from proteolytic digestion by trypsin

Abstract

The tryptic cleavage pattern of transducin (G(t)) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light- activated rhodopsin, metarhodopsin II (Rh*). When G(t) was in the high affinity complex with Rh*, the α(t) subunit was almost completely protected from proteolysis. The protection of α(t) at Arg310 was complete, while Arg204 was substantially protected. The cleavage of α(t) at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of β(t) was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*·G(t) complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between G(t) and Rh*, the protection at Arg310 and Arg204 was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg204 and Arg310 or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg204 is involved in the switch interface with βγ(t) (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that βγ(t) is implicated in protecting this cleavage site in the receptor- bound, stabilized heterotrimer. Arg310 is not near the βγ(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, Ar310, only a few angstroms away from the carboxyl terminus of α(t), which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys18 in the presence of phospholipid vesicles suggests that the amino-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 379, 311-319).