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Volume 271, Number 47, Issue of November 22, 1996 pp. 30034-30040
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Interaction of Transducin with Light-activated Rhodopsin Protects It from Proteolytic Digestion by Trypsin

(Received for publication, August 9, 1996, and in revised form, September 9, 1996)

Maria R. Mazzoni and Heidi E. Hamm ^¶

From  Istituto Policattedra di Discipline Biologiche, University of Pisa, 56126 Pisa, Italy and the ^¶ Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

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 _t subunit was almost completely protected from proteolysis. The protection of _t at Arg³¹⁰ was complete, while Arg²⁰⁴ was substantially protected. The cleavage of _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 _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 _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 _t is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg³¹⁰ is not near the _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, Arg³¹⁰, only a few angstroms away from the carboxyl terminus of _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).

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