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

本研究利用原代培养的小鼠大脑皮层神经元，对GTP结合蛋白生物合成调控的分子机制进行了研究，获得了以下结果。神经元长期暴露于M受体拮抗剂阿托品后，M受体和G蛋白的表达增加。[~3H]GppNHp结合量和GTP酶活性的测定证实了后者的增加。本研究所用神经元具有β-肾上腺素能受体和环磷酸腺苷(CAMP)生成系统，功能上与β-肾上腺素能受体偶联。此外，在原代培养的神经元中发现了β-肾上腺素能受体刺激下参与cAMP形成的Gsalpha蛋白的mRNA的存在，其含量在神经元发育的早期达到平台期。长期接触心得安(一种β-肾上腺素能受体的特异性拮抗剂)可引起β-肾上腺素能受体数量的增加。在此条件下，与未处理的神经元相比，GppNHp(GppNHp，一种非水解型GTP类似物)可显著增加cAMP的生成。在普萘洛尔处理的神经元中，在霍乱毒素存在的情况下，45 KDa蛋白与[~3H]NAD的ADP-核糖化作用也增加。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。而Gsalpha蛋白的信使核糖核酸没有明显变化。根据这些结果，推测有必要在心得安暴露的早期阶段检测Gsalpha蛋白的mRNA的变化，因为mRNA的周转被认为是短暂的。从这一角度出发，本实验室正在对心得安暴露后基因表达变化的时程进行研究。

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.