Analyses on the secretory granule formation by using a yeast two hybrid system

利用酵母二元杂交系统分析分泌颗粒的形成

基本信息

批准号：
11670011
负责人：
WATANABE Tsuyoshi
金额：
$ 2.3万
依托单位：
Asahikawa Medical College (2000-2001)Osaka University (1999)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1999
资助国家：
日本
起止时间：
1999 至 2001
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11670011/
关键词：
Chromogranin Secretogranin Secretory granules sorting mechanism Yeast two hybrid system endocrine cells immunocyto chemistry rat セレクトグラニン Yeast two hybrid法 Yeast-two hybrid法グラニン蛋白群蛋白選別輸送クロモグラニンA セクレトグラニンIII 免疫組織化学法セクレトグ

项目摘要

Granins (chromogranins/secretogranins) are acidic secretory proteins which are specifically localized to secretory granules in various endocrine cells with peptide hormones. As we previously demonstrated, two representative granins, chromogranin A (CgA) and secretogranin II (SgII), are separately localized to two distinct subsets of secretory granules in rat pituitary gonadotropes, and the sizes and composition of secretory granules in gonadotropes are closely associated with the expression levels of these granins. Based on these findings it is suggested that granins function as fundamental matrices of secretory granules, although the targeting mechanisms of the granins for secretory granules still remain unexplained.During attempts to discover the molecules responsible for the transport of granins to secretory granules, secretogranin III (SgIII), another member of the granin family, was identified as a binding partner of CgA with yeast two-hybrid screens and assays. The interaction between SgIII and CgA was augmented under low pH and high calcium conditions that mimic the luminal milieu at the trans-Golgi network. Immmunocytochemical analyzes demonstrated that both proteins were co-localized in secretory granules of endocrine cells in vivo , and that the deletion of the SgIII -binding domain of CgA impaired its targeting for secretory granules in AtT-20 cells. These findings suggest that SgIII acts as an interface molecule between CgA and a putative sorting receptor destined for secertory granules. These heterophilic complex of two or more granins might also contribute to the organization of secretory granules in general, by providing multiple anchor sites for both regulated secretory proteins and membrane-bound proteins such as processing enzymes, ion channels, amine transporters, and fusion machineries required for regulated exocytosis.

颗粒蛋白（嗜铬粒蛋白/分泌粒蛋白）是酸性分泌蛋白，与肽激素一起特异性定位于各种内分泌细胞中的分泌颗粒。正如我们之前所证明的，两种代表性的颗粒蛋白，嗜铬粒蛋白 A (CgA) 和分泌粒蛋白 II (SgII)，分别定位于大鼠垂体促性腺激素中两个不同的分泌颗粒子集，并且促性腺激素中的分泌颗粒的大小和组成与促性腺激素密切相关。这些颗粒的表达水平。基于这些发现，表明颗粒蛋白作为分泌颗粒的基本基质发挥作用，尽管颗粒蛋白对于分泌颗粒的靶向机制仍不清楚。在尝试发现负责将颗粒蛋白转运至分泌颗粒的分子期间，分泌颗粒蛋白III（ SgIII) 是粒蛋白家族的另一个成员，通过酵母双杂交筛选和测定，被鉴定为 CgA 的结合配偶体。 SgIII 和 CgA 之间的相互作用在低 pH 和高钙条件下增强，模拟跨高尔基体网络的管腔环境。免疫细胞化学分析表明，这两种蛋白在体内共定位于内分泌细胞的分泌颗粒中，并且CgA的SgIII结合结构域的缺失削弱了其对AtT-20细胞中分泌颗粒的靶向作用。这些发现表明，SgIII 充当 CgA 和假定的分泌颗粒分选受体之间的界面分子。这些两种或多种颗粒蛋白的异嗜性复合物也可能有助于分泌颗粒的组织，为调节的分泌蛋白和膜结合蛋白提供多个锚定位点，例如所需的加工酶、离子通道、胺转运蛋白和融合机制用于调节胞吐作用。