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

小GTP酶Rab是一个可能的膜转运蛋白大家族，每个成员被认为调节一种特定类型的膜转运(S)。然而，Rab蛋白(S)是否参与外分泌细胞分泌颗粒的胞吐作用，以及该过程的分子机制尚不清楚。在这里，我们发现了与Rab27A密切相关的亚型Rab27B，它调节细胞毒性T淋巴细胞中溶酶体相关颗粒的胞吐，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。我们还鉴定了大鼠腮腺中可能的Rab27B效应蛋白Slac2-c(SLP同源缺少C2结构域-c)/MyRIP，它最初被描述为一种肌球蛋白Va/VIIa和肌动蛋白结合蛋白。亚细胞分级、免疫沉淀和免疫组织化学研究结果表明，在体内分泌颗粒上形成了Rab27B-Slac2-c复合体。引入特定的Rab27结合域(即Slac2-b的重组SLP同源结构域，它能特异性结合Rab27A/B，但不能与其他RAB结合)或在体外特异性阻断Rab27B-Slac2-c复合体的功能阻断抗体的引入，强烈地抑制了异丙肾上腺素刺激的链溶素O通透性腮腺腺泡细胞的淀粉酶释放。我们的结果表明，Rab27B-Slac2-c复合体是腮腺腺泡细胞分泌颗粒吐出的重要成分。

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.