Traffic of catecholamine storage vesicle proteins

描述（由申请人提供）： 嗜铬粒蛋白/分泌粒蛋白（或“粒蛋白”）是普遍存在于胺和肽激素和神经递质分泌囊泡的核心中的调节分泌蛋白家族。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，已记录了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，特别是其索引成员嗜铬粒蛋白A（CgA）。颗粒蛋白还作为激素原发挥作用，通过蛋白水解加工产生大量肽片段，已经在体外、离体和最近的体内证明了这些肽片段的各种自分泌、旁分泌和内分泌活性。例如，CgA片段vasostatin和catestatin控制血管反应性和儿茶酚胺释放，CgA片段pancreastatin升高血糖。本研究提出了两个具体的目的，即利用新的颗粒蛋白嵌合蛋白，原位研究嗜铬颗粒蛋白（CgA）被分选到交感肾上腺嗜铬细胞的儿茶酚胺储存颗粒中，并与分泌颗粒的其他分泌蛋白或膜蛋白相互作用的机制。待研究的反应是由蛋白质-蛋白质相互作用引起的胞吐和FRET（荧光共振能量转移）分子通讯。目的1：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，在原位鉴定CgA一级结构（顺式决定簇）中介导CgA靶向嗜铬颗粒的分选信号。在目标2中，颗粒蛋白嵌合蛋白将用于研究在活细胞中，哪些分泌器的决定簇（反式决定簇）与CgA相互作用以影响颗粒蛋白进入受调节的分泌途径的分选及其在儿茶酚胺储存颗粒的核心内的储存。这些研究的结果将提高我们的知识，交感神经刺激过程中的儿茶酚胺/颗粒的存储和释放，从而更好地了解神经内分泌颗粒的生物发生。

DESCRIPTION (provided by applicant): The chromogranin/secretogranins (or "granins") are a family of regulated secretory proteins ubiquitously found in the cores of amine and peptide hormone and neurotransmitter secretory vesicles. 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, especially for its index member, chromogranin A (CgA). Granins also function as pro-hormones, giving rise by proteolytic processing to a large array of peptide fragments for which diverse autocrine, paracrine, and endocrine activities have been demonstrated in vitro, ex vivo and more recently in vivo. For example, CgA fragments vasostatin and catestatin control vasoreactivity and catecholamine release, and the CgA fragment pancreastatin elevates blood glucose. This proposal develops 2 specific aims, employing novel granins chimeric proteins, to investigate in situ, mechanisms whereby chromogranin (CgA) is sorted into catecholamine storage granule of sympathoadrenal chromaffin cells, and interacts with other secreted or membrane proteins of the secretory granule. The responses to be investigated are exocytosis and FRET (fluorescence resonance energy transfer) molecular communication resulting from protein-protein interaction. In aim 1, we will use a series of CgA domains tagged with green fluorescent protein (GFP) or embryonic alkaline phosphatase (EAP), to identify in situ, the putative sorting signals within the primary structure of CgA (cis determinant) that mediate CgA's targeting to the chromaffin granule. In aim 2, granin chimeric proteins will be used to investigate in the living cell, which determinants of the secretory apparatus (trans determinants) interact with CgA to influence sorting of the granin into the regulated secretory pathway and its storage within the core of the catecholamine storage granule. The results of these studies will enhance our knowledge of catecholamine/granins storage and release during sympathetic stimulation, leading to a better understanding of neuroendocrine secretory granule biogenesis .