Traffic of catecholamine storage vesicle proteins

儿茶酚胺储存囊泡蛋白的运输

• 批准号: 6772916
• 负责人: LAURENT TAUPENOT
• 金额: $ 12.18万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-06-01 至 2006-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6772916
• 关键词: PC12 cells; biological signal transduction; catecholamines; chimeric proteins; chromogranins; exocytosis; fluorescence resonance energy transfer; genetic transcription; granule; green fluorescent proteins; intracellular transport; protein protein interaction; secretion; stimulus /response

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 .

描述（由申请人提供）： 嗜铬粒蛋白/分泌粒蛋白（或“粒素”）是一类调节分泌蛋白，普遍存在于胺和肽激素和神经递质分泌囊泡的核心中。现在已经收集到支持该蛋白质家族的细胞内和细胞外功能的证据。在原始细胞内，激素或神经递质分泌调节途径中的颗粒生成或分选作用已被记录，特别是其指标成员嗜铬粒蛋白 A (CgA)。颗粒蛋白还具有前激素的功能，通过蛋白水解加工产生大量肽片段，这些肽片段的多种自分泌、旁分泌和内分泌活性已在体外、离体和最近的体内得到证实。例如，CgA 片段血管抑素和儿茶酚素控制血管反应性和儿茶酚胺释放，而 CgA 片段胰抑素则升高血糖。该提案提出了两个具体目标，采用新型颗粒嵌合蛋白，原位研究嗜铬粒蛋白（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。要研究的反应是胞吐作用和蛋白质-蛋白质相互作用产生的 FRET（荧光共振能量转移）分子通讯。在目标 1 中，我们将使用一系列用绿色荧光蛋白 (GFP) 或胚胎碱性磷酸酶 (EAP) 标记的 CgA 结构域来原位识别 CgA 一级结构（顺式决定簇）内的推定分选信号，这些信号介导 CgA 靶向嗜铬颗粒。在目标 2 中，颗粒嵌合蛋白将用于研究活细胞中的分泌装置决定簇（反式决定簇）与 CgA 相互作用，影响颗粒蛋白进入受调节分泌途径的分类及其在儿茶酚胺储存颗粒核心内的储存。这些研究的结果将增强我们对交感神经刺激期间儿茶酚胺/颗粒储存和释放的了解，从而更好地了解神经内分泌分泌颗粒的生物发生。