Mazzoni M R, Hamm H E
Istituto Policattedra di Discipline Biologiche, University of Pisa, 56126 Pisa, Italy.
J Biol Chem. 1996 Nov 22;271(47):30034-40. doi: 10.1074/jbc.271.47.30034.
The tryptic cleavage pattern of transducin (Gt) 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 Gt was in the high affinity complex with Rh*, the alphat subunit was almost completely protected from proteolysis. The protection of alphat at Arg310 was complete, while Arg204 was substantially protected. The cleavage of alphat at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of betat was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*. Gt complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between Gt 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 betagammat (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 betagammat is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg310 is not near the betagammat subunit, thus we believe that the high affinity binding of Gt to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg310, only a few angstroms away from the carboxyl terminus of alphat, 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).
将溶液中的转导蛋白（Gt）的胰蛋白酶切割模式与存在磷脂囊泡、黑暗中保存的视杆外段（ROS）膜或含有光激活视紫红质（变视紫红质II，Rh*）的ROS膜时的切割模式进行了比较。当Gt与Rh形成高亲和力复合物时，αt亚基几乎完全受到蛋白水解的保护。对αt亚基上Arg310的保护是完全的，而Arg204也受到了显著保护。在存在磷脂囊泡、黑暗中保存的ROS膜或含有Rh的ROS膜时，αt亚基在Lys18处的切割受到保护。在存在ROS膜或磷脂囊泡时，βt亚基的切割较慢。当Rh*.Gt复合物与鸟苷-5'-硫代磷酸（一种已知可释放Gt与Rh之间高亲和力相互作用的鸟嘌呤核苷酸类似物）一起孵育时，对Arg310和Arg204的保护作用减弱。根据我们的结果，我们提出Rh要么物理性地阻止胰蛋白酶接近Arg204和Arg310，要么使异源三聚体保持在这样一种构象，即这些切割位点不可用。由于Arg204参与了与βγt的开关界面（兰布赖特，D.G.，桑德克，J.，博姆，A.，斯基巴，N.P.，哈姆，H.E.，和西格勒，P.B.（1996年）《自然》379，311-319），可能是βγt在受体结合的、稳定的异源三聚体中参与保护这个切割位点。Arg310不在βγt亚基附近，因此我们认为Gt与Rh的高亲和力结合在物理上或空间上阻止了胰蛋白酶接近这个位点。因此，距离已知直接与Rh结合的αt亚基羧基末端仅几埃的Arg310，可能也是Rh*结合位点的一部分。这与其他研究一致，并对受体催化G蛋白释放GDP的机制有影响。在存在磷脂囊泡时对Lys18的保护表明氨基末端区域与膜接触，这与异源三聚体的晶体结构一致（兰布赖特，D.G.，桑德克，J.，博姆，A.，斯基巴，N.P.，哈姆，H.E.，和西格勒，P.B.（1996年）《自然》379，311-319）。
