Traffic of catecholamine storage vesicle proteins

描述(由申请人提供)： 嗜铬粒蛋白/分泌颗粒蛋白(或称颗粒蛋白)是一类受调节的分泌蛋白家族，广泛存在于胺、肽类激素和神经递质分泌囊泡的核心中。现在已经收集的证据支持该蛋白家族的细胞内和细胞外功能。在起源的细胞中，激素或神经递质分泌的调节途径中有粒形成或分选作用，特别是其指标成员嗜铬粒蛋白A(CGA)。Granins还可以作为激素前体，通过蛋白质分解过程产生大量的多肽片段，这些片段的自分泌、旁分泌和内分泌活性在体外、体外和最近的体内都得到了证明。例如，CGA片段血管抑素和儿茶酚胺抑制素控制血管反应性和儿茶酚胺的释放，而CGA片段胰腺抑素则升高血糖。本研究利用新型颗粒蛋白嵌合蛋白，建立了两个特定的目标，以原位研究嗜铬颗粒(CGA)被分选到交感肾上腺嗜铬细胞的儿茶酚胺储存颗粒中，并与分泌颗粒中的其他分泌或膜蛋白相互作用的机制。被研究的反应是由蛋白质-蛋白质相互作用引起的胞吐作用和FRET(荧光共振能量转移)分子通信。在目标1中，我们将使用一系列标记了绿色荧光蛋白(GFP)或胚胎碱性磷酸酶(EAP)的CGA结构域，来原位鉴定CGA(顺式决定簇)一级结构中可能的分选信号，这些信号介导CGA靶向嗜铬粒。在目标2中，Granin嵌合蛋白将被用于研究活细胞中哪些分泌器官的决定因素(反式决定因素)与CGA相互作用，以影响Granin分选到调节的分泌途径中，并将其储存在儿茶酚胺储存颗粒的核心中。这些研究结果将加深我们对交感刺激中儿茶酚胺/颗粒的储存和释放的认识，从而更好地理解神经内分泌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 .