The formation mechanisms of secretory granules and synaptic-like micro-vesicles by the lead of cholesterol in endocrine cells.

神经内分泌细胞的分泌颗粒(SGS)除了含有多肽激素和前激素转换酶外，还含有颗粒蛋白。颗粒蛋白家族蛋白包括嗜铬颗粒蛋白A(CgA)、嗜铬颗粒蛋白B、分泌颗粒蛋白II、分泌颗粒蛋白III(SgIII)和7B2，它们被认为是将多肽激素浓缩成SGS的蛋白质。为了研究多肽激素对SGS的分选机制，我们选择CGA作为模型蛋白。然后，我们讨论了以下四个发现。1)以CGA为诱饵，采用酵母双杂交系统筛选了大鼠脑cDNA文库。虽然最初预期的是膜结合蛋白，但鉴定出了一种可溶性分泌蛋白SgIII。以下分析表明，CGA需要SgIII结合域来靶向SGS，而SgIII不需要CGA结合域来靶向SGS。2)SgIII定位于分泌颗粒膜(SGM)，但缺少跨膜区。SgIII直接与SGM的胆固醇组分结合，并将CGA靶向SGS。3)SgIII和羧基肽酶E(CPE；前激素分选受体的不同分子)通过锚定到富含胆固醇的分泌颗粒膜上，以分离的功能分选复合体为核心，POMC衍生的多肽从CPE转移到SGIII，进而转移到CGA。利用这笔资金，我研究了内分泌细胞在胆固醇的引导下，分泌颗粒和突触样微泡的形成机制。此外，我们还发现SGIII可以结合聚集形式的CGA，这表明少量的SGIII足以将大量的CGA靶向分泌颗粒。

The secretory granules (SGs) of neuroendocrine cells contain granin proteins in addition to peptide hormones and prohormone convertases. Granin-family proteins, including chromogranin A (CgA), chromogranin B, secretogranin II, secretogranin III (SgIII), and 7B2, are thought to condense peptide hormones to SGs. To investigate the sorting mechanisms of peptide hormones to the SGs, we selected CgA as a model protein. Then we addressed following four findings. 1) We have screened a rat brain cDNA library with a yeast two-hybrid system using CgA as a bait. Although membrane-bound proteins were initially expected, a soluble secretory protein, SgIII, was identified. Following analyses demonstrated that CgA requires the SgIII-binding domain for its targeting to the SGs, whereas SgIII does not need the CgA-binding domain to target to the SGs. 2) SgIII was localized to the secretory granule membrane (SGM) despite lacking the transmembrane region. SgIII directly binds to cholesterol components of the SGM and targets CgA to SGs. 3) SgIII and carboxypeptidase E (CPE; a different molecule of a sorting receptor for prohormones) core the separate functional sorting complex by anchoring to cholesterol-rich secretory granule membranes, and POMC-derived peptides are transferred from CPE to SgIII, and further to CgA. With this grant, I have studied the formation mechanisms of secretory granules and synaptic-like micro-vesicles by the lead of cholesterol in endocrine cells. Moreover, we have shown that SgIII can bind the aggregated form of CgA, suggesting that a small quantity of SgIII is enough to target a large quantity of CgA to the secretory granules.