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

• 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

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.

本研究利用小鼠大脑皮层原代培养的神经元，对GTP结合蛋白生物合成调控的分子机制进行了研究，得到了以下结果： 1．神经元长期接触阿托品（一种毒蕈碱受体拮抗剂）会导致毒蕈碱受体和 G 蛋白增加。后者的增加通过[^3H]GppNHp 结合和GTPase 活性的测量得到澄清。2．本研究中使用的神经元拥有 β 肾上腺素受体和与 β 肾上腺素受体功能耦合的环 AMP (CAMP) 生成系统。此外，在原代培养的神经元中阐明了与 CAMP 形成有关的 Gsα 蛋白 mRNA 的存在，该蛋白是由β-肾上腺素能受体的刺激诱导的，并且其含量在神经元发育的早期阶段达到一个平台期。 .3.长期接触普萘洛尔（一种β-肾上腺素能受体特异性拮抗剂）会导致β-肾上腺素能受体数量增加。在这种条件下，与未处理的神经元相比，在 GppNHp（GTP 的非水解类似物）存在下，cAMP 的形成显示出显着增加。在存在霍乱毒素的情况下，在普萘洛尔处理的神经元中也检测到 45 KDa 蛋白与 [^3H] NAD 的 ADP 核糖基化增加。这些结果表明Gsα蛋白的增加与β-肾上腺素受体的上调有关。另一方面，Gsalpha蛋白的mRNA没有显着变化。基于这些结果，认为有必要在接触普萘洛尔的早期阶段检查Gsα蛋白的mRNA变化，因为mRNA的更新被认为是短暂的。从这些观点来看，我们实验室正在对普萘洛尔暴露后mRNA变化的时间过程进行研究。