Verrall S, Ishii M, Chen M, Wang L, Tram T, Coughlin S R
Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130, USA.
J Biol Chem. 1997 Mar 14;272(11):6898-902. doi: 10.1074/jbc.272.11.6898.
Thrombin activates human platelets and other cells in part by cleaving an unusual G protein-coupled receptor. Thrombin cleavage of this receptor's amino-terminal exodomain unmasks a new amino terminus. This then binds intramolecularly to the body of the receptor to trigger transmembrane signaling and activation of Gi- and Gq-like G proteins. Toward identifying the domains responsible for thrombin receptor-G protein interactions, we examined the signaling properties of chimeric receptors in which thrombin receptor cytoplasmic sequences replaced the cognate sequences in the Gs-coupled beta2-adrenergic receptor (beta2AR) or the Gi-coupled dopamine D2 receptor (D2R). In Xenopus oocytes, a chimeric beta2AR bearing the thrombin receptor second cytoplasmic (C2) loop gained the ability to trigger intracellular Ca2+ release in response to adrenergic agonist, whereas a beta2AR bearing the cognate C2 loop from the D2R did not. Similarly, in COS-7 cells, a chimeric D2R bearing the thrombin receptor C2 loop gained the ability to trigger phosphoinositide hydrolysis in response to dopaminergic agonist, apparently by coupling to a Gq-like G protein. No detectable Gs coupling was seen. Thus, the thrombin receptor C2 loop was able to confer Gq-like coupling in several different receptor contexts. These observations suggest that the thrombin receptor C2 loop specifies Gq coupling by directly contacting Gq or by contributing to a structure required for Gq coupling. The ability of the thrombin receptor C2 loop to function in the context of the D2R and beta2AR strongly suggests that the transmembrane switching and G protein activation strategies used by the thrombin receptor must be very similar to those used by the D2R and beta2AR despite the thrombin receptor's strikingly different liganding mechanism.
凝血酶部分通过裂解一种不同寻常的G蛋白偶联受体来激活人血小板和其他细胞。该受体氨基末端胞外域被凝血酶裂解后暴露出一个新的氨基末端。然后，这个新的氨基末端在分子内与受体主体结合，从而触发跨膜信号传导并激活Gi样和Gq样G蛋白。为了确定负责凝血酶受体与G蛋白相互作用的结构域，我们研究了嵌合受体的信号传导特性，其中凝血酶受体的胞质序列取代了Gs偶联的β2肾上腺素能受体（β2AR）或Gi偶联的多巴胺D2受体（D2R）中的相应序列。在非洲爪蟾卵母细胞中，带有凝血酶受体第二个胞质（C2）环的嵌合β2AR获得了响应肾上腺素能激动剂触发细胞内Ca2+释放的能力，而带有来自D2R的相应C2环的β2AR则没有。同样，在COS-7细胞中，带有凝血酶受体C2环的嵌合D2R获得了响应多巴胺能激动剂触发磷酸肌醇水解的能力（显然是通过与Gq样G蛋白偶联），未观察到可检测到的Gs偶联。因此，凝血酶受体C2环能够在几种不同受体背景下赋予Gq样偶联能力。这些观察结果表明，凝血酶受体C2环通过直接接触Gq或通过促成Gq偶联所需的结构来指定Gq偶联作用。凝血酶受体C2环在D2R和β2AR背景下发挥作用的能力强烈表明，尽管凝血酶受体具有截然不同的配体结合机制，但其使用的跨膜转换和G蛋白激活策略必定与D2R和β2AR所使用策略非常相似。
