Trafficking of catecholamine storage vesicle proteins

嗜铬粒蛋白/分泌粒蛋白（或“粒蛋白”）是在胺和肽激素和神经递质分泌囊泡的核心中发现的调节分泌蛋白家族。该蛋白质家族包括嗜铬粒蛋白A（CgA）、嗜铬粒蛋白B（Cg B）和分泌粒蛋白II（Sgll）。现在已经收集了支持该蛋白质家族的细胞内和细胞外功能的证据。在起源的细胞内，在激素或神经递质分泌的调节途径中的颗粒形成或分选作用已被记录。颗粒蛋白还作为激素原发挥作用，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释放，并且片段pancreastatin升高血糖。产生活性颗粒蛋白衍生肽的激素原加工机制可能涉及囊泡PC 1和PC 2激素原转化酶和分泌颗粒组织蛋白酶L。利用一系列新的颗粒蛋白嵌合体，本项目开发了4个具体的目标，旨在了解，在原位，运输和储存 颗粒素转化为儿茶酚胺分泌颗粒，以及对颗粒素动力学的理解， 囊内pH值及其在分泌过程中的作用。目的一：利用一系列标记有绿色荧光蛋白（GFP）或胚胎碱性磷酸酶（EAP）的CgA结构域，鉴定CgA中介导嗜铬颗粒靶向CgA的分选信号（顺式决定簇）。目的二是通过沉默CgA基因的表达来破坏嗜铬颗粒的生物合成，并利用一系列标记有GFP或EAP的CgA结构域来拯救或诱导分泌颗粒的形成，并鉴定CgA的颗粒形成决定簇。在目标3中，将采用颗粒蛋白嵌合体来研究分泌器的哪些特征（反式决定簇）与CgA相互作用以影响其分选和其在嗜铬颗粒内的储存。在目的4中，我们将使用pH敏感的嵌合CgA光蛋白来研究囊泡内pH的动态变化及其在由生理促分泌素或拟交感神经胺触发的分泌过程中的作用。这些研究结果将增强我们对大致密核心分泌颗粒生物发生的理解， 交感神经刺激过程中儿茶酚胺颗粒素的储存和释放。

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.