CELLULAR ROLE OF GRANINS IN HORMONE AND NEUROTRANSMITTER STORAGE AND R

谷粒蛋白在激素和神经递质储存和 R 中的细胞作用

基本信息

批准号：
7957615
负责人：
LAURENT TAUPENOT
金额：
$ 0.78万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN DIEGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-06-15 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7957615
关键词：
Adrenal Medulla Biogenesis C-terminal Cells Chimera organism Chromaffin Cells Chromaffin granule Chromogranin A Chromogranins Computer Retrieval of Information on Scientific Projects Database Endocrine Exocytosis Family Funding Grant Hormones Image Analysis Institution Luminescent Proteins Mediating Membrane Molecular Mus Neuroendocrine Cell Neurons Neurosecretory Systems Neurotransmitters Organelles PHluorin Pathway interactions Peptide Fragments Protein Region Proteins Proteolytic Processing Recruitment Activity Reporting Research Research Personnel Resources Role Secretory Vesicles Series Sorting - Cell Movement Source Structure Sympathetic Nervous System Testing Total Internal Reflection Fluorescent United States National Institutes of Health Variant chromogranin B chromogranin C novel response secretogranins secretory protein trafficking vacuolar H+-ATPase

项目摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The chromogranins/secretogranins (granins) are a family of regulated secretory proteins widely distributed in dense-core secretory granules (DCGs) of neuroendocrine, endocrine, and neuronal cells, and includes chromogranin A (CgA), chromogranin B (CgB), and secretogranin II (SgII). Granins function as pro-hormones, giving rise by proteolytic processing to biologically active peptide fragments. Recent evidence suggests that granins may be required for the biogenesis of DCGs, and hormone sequestration in neuroendocrine cells. Using a series of novel granin chimeric photoproteins, we aim to provide a molecular definition of the cellular role of granins in hormone and neurotransmitter storage and release within the sympathetic nervous system, and the adrenal medulla. This project aims to characterize the contribution of CgA to the initiation and formation of DCGs in sympathoadrenal chromaffin cells, and to identify putative granulogenic determinant(s) within the primary structure of this granin. We previously reported that information necessary for CgA trafficking within the regulated pathway of chromaffin cells is contained in the amino-terminal region (CgA1-115) of the granin1. More recently, we have shown that a V-ATPase-mediated acidification of the TGN and/or the immature chromaffin granule contributes to the sorting of CgA to the regulated secretory pathway2 and to the biogenesis of DCGs by a mechanism that may recruit a sorting/granulogenic determinant located within CgA1-115, but not the C-terminal region of the protein 2, 3 We will test the hypothesis that CgA may rescue the formation of secretory granules in secretory organelle-deficient chromaffin cells, and a particular motif (cis determinant) within the primary structure of CgA is essential for such granulogenic role. We propose to express a series of CgA domains fused to EGFP or pHluorin in secretory organelle-deficient chromaffin cells, to investigate and characterize granulogenic cis determinant(s) within the primary structure of CgA. Responses to be investigated include secretagogue-stimulated exocytosis of transiently transfected CgA chimera by TIRF microscopy. The studies will be initially conducted in the sympathoadrenal cell variant A35C, which lacks DCGs and a regulated secretory pathway, and does not express several membrane or soluble secretory granule proteins, including CgA, CgB, and SgII 4. We will extend these studies to primary culture of chromaffin cells isolated from CgA-/- KO mice, which show reduced DCGs number.

该子项目是利用该技术的众多研究子项目之一资源由 NIH/NCRR 资助的中心拨款提供。子项目及研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金，因此可以在其他 CRISP 条目中表示。列出的机构是对于中心来说，它不一定是研究者的机构。嗜铬粒蛋白/分泌粒蛋白（颗粒）是一类广泛分布于神经内分泌、内分泌和神经元细胞的致密核心分泌颗粒（DCG）中的调节分泌蛋白家族，包括嗜铬粒蛋白A（CgA）、嗜铬粒蛋白B（CgB）和分泌粒蛋白II (SgII)。颗粒蛋白起到激素原的作用，通过蛋白水解加工产生具有生物活性的肽片段。最近的证据表明，颗粒蛋白可能是 DCG 的生物发生和神经内分泌细胞中激素的隔离所必需的。使用一系列新型颗粒嵌合发光蛋白，我们的目标是提供颗粒在交感神经系统和肾上腺髓质内激素和神经递质储存和释放中的细胞作用的分子定义。该项目旨在表征 CgA 对交感肾上腺嗜铬细胞中 DCG 的启动和形成的贡献，并鉴定该粒蛋白一级结构内推定的粒形成决定簇。我们之前报道过，在嗜铬细胞调控途径中 CgA 运输所需的信息包含在 granin1 的氨基末端区域 (CgA1-115) 中。最近，我们发现 V-ATP 酶介导的 TGN 和/或未成熟嗜铬颗粒的酸化有助于将 CgA 分选至受调节的分泌途径2，并通过可能招募分选/的机制促进 DCG 的生物发生。颗粒形成决定簇位于 CgA1-115 内，但不位于蛋白质 2、3 的 C 末端区域我们将检验以下假设：CgA 可以挽救分泌细胞器缺陷的嗜铬细胞中分泌颗粒的形成，并且 CgA 一级结构内的特定基序（顺式决定簇）对于这种颗粒形成作用至关重要。我们建议在分泌细胞器缺陷的嗜铬细胞中表达一系列与 EGFP 或 pHluorin 融合的 CgA 结构域，以研究和表征 CgA 一级结构内的颗粒生成顺式决定簇。待研究的反应包括通过 TIRF 显微镜观察促分泌素刺激的瞬时转染 CgA 嵌合体的胞吐作用。这些研究最初将在交感肾上腺细胞变体 A35C 中进行，该细胞缺乏 DCG 和受调节的分泌途径，并且不表达多种膜或可溶性分泌颗粒蛋白，包括 CgA、CgB 和 SgII 4。我们将把这些研究扩展到初级细胞从 CgA-/- KO 小鼠中分离的嗜铬细胞培养物，显示 DCG 数量减少。