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Study of secretory granule proteins associated with syntaxin 2 in the rat parotid gland

大鼠腮腺中突触蛋白2相关分泌颗粒蛋白的研究

基本信息

批准号：
14571780
负责人：
IMAI Akane
金额：
$ 2.05万
依托单位：
The Nippon Dental University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2004
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14571780/
关键词：
parotid gland secretory granule Munc18 syntaxin Rab27 Slac2-c Slp4-a amylase release Munc18-3 SNARE Rabタンパク質 VAMP 細胞内局在相互作用

项目摘要

Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechanism underlying this process. Here we show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation, and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e., a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.

小GTPase Rab是一个假定的膜运输蛋白大家族，每个成员被认为调节一种特定类型的膜运输。然而，对于Rab蛋白参与外分泌细胞的分泌颗粒胞吐或这一过程的分子机制知之甚少。在这里，我们发现Rab27B是Rab27A的一个密切相关的异构体，可以调节细胞毒性T淋巴细胞中溶酶体相关的颗粒胞吐，Rab27B在大鼠腮腺腺泡细胞中含有淀粉酶的分泌颗粒上大量表达。我们还在大鼠腮腺中鉴定了推定的Rab27B效应蛋白Slac2-c (Slp同源物缺乏C2结构域-c)/MyRIP，该蛋白最初被描述为肌凝蛋白Va/VIIa和肌动蛋白结合蛋白。亚细胞分离、免疫沉淀和免疫组织化学研究结果表明，体内分泌颗粒上形成Rab27B-Slac2-c复合物。在体外引入特异性Rab27结合域（即特异性结合Rab27A/B而不结合其他Rabs的重组Slp同源域）或特异性破坏Rab27B-Slac2-c复合物的功能阻断抗体，均能强烈抑制异丙肾上腺素刺激的淀粉酶从溶血素o渗透性腮腺腺泡细胞释放。结果表明，Rab27B-Slac2-c复合物是腮腺腺泡细胞分泌颗粒胞吐的重要组成部分。