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Molecular Mechanisms of Exocytotic Prpces and Its Spatio-temporal Regulation

胞吐过程的分子机制及其时空调控

基本信息

批准号：
15200030
负责人：
KUMAKURA Konosuke
金额：
$ 27.79万
依托单位：
Sophia University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

The aim of this research project is to elucidate the molecular mechanism of exocytotic process and its spatio-temporal regulation, and we have obtained the following results.1) Roles of PKC in vesicle recruitment.The facilitation of the vesicle recruitment from the reserve pool to the releasable pool by protein kinase C (PKC) is mediated predominantly by PKCα. PKCβ also increases the secretory function, but by a distinct mechanism. Vesicle recruitment was increased by PKC through the vesicle movement at the rate of 0.03-0.06 μm/s, and the facilitation was detected as an increase in the frequency of exocytotic events in the sustained response. The facilitation of the vesicle recruitment by PKC is accompanied with a substantial increase in the vesicle numbers beneathe the plasma membrane. The receptor molecule for the activated PKC, RACK1, seemed to have roles as anchoring and signaling molecules in the regulation.2) Roles of motor proteins in vesicle recruitment.The important role of actin-myosin interaction in the recruitment of chromaffin granules to the release sites has been suggested. In the present research, it was suggested that myosin-V is specifically involved in the vesicle recruitment, while myosin-I is involved in the general movement of the vesicles. In addition, myosin-ATPase was shown to be essential for the regulation of the vesicle recruitment in chromaffin cells. The inhibition of myosin-ATPase resulted a decrease in the vesicle numbers beneathe the plasma membrane.3) Regulation of SNARE complex.Binding of calmodulin to C-teminus of VAMP-2, and binding between SyntaxinlA and CaMkinase II regulate SNARE complex and are essential for exocytosis.

本研究的目的是阐明胞吐过程及其时空调控的分子机制，得到以下结果：1)蛋白激酶C在囊泡募集中的作用，蛋白激酶Cα主要介导蛋白激酶C促进囊泡从储备池向可释放池的募集。PKCβ也能增强细胞的分泌功能，但机制不同。PKC以0.03~0.06μm/S的速率通过囊泡运动促进囊泡募集，其易化作用表现为持续反应中胞吐事件频率的增加。在PKC促进囊泡募集的同时，质膜上的囊泡数量也显著增加。激活的PKC的受体分子RACK1似乎在调节中起着锚定和信号分子的作用。2)运动蛋白在囊泡募集中的作用。肌动蛋白-肌球蛋白相互作用在嗜铬细胞颗粒向释放部位募集中起重要作用。在目前的研究中，有人认为肌球蛋白-V特异性地参与囊泡的募集，而肌球蛋白-I参与囊泡的一般运动。此外，肌球蛋白-ATPase被证明对嗜铬细胞囊泡募集的调节是必不可少的。3)SNARE复合体的调节，钙调蛋白与VAMP-2的C-teminus结合，Synaxin1A与CaMkinII结合调节SNARE复合体，是胞吐所必需的。