CELLULAR ROLE OF GRANINS IN HORMONE AND NEUROTRANSMITTER STORAGE AND R

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 嗜铬粒蛋白/分泌粒蛋白（粒蛋白）是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，并且包括嗜铬粒蛋白A（CgA）、嗜铬粒蛋白B（Cg B）和分泌粒蛋白II（Sg II）。颗粒蛋白作为激素原发挥作用，通过蛋白水解加工产生生物活性肽片段。最近的证据表明，颗粒蛋白可能需要DCG的生物合成，和神经内分泌细胞中的激素螯合。使用一系列新的颗粒蛋白嵌合发光蛋白，我们的目标是提供一个分子定义的颗粒蛋白在激素和神经递质的储存和释放交感神经系统内的细胞作用，肾上腺髓质。 该项目旨在描述CgA对交感肾上腺嗜铬细胞中DCG的启动和形成的贡献，并确定这种颗粒蛋白的一级结构中的推定颗粒形成决定簇。 我们以前报道过，在嗜铬细胞的调节途径内的CgA运输所需的信息包含在氨基末端区域（CgA 1 -115）的颗粒蛋白1。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CgA分选到受调节的分泌途径2，并通过一种机制促进DCG的生物发生，该机制可能募集位于CgA 1 -115内的分选/颗粒形成决定簇，但不募集蛋白质的C末端区域2，3 我们将测试的假设，CGA可能会拯救分泌细胞器缺陷的嗜铬细胞中的分泌颗粒的形成，和一个特定的主题（顺式决定簇）内的CGA的一级结构是必不可少的颗粒的作用。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一级结构内的颗粒形成顺式决定簇。要研究的反应包括促分泌素刺激的瞬时转染CgA嵌合体的胞吐作用，通过TIRF显微镜。 这些研究最初将在交感肾上腺细胞变体A35 C中进行，该变体缺乏DCG和受调节的分泌途径，并且不表达几种膜或可溶性分泌颗粒蛋白，包括CgA，CgB和SgII 4。我们将这些研究扩展到从CgA-/- KO小鼠分离的嗜铬细胞的原代培养，其显示DCG数量减少。

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.