Baek K J, Das T, Gray C D, Desai S, Hwang K C, Gacchui R, Ludwig M, Im M J
Department of Molecular Cardiology, Research Institute, The Cleveland Clinic Foundation, Ohio 44195, USA.
Biochemistry. 1996 Feb 27;35(8):2651-7. doi: 10.1021/bi9522965.
Regulation of cellular response is an important mechanism for controlling cellular functions. The transmembrane signaling of the hormone receptors is regulated by GTP-binding proteins (GTPases) and their associated proteins. Our previous studies demonstrated that the bifunctional GTP-binding protein, G alpha h (transglutaminase II), consistently copurified with an approximately 50 kDa protein (G Beta h) which is dissociated from G alpha h upon activation with GTP gamma S or AlF4-. Present immunological and biochemical studies on the regulation of the GTPase cycle of G alpha h, which involves the alpha 1-adrenoceptor and 50 KDa G beta h, reveal that the 50 kDa protein is indeed a G alpha h-associated protein and down regulates functions of G alpha h. Thus, polyclonal antibody against G Beta h coimmunoprecipitates GDP-bound G alpha h but not the GDP-AlF4--bound form. The GTP gamma S binding and GTPase activity of G alpha h are inhibited in a G beta h concentration dependent manner. Supporting this notion, G beta h accelerated GTP gamma S release from G alpha h and changes the affinity of G alpha h from GTP to GDP. Moreover, the ternary complex preparation exhibits TGase activity that is inhibited in the presence of the alpha 1-agonist and GTP. The GTP gamma S binding by the ternary complex, consisting of the alpha 1-agonist, the receptor, and Gh, is also inhibited by G beta h. The inhibition of GTP gamma S binding with the ternary complex requires a > or = 2.7-fold higher concentration of G beta h than the G alpha h alone, indicating that the receptor enhances the affinity of G alpha h for GTP. In addition, G beta h copurifies with an alpha 1-agonist, adrenoceptor, and G alpha h ternary complex, showing that the complex is a heterotetramer. Our data also suggest that G beta h does not directly interact with alpha 1-adrenoceptor. These findings clearly demonstrate that G alpha h associates with a novel protein which modulates the affinity of G alpha h for guanine nucleotides and that the GDP-bound Gh is the ground state for the counterpart activator, the alpha 1-adrenoceptor, in this signaling system.
细胞反应的调节是控制细胞功能的重要机制。激素受体的跨膜信号传导由GTP结合蛋白（GTP酶）及其相关蛋白调节。我们之前的研究表明，双功能GTP结合蛋白Gαh（转谷氨酰胺酶II）始终与一种约50 kDa的蛋白（Gβh）共纯化，该蛋白在被GTPγS或AlF4-激活后会与Gαh解离。目前关于Gαh的GTP酶循环调节的免疫学和生化研究涉及α1肾上腺素能受体和50 kDa的Gβh，结果表明该50 kDa蛋白确实是一种与Gαh相关的蛋白，并下调Gαh的功能。因此，针对Gβh的多克隆抗体可共免疫沉淀结合GDP的Gαh，但不能沉淀结合GDP-AlF4-的形式。Gαh的GTPγS结合和GTP酶活性以Gβh浓度依赖性方式受到抑制。支持这一观点的是，Gβh加速了GTPγS从Gαh的释放，并改变了Gαh对GTP和GDP的亲和力。此外，三元复合物制剂表现出转谷氨酰胺酶活性，在α1激动剂和GTP存在时受到抑制。由α1激动剂、受体和Gh组成的三元复合物对GTPγS的结合也受到Gβh的抑制。与单独的Gαh相比，抑制三元复合物与GTPγS的结合需要高出≥2.7倍浓度的Gβh，这表明受体增强了Gαh对GTP的亲和力。此外，Gβh与α1激动剂、肾上腺素能受体和Gαh三元复合物共纯化，表明该复合物是一种异源四聚体。我们的数据还表明，Gβh不直接与α1肾上腺素能受体相互作用。这些发现清楚地表明，Gαh与一种新型蛋白结合，该蛋白调节Gαh对鸟嘌呤核苷酸的亲和力，并且结合GDP的Gh是该信号系统中对应激活剂α1肾上腺素能受体的基态。
