Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner
Slp4-a/granuphilin-a interacts with syntaxin-2/3 in a Munc18-2-dependent manner
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DOI：
10.1074/jbc.m505759200
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发表时间：
2005-11-25
影响因子：
4.8
通讯作者：
Shimomura, H
中科院分区：
文献类型：
--
作者：
Fukuda, M;Imai, A;Shimomura, H
Slp4-a/granuphilin-a 最初被描述为与胰腺 β 细胞中含胰岛素颗粒特异性相关的蛋白质，但随后发现它存在于腮腺腺泡细胞中含淀粉酶颗粒上。尽管Slp4-a被认为通过与syntaxin-1a和/或Munc18-1相互作用来控制胰岛素分泌，但对于腮腺腺泡细胞释放淀粉酶过程中Slp4-a的结合伴侣一无所知，腮腺腺泡细胞不内源表达syntaxin-1a或Munc18-1。在本研究中，我们使用COS-7细胞通过免疫共沉淀法系统地研究了syntaxin-1-5和Munc18-1-3之间的相互作用，发现Slp4-a以Munc18-2依赖性方式与syntaxin-2/3的闭合构象相互作用，而Munc18-2本身几乎不与Slp4-a相互作用。相比之下，无论是否存在 Syntaxin-2/3，Slp4-a 都会与 Munc18-1 强烈相互作用，并且只有在 Munc18-1/2 存在的情况下，syntaxin-2/3 才会与 Slp4-a 发生免疫共沉淀。删除分析显示 Syntaxin-2/3（或 Munc18-1/2）结合位点是 Slp4a（氨基酸残基 144-354）的接头结构域，Slp4a 是位于 N 端 Rab27A 结合结构域和 C2A 结构域之间的先前未表征的区域。我们还发现，Slp4-a(.)syntaxin-2 复合物实际上存在于大鼠腮腺中，并且将针对 Slp4-a 连接结构域的抗体引入链球菌溶血素 O 通透的腮腺腺泡细胞中，会严重减弱异丙肾上腺素刺激的淀粉酶释放，这可能是通过破坏 Slp4-a 和 Syntaxin-2/3（或 Munc18-2）之间的相互作用来实现的。这些结果表明，Slp4-a 通过与顶端质膜上的 Syntaxin-2/3 相互作用来调节腮腺腺泡细胞的淀粉酶释放。
Slp4-a/granuphilin-a was originally described as a protein specifically associated with insulin-containing granules in pancreatic beta-cells, but it was subsequently found to be present on amylase-containing granules in parotid acinar cells. Although Slp4-a has been suggested to control insulin secretion through interaction with syntaxin-1a and/or Munc18-1, nothing is known about the binding partner(s) of Slp4-a during amylase release from parotid acinar cells, which do not endogenously express either syntaxin-1a or Munc18-1. In this study we systematically investigated the interaction between syntaxin-1-5 and Munc18-1-3 by co-immunoprecipitation assay using COS-7 cells and discovered that Slp4-a interacts with a closed conformation of syntaxin-2/3 in a Munc18-2-dependent manner, whereasMunc18-2itself hardly interacts with Slp4-a at all. By contrast, Slp4-a was found to strongly interact with Munc18-1 regardless of the presence of syntaxin-2/3, and syntaxin-2/3 co-immunoprecipitated with Slp4-a only in the presence of Munc18-1/2. Deletion analysis showed that the syntaxin-2/3 (or Munc18-1/2)-binding site is a linker domain of Slp4a (amino acid residues 144-354), a previously uncharacterized region located between the N-terminal Rab27A binding domain and the C2A domain. We also found that the Slp4-a(.)syntaxin-2 complex is actually present in rat parotid glands and that introduction of the antibody against Slp4-a linker domain into streptolysin O-permeabilized parotid acinar cells severely attenuates isoproterenol-stimulated amylase release, possibly by disrupting the interaction between Slp4-a and syntaxin-2/3 (or Munc18-2). These results suggest that Slp4-a modulates amylase release from parotid acinar cells through interaction with syntaxin-2/3 on the apical plasma membrane.