Trafficking of catecholamine storage vesicle proteins

儿茶酚胺储存囊泡蛋白的贩运

• 批准号: 7844957
• 负责人: LAURENT TAUPENOT
• 金额: $ 24.11万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7844957
• 关键词: Adenylate Cyclase; Alkaline Phosphatase; Alkalinization; Amines; Binding; Biogenesis; Blood Glucose; Blood Pressure; CHGA gene; CHGB gene; Catecholamines; Cathepsin L; Cells; Chimera organism; Chromaffin Cells; Chromaffin granule; Chromogranin A; Chromogranins; Code; Complement; Comprehension; Cytoplasmic Granules; Embryo; Endocrine; Engineering; Event; Exocytosis; Family; Fluorescence; Fluorescence Resonance Energy Transfer; Genetic Polymorphism; Green Fluorescent Proteins; Hormones; Human; In Situ; In Vitro; Knowledge; Luminescent Proteins; Mediating; Membrane Transport Proteins; Neuroeffector Junction; Neurotransmitters; Nicotine; Pathway interactions; Peptide Fragments; Peptides; Physiological; Pituitary Gland; Process; Proprotein Convertase 1; Proprotein Convertase 2; Protein Family; Proteins; Proteolytic Processing; Regulation; Role; Route; Secretory Vesicles; Series; Signal Transduction; Single Nucleotide Polymorphism; Sorting - Cell Movement; Structure; Subtilisins; Sympathomimetic Amines; Variant; Vesicle; autocrine; blood pressure regulation; cholinergic; chromogranin A (344-364); chromogranin B; chromogranin C; extracellular; in vivo; indexing; member; novel; pancreastatin; paracrine; peptide hormone; polypeptide; programs; prohormone; prohormone thiol protease; response; secretogranins; secretory protein; sensor; trafficking; vasostatin

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/分泌粒蛋白（或“颗粒”）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。该蛋白家族包括嗜铬粒蛋白 A (CgA)、嗜铬粒蛋白 B (CgB) 和分泌粒蛋白 II (Sgll)。现已收集的证据支持细胞内和分泌蛋白 该蛋白质家族的细胞外功能。在原始细胞内，激素或神经递质分泌的调节途径中的颗粒生成或分选作用已被记录。颗粒蛋白还起到激素原的作用，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 片段血管抑素和儿茶素对照 血管反应性和儿茶酚胺释放，胰酶片段会升高血糖。产生活性谷物衍生肽的激素原加工机制可能涉及囊泡 PC1 和 PC2 激素原转化酶以及分泌颗粒组织蛋白酶 L。利用一系列新型谷物蛋白嵌合体，该项目开发了 4 个具体目标，旨在原位了解 贩运和储存 将颗粒转化为儿茶酚胺分泌颗粒，并了解其动力学 囊泡内 pH 值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 结构域来识别 CgA（顺式决定簇）中介导嗜铬颗粒靶向 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，并使用一系列带有GFP或EAP标记的CgA结构域来拯救或诱导分泌颗粒的形成，并鉴定CgA的颗粒发生决定因素。在目标 3 中，颗粒嵌合体将用于研究分泌装置（反式决定簇）的哪些特征与 CgA 相互作用以影响其分类及其在嗜铬颗粒内的存储。在目标 4 中，我们将使用 pH 敏感的嵌合 CgA 光蛋白来研究囊泡内 pH 的动态及其在由生理促泌剂或拟交感胺触发的分泌过程中的作用。这些研究的结果将增强我们对大致密核心分泌颗粒生物发生和 儿茶酚胺颗粒在交感神经刺激期间储存和释放。