STIMULUS-EVENT COUPLING IN CHROMAFFIN SECRETORY GRANULES

嗜铬分泌颗粒中的刺激事件耦合

• 批准号: 6322497
• 负责人: LAURENT TAUPENOT
• 金额: $ 8.48万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-06-01 至 2004-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6322497
• 关键词: PC12 cells; acid base balance; biological signal transduction; catecholamines; chimeric proteins; chromogranins; genetic transcription; green fluorescent proteins; intracellular transport; protein protein interaction; secretion; stimulus /response

DESCRIPTION (adapted from the application) In secretory granules of sympathoadrenal chromaffin cells, co-stored catecholamines and peptides are cosecreted by exocytosis in response to acetylcholine or to the novel non-cholinergic neurotransmitter pituitary adenylyl cyclase-activating polypeptide (PACAP). The chromogranins/ secretogranins (Cg/Sg), a family of acidic, soluble proteins found in the cores of amine and peptide hormone and neurotransmitter secretory vesicles, have a virtually ubiquitous occurrence in such vesicles throughout the neuroendocrine system. Their proteolytic fragments exert effects on hormone or neurotransmitter release as well as target cells at a variety of sites. For example, chromogranin A (CgA) fragments vasostatin and catestatin control vasoreactivity and catecholamine release, and CgA fragment pancreastatin elevates blood glucose. This proposal develops 3 specific Aims in seeking to understand secretory granule events which occur in response to secretory stimuli in neuroendocrine cells such as the chromaffin cell. The responses to be investigated are secretion and gene expression (transcription). In Aim 1, we will investigate how catecholamine secretion and CgA gene transactivation responses desensitize following pre-ganglionic stimulation of chromaffin cells by PACAP and whether desensitization of these responses involves G protein-coupled receptor kinases (GRK), beta-arrestins and/or second messenger-activated kinases. In Aim 2, we will use the fluorescence of a novel CgA-EGFP chimera (Enhanced Green Fluorescent Protein) to determine in situ whether discrete CgA primary structural domains are responsible for the trafficking of CgA into the regulated pathway of secretion, and how CgA interacts with other secreted or membrane proteins of the secretory granule. Finally, we will use this chimeric CgA-EGFP protein as a calibrated H+ (pH) sensor (Aim 3) to investigate in situ, the dynamics of intravesicular pH and its role in the secretory process triggered by the physiologic secretagogues, such as PACAP or acetylcholine, or by sympathomimetic amines.

描述（改编自应用程序） 在交感肾上腺嗜铬细胞的分泌颗粒中，共同储存 儿茶酚胺和肽通过胞吐作用共同分泌 乙酰胆碱或新型非胆碱能神经递质垂体 腺苷酸环化酶激活多肽（PACAP）。嗜铬粒蛋白/ 分泌颗粒素 (Cg/Sg)，是核心中发现的酸性可溶性蛋白质家族 胺和肽激素和神经递质分泌囊泡，具有 这种囊泡在整个神经内分泌系统中几乎普遍存在 系统。它们的蛋白水解片段对激素或 神经递质释放以及多个位点的靶细胞。为了 例如，嗜铬粒蛋白 A (CgA) 片段 vasostatin 和 catestatin 对照 血管反应性和儿茶酚胺释放，以及 CgA 片段胰蛋白酶 升高血糖。该提案制定了 3 个具体目标 了解响应分泌而发生的分泌颗粒事件 神经内分泌细胞（例如嗜铬细胞）的刺激。对以下问题的回应 所研究的是分泌和基因表达（转录）。在目标 1 中，我们 将研究儿茶酚胺分泌和CgA基因反式激活如何 嗜铬细胞节前刺激后反应脱敏 通过 PACAP 以及这些反应的脱敏是否涉及 G 蛋白偶联受体激酶 (GRK)、β-抑制蛋白和/或第二 信使激活激酶。在目标 2 中，我们将使用小说的荧光 CgA-EGFP嵌合体（增强型绿色荧光蛋白）原位测定 离散的 CgA 一级结构域是否负责 CgA 转运到分泌调节途径，以及 CgA 如何 与分泌颗粒的其他分泌蛋白或膜蛋白相互作用。 最后，我们将使用这种嵌合 CgA-EGFP 蛋白作为校准的 H+ (pH) 传感器（目标 3）原位研究囊泡内 pH 值和 它在生理促分泌剂触发的分泌过程中的作用， 例如 PACAP 或乙酰胆碱，或拟交感胺。