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

TRH敏感磷脂酶C激活机制中GTP结合蛋白的结构和功能研究

基本信息

批准号：
62570529
负责人：
YAJIMA Yukiko
金额：
$ 1.15万
依托单位：
The Tokyo Metroplotan Institute of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

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.

下丘脑三肽 TRH（促甲状腺激素释放激素）在几秒钟内与垂体靶细胞上的特定高亲和力受体结合，刺激磷脂酶 C 降解多磷酸肌醇，并同时形成磷酸肌醇和二酰甘油。最近，鸟嘌呤核苷酸结合蛋白 G 蛋白被提议将细胞表面受体与磷脂酶 C 介导的多磷酸肌醇分解偶联。为了确定G蛋白偶联TRH受体的结构和功能，我们表征了GH3克隆垂体细胞和细胞膜中TRH受体和G蛋白之间的关系，这些细胞和膜经常用于研究TRH作用机制。我们发现TRH利用外源底物[^3H]磷脂酰肌醇-4,5二磷酸刺激磷脂酶C的活性与这些细胞膜中鸟嘌呤核苷酸的依赖性。我们还发现，霍乱毒素处理的膜表现出 TRH 诱导的低 Km GTP 酶活性部分降低，并且调节 TRH 受体对 TRH 配体亲和力所需的鸟嘌呤核苷酸浓度增加 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。一份报告称，霍乱毒素通过 cAMP 独立机制抑制促胰液素介导的多磷酸肌醇水解增加。最近发现腺苷酸环化酶的刺激G蛋白Gs可以刺激ca通道的活性。因此，霍乱毒素敏感的G蛋白将被认为是信号转导系统的新作用，我们的结果将有助于研究这些目的。