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Molecular biological analysis on regulatory mechanisms underlying the biosynthesis of GTP binding protein

GTP结合蛋白生物合成调控机制的分子生物学分析

基本信息

批准号：
02807021
负责人：
OHKUMA Seitaro
金额：
$ 1.02万
依托单位：
Kyoto Prefectural University of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-02807021/
关键词：
G Protein beta-Adrenergic Receptor Adenylate Cyclase Muscarinic Acetylcholine Receptor PI Turnover Primary Culture Cerebral Cortical Neuron 細胞内情報伝達系

项目摘要

In this research, molecular mechanisms underlying the regulation of the biosynthesis of GTP-binding protein were investigated using primary cultured neurons prepared from the mouse cerebral cortex and the following results were obtained.1. A long-term exposure of neurons to atropine, a muscarinic receptor antagonist, produced the increase in muscarinic receptor as well as G protein. The latter increase was clarified by the measurement of [ ^3H] GppNHp binding and GTPase activity.2. Neurons used in this study possessed beta-adrenergic receptor and cyclic AMP (CAMP) generating system functionally coupled with beta-adrenergic receptor. In addition, the presence of MRNA for Gsalpha protein which is involved in the formation of CAMP, induced by the stimulation of beta-adrenergic receptor, was clarified in primary cultured neurons and its content was reached to a plateau in the early stage of neuronal development.3. A long-term exposure to propranolol, an antagonist specific for, beta-adrenergic receptor, induced an increase in the number of beta-adrenergic receptor. Under such conditions, cAMP formation in the presence of GppNHp, a non-hydrolyzed analogue of GTP, showed a significant increase in comparison with that in non-treated neurons. The increase in ADP-ribosylation of 45 KDa protein with [ ^3H] NAD in the presence of cholera toxin was also detected in propranolol-treated neurons. These results indicate that the increase of Gsalpha protein is associated with the up-regulation of beta-adrenergic receptor. On the other hand, the MRNA for Gsalpha protein had no significant changes. Based on these results, it is assumed to be necessary to examine the alteration of MRNA for Gsalpha protein in an early stage of the exposure to propranolol, because the turnover of MRNA is considered to be short. From these viewpoints, studies on the time course of the change of MRNA after the exposure to propranolol are underway in our laboratory.

本研究以小鼠大脑皮层原代培养的神经元为材料，研究了GTP结合蛋白生物合成调控的分子机制，取得了以下结果.长期暴露于毒蕈碱受体拮抗剂阿托品，可引起毒蕈碱受体和G蛋白的增加。通过测量[ ^3H] GppNHp结合和GTbR活性来阐明后一种增加。本研究中使用的神经元具有β-肾上腺素能受体和与β-肾上腺素能受体功能偶联的环腺苷酸（cAMP）生成系统。此外，在原代培养的神经元中，β-肾上腺素能受体刺激诱导CAMP形成过程中，Gsalpha蛋白mRNA的存在得到了证实，其含量在神经元发育的早期阶段达到一个平台.长期暴露于β-肾上腺素能受体特异性拮抗剂普萘洛尔可诱导β-肾上腺素能受体数量增加。在这样的条件下，cAMP的形成在GppNHp，GTP的非水解类似物的存在下，表现出显着的增加相比，在未处理的神经元。在普萘洛尔处理的神经元中，在霍乱毒素存在的情况下，[ ^3H] NAD对45 KDa蛋白的ADP核糖基化作用也有所增加。这些结果表明，Gsalpha蛋白的增加与β-肾上腺素能受体的上调有关。另一方面，Gsalpha蛋白的mRNA没有显着变化。基于这些结果，认为有必要在普萘洛尔暴露的早期阶段检查Gsalpha蛋白的mRNA变化，因为认为mRNA的周转时间较短。从这些观点出发，我们实验室正在研究普萘洛尔暴露后mRNA变化的时间过程。