Trafficking of catecholamine storage vesicle proteins

儿茶酚胺储存囊泡蛋白的贩运

• 批准号: 7122644
• 负责人: LAURENT TAUPENOT
• 金额: $ 21.55万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7122644
• 关键词: PC12 cells; acidity /alkalinity; alkaline phosphatase; blood pressure; catecholamines; chimeric proteins; chromaffin cells; chromogranins; density gradient ultracentrifugation; enzyme activity; fluorescence resonance energy transfer; gene expression; gene induction /repression; green fluorescent proteins; neurotransmitter transport; prohormone convertase; protein localization; protein protein interaction; sympathetic nervous system

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.

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，存在于胺、肽类激素和神经递质分泌囊泡的核心中。这个蛋白质家族包括嗜铬粒蛋白A(CGA)、嗜铬粒蛋白B(CGB)和分泌颗粒蛋白II(SG11)。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的粒形成或分选作用已有文献记载。Granins还具有激素前体的功能，通过蛋白质分解处理产生多肽片段，其活性已在体外和体内得到证明。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，而胰岛抑素片段则升高血糖。产生活性颗粒衍生多肽的激素前处理机制可能涉及囊泡PC1和PC2前激素转换酶和分泌颗粒组织蛋白L。利用一系列新的颗粒嵌合体，本项目开发了四个特定的目标，旨在原位了解运输和储存 将颗粒物转化为儿茶酚胺分泌颗粒，并对其动力学进行了理解。 囊泡内pH及其在分泌过程中的作用。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，来鉴定CGA(顺式决定簇)中介导嗜铬粒靶向CGA的分选信号。在目标2中，我们将通过沉默CGA的表达来损害嗜铬颗粒的生物发生，并利用一系列标记了GFP或EAP的CGA结构域来挽救或诱导分泌颗粒的形成，并鉴定CGA的颗粒发生决定因素。在目标3中，将使用Granin嵌合体来研究分泌器官的哪些特征(反式决定因素)与CGA相互作用，以影响其分选和在嗜铬细胞颗粒中的储存。在目标4中，我们将使用pH敏感的嵌合CGA光蛋白来研究由生理性分泌物或交感神经胺触发的分泌过程中泡内pH的动态及其变化。这些研究结果将加深我们对大的致密核分泌颗粒生物发生的理解，以及对 交感神经刺激中儿茶酚胺颗粒的储存和释放。