The study of the structure and the function of GTP-binding protein which involved in the mechanism of TRH-sensitive phospholipase C activation

下丘脑三肽TRH（促甲状腺激素释放激素）在数秒内与垂体靶细胞上的特异性高亲和力受体结合，刺激磷脂酶C降解聚磷酸肌醇，并伴随形成肌醇磷酸和甘油二酯。最近，鸟嘌呤核苷酸结合蛋白，G蛋白已被提出耦合细胞表面受体磷脂酶C介导的多磷酸肌醇的分解。为了确定G蛋白偶联TRH受体的结构和功能，我们在研究TRH作用机制的常用细胞膜和GH 3克隆垂体细胞中，对TRH受体和G蛋白的关系进行了表征，发现TRH通过外源底物[^3H]磷脂酰肌醇-4刺激磷脂酶C的活性，在这些细胞膜中依赖于鸟嘌呤核苷酸。我们还发现，霍乱毒素处理的膜表现出TRH诱导的低Km GT酶活性的部分降低，并且在调节TRH-受体对TRH-配体亲和力的半最大效应中所需的鸟嘌呤核苷酸浓度增加了10倍。这些数据表明，霍乱毒素可能直接作用于与TRH受体相关的G蛋白。有一份报告指出，霍乱毒素通过cAMP非依赖性机制抑制分泌素介导的聚磷酸肌醇水解增加。近年来研究发现，腺苷酸环化酶的刺激性G蛋白Gs可刺激钙通道的活性。因此，霍乱毒素敏感G蛋白将被认为是一种新的信号转导系统，我们的研究结果将有助于研究这些目的。

The hypothalamic tripetide TRH (thyrotropin-releasing hormone) binds to specific high affinity receptors on target cells of the pituitary within seconds, stimulates the degradation of polyphosphoinositides by phospholipase C and the concomitant formation of inositol phosphates and diacylglycerol. Recently, guanine nucleotide-binding protein, G protein has been proposed to coupled cell-surface receptors to phospholipase C-mediated polyphosphoinositide breakdown. In order to determine the structure and the function of G protein coupled TRH-receptor, we have characterized the relationship between TRH-receptor and G protein in GH3 clonal pituitary cells and membranes which are frequently used to study for the mechanism of TRH action.We found that TRH stimultes the activity of phospholipase C using exogenous substrate [^3H]phosphatidylinositol-4,5 bisphosphate with the dependency of guanine nucleotide in these cell membranes. And also we found that the cholera toxin-treated membranes demonstrated a partial reduction in the activity of TRH-induced low Km GTPase activity and a 10-fold increase in the concentration of guanine nuclerotide required for a half-maxmimal effect in regulating the TRH-receptor affinity for TRH-ligand. These data suggested that cholera toxin may act directly on a G rotein that is associated with TRH-receptors. There have been one report that cholera toxin inhibits secretin-mediated increase in the hydrolysis of polyphosphoinositide by a cAMP-independent mechanism. Recently it was found that Gs, the stimulatory G protein for adenylate cyclase, can stimulete the activity of ca channel. Therefore, cholera toxin-sensitive G protein will be considered about a new role of the signal transduction system and our results will help to study these purpose.