CELLULAR ROLE OF GRANINS IN HORMONE AND NEUROTRANSMITTER STORAGE AND R

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 嗜铬颗粒蛋白/分泌颗粒蛋白是一类广泛分布于神经内分泌、内分泌和神经细胞的致密核分泌颗粒(DCGS)的分泌蛋白家族，包括嗜铬颗粒蛋白A(CgA)、嗜铬颗粒蛋白B(CgB)和分泌颗粒蛋白II(SgII)。Granins作为激素前体，通过蛋白质分解过程产生具有生物活性的多肽片段。最近的证据表明，颗粒素可能是DCGS的生物发生和神经内分泌细胞中的激素隔离所必需的。使用一系列新的颗粒素嵌合光蛋白，我们的目标是提供颗粒素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。 本项目旨在研究CgA在交感肾上腺嗜铬细胞DCGS的形成和启动中的作用，并在该颗粒的一级结构中确定可能的粒系决定因素(S)。 我们以前报道过，在嗜铬细胞的调控途径中，CGA运输所需的信息包含在颗粒蛋白1的氨基末端区域(CgA1-115)。最近，我们已经证明，V-ATPase介导的TGN和/或未成熟嗜铬粒的酸化有助于CGA对调节的分泌途径2的分选和DCGS的生物发生，其机制可能是招募位于CgA1-115内的分选/粒形成决定因素，而不是蛋白质2，3的C-末端区域 我们将检验这一假设，即CGA可能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初级结构中的一个特定基序(顺式决定簇)对于这种颗粒发生作用是必不可少的。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一级结构中的粒细胞顺式决定簇(S)。在TIRF显微镜下研究的反应包括促分泌剂刺激瞬时转染的CGA嵌合体的胞吐作用。 这些研究将首先在交感肾上腺细胞变异体A35C中进行，它缺乏DCGS和调节的分泌途径，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。我们将把这些研究扩展到从CGA-/-KO小鼠分离的嗜铬细胞的原代培养，这些细胞显示DCGS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.