Trafficking of catecholamine storage vesicle proteins

嗜铬粒蛋白/分泌粒蛋白（granins /secretogranins）是一类受调节的分泌蛋白，存在于胺、肽激素和神经递质分泌囊泡的核心。该蛋白家族包括嗜铬粒蛋白A （CgA）、嗜铬粒蛋白B （CgB）和分泌粒蛋白II （sll）。现在已经有证据支持该蛋白家族的细胞内和细胞外功能。在起源细胞内，在激素或神经递质分泌的调节途径中，颗粒形成或分选作用已被证明。颗粒蛋白还具有促激素的功能，通过蛋白水解加工产生肽片段，其活性已在体外和体内得到证实。例如，CgA片段vasostatin和catestatin控制血管反应性和儿茶酚胺的释放，胰抑素片段升高血糖。产生活性颗粒衍生肽的激素原加工机制可能涉及囊泡PC1和PC2激素原转化酶以及分泌颗粒组织蛋白酶L.利用一系列新的颗粒嵌合体，本项目开发了4个特定目标，旨在了解颗粒的原位运输和储存

The chromogranin/secretogranins (or "granins') are a family of regulated secretory proteins found in the cores of amine and peptide hormone and neurotransmitter secretory vesicles. This family of proteins includes chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (Sgll). Evidence has now been gathered in support of both intracellular and extracellular functions for this protein family. Within the cells of origin, a granulogenic or sorting role in the regulated pathway of hormone or neurotransmitter secretion has been documented. Granins also function as pro-hormones, giving rise by proteolytic processing to peptide fragments for which activities have been demonstrated in vitro and in vivo. For instance, CgA fragments vasostatin and catestatin control vasoreactivity and catecholamine release, and the fragment pancreastatin elevates blood glucose. Prohormone processing mechanisms that generate active granin-derived peptides may involve the vesicular PC1 and PC2 prohormone convertases and the secretory granule cathepsin L. Using a series of novel granins chimeras, this project develop 4 specific aims directed to the understanding, in situ, of the trafficking and the storage of granins into catecholamine secretory granules, and to the comprehension of the dynamics of intravesicular pH and its role in the secretory process. In aim 1, we will use a series of CgA domains tagged with green fluorescent protein (GFP) or with embryonic alkaline phosphatase (EAP), to identify the sorting signals in CgA (cis determinant) that mediate chromaffin granule targeting of CgA. In aim 2, we will impair the biogenesis of chromaffin granules by silencing the expression of CgA, and use a series of CgA domains tagged with GFP or EAP to rescue or induce the formation of secretory granules, and identify CgA's granulogenic determinants. In aim 3, granin chimeras will be employed to investigate which features of the secretory apparatus (trans determinants) interact with CgA to influence its sorting, and its storage within the chromaffin granule. In aim 4, we wilt use pH-sensitive chimeric CgA photoproteins to investigate the dynamics of intravesicular pH and its rote in the secretory process triggered by the physiologic secretagogues or by sympathomimetic amines. The results of these studies will enhance our understanding of large dense-core secretory granule biogenesis, and of catecholaminelgranins storage and release during sympathetic stimulation.