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（促甲状腺素释放激素）在几秒钟内结合到垂体靶细胞上的特定高亲和力受体，刺激磷脂酶C降解多磷酸肌醇，并伴随形成磷酸肌醇和二酰基甘油。最近，鸟嘌呤核苷酸结合蛋白G蛋白被提出与细胞表面受体偶联以参与磷脂酶c介导的多磷酸肌肽分解。为了确定G蛋白偶联TRH受体的结构和功能，我们对经常用于研究TRH作用机制的GH3克隆垂体细胞和膜中TRH受体与G蛋白的关系进行了表征。我们发现TRH利用外源性底物[^3H]磷脂酰肌醇-4,5二磷酸刺激磷脂酶C的活性，并依赖于这些细胞膜中的鸟嘌呤核苷酸。我们还发现，经霍乱毒素处理的膜显示出trh诱导的低Km GTPase活性的部分降低，而在调节trh受体对trh配体的亲和力方面，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。有报道称霍乱毒素通过一种不依赖于camp的机制抑制分泌素介导的多磷酸肌肽水解的增加。近年来研究发现，腺苷酸环化酶的刺激蛋白G可以刺激ca通道的活性。因此，霍乱毒素敏感的G蛋白将被考虑在信号转导系统中发挥新的作用，我们的研究结果将有助于这些目的的研究。

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.