Imai Akane, Yoshie Sumio, Nashida Tomoko, Shimomura Hiromi, Fukuda Mitsunori
Department of Biochemistry, The Nippon Dental University, School of Dentistry at Niigata, 1-8, Hamaura-cho, Niigata 951-8580, Japan.
J Cell Sci. 2004 Apr 15;117(Pt 10):1945-53. doi: 10.1242/jcs.01048. Epub 2004 Mar 23.
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. 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GTP酶Rab是一个假定的膜运输蛋白大家族，每个成员被认为调控特定类型的膜运输。然而，关于Rab蛋白在外分泌细胞分泌颗粒胞吐作用中的参与情况或该过程的分子机制知之甚少。我们发现，Rab27B是Rab27A的密切相关异构体，在细胞毒性T淋巴细胞中调控溶酶体相关颗粒胞吐作用，在大鼠腮腺腺泡细胞含淀粉酶的分泌颗粒上大量表达。我们还在大鼠腮腺中鉴定出假定的Rab27B效应蛋白Slac2-c（缺乏C2结构域的Slp同源物-c）/MyRIP，它最初被描述为肌球蛋白Va/VIIa和肌动蛋白结合蛋白。亚细胞分级分离、免疫沉淀和免疫组织化学研究结果表明，Rab27B-Slac2-c复合物在体内分泌颗粒上形成。在体外引入特异性Rab27结合结构域（即Slac2-b的重组Slp同源结构域，其特异性结合Rab27A/B而非其他Rab）或特异性破坏Rab27B-Slac2-c复合物的功能阻断抗体，强烈抑制异丙肾上腺素刺激的淀粉酶从链球菌溶血素O通透的腮腺腺泡细胞中释放。我们的结果表明，Rab27B-Slac2-c复合物是腮腺腺泡细胞分泌颗粒胞吐作用的重要组成部分。
