MECHANISMS FOR SORTING OF CHROMOGRANIN A

内分泌和神经内分泌细胞储存肽类激素和神经肽 在调节分泌途径的分泌颗粒中， 组成型分泌蛋白直接分泌， 细胞内储存将肽前体分选到受调节的 分泌途径对于生物活性物质的刺激释放至关重要 肽，但分选机制仍然在很大程度上未知。钙和 低pH诱导的聚集和膜结合被认为是 在不同调节分泌蛋白的分选中的作用。然而，在这方面， 它们对分类的相对贡献尚不清楚。长期目标 这项研究的目的是确定膜结合和蛋白质的作用， 用于分选调节分泌蛋白的聚集。染色粒素A (CgA)与肽激素共同储存在大多数 内分泌和神经内分泌细胞。我们最近发现了 CgA中可能在膜结合和聚集中起作用的结构域， 分别因此，CgA提供了一个独特的机会，以确定 膜结合和聚集对分选的相对贡献 单个蛋白质。根据这些观察，我们建议， 膜结合和聚集位点作为冗余的分选结构域 用于将CgA分选到内分泌细胞的调节分泌途径。 为了验证这一假设，提出了以下具体目标：1。 确定CgA的N-末端结构域是否在膜结合中起作用 并分选至受调节的分泌途径; 2.确定哪一部分 CgA的C-末端结构域在聚集和分选中起作用， 神经内分泌细胞中受调节的分泌途径。解决这些 问题，我们将删除假定的膜结合和聚集 结构域，并确定对分选，膜结合和 CgA的聚集。将构建转移嵌合体，以测试这些 结构域足以进行分选、膜结合和聚集， 内分泌细胞拟议的研究将提供一个框架， 了解内分泌细胞如何传递的生理学和病理学 生物活性肽的循环。

Endocrine and neuroendocrine cells store peptide hormone and neuropeptides in secretory granules in the regulated secretory pathway while constitutive secretory proteins are directly secreted without intracellular storage. Sorting of the peptide precursors to the regulated secretory pathway is critical for the stimulated release of bioactive peptides but the sorting mechanisms remain largely unknown. Calcium-and low pH-induced aggregation and membrane binding have been proposed to play a role in the sorting of different regulated secretory proteins. However, their relative contributions to sorting is not clear. The long term goal of this research is to determine the roles of membrane binding and protein aggregation for sorting of regulated secretory proteins. Chromagranin A (CgA) is co-stored with peptide hormones in secretory granules of most endocrine and neuroendocrine cells. We have recently identified distinct domains in CgA that may function in membrane binding and aggregation, respectively. Thus, CgA offer a unique opportunity to determine the relative contributions of membrane binding and aggregation to sorting of an individual protein. Based on these observation we propose that separate membrane binding and aggregation sites act as redundant sorting domains for sorting of CgA to the regulated secretory pathways of endocrine cells. To test this hypothesis, the following specific aims are proposed: 1. Determine if the N-terminal domain of CgA plays a role in membrane binding and sorting to the regulated secretory pathway; 2. Determine which part of the C-terminal domain of CgA plays a role in aggregation and sorting to the regulated secretory pathway in neuroendocrine cells. To address these questions, we will delete the putative membrane-binding and aggregation domains and determine the effect on sorting, membrane-binding and aggregation of CgA. Transfer chimeras will be constructed to test if these domains are sufficient for sorting, membrane binding and aggregation in endocrine cells. The proposed studies will provide a framework for understanding the physiology and pathology of how endocrine cells deliver bioactive peptides to the circulation.