MECHANISMS FOR SORTING OF CHROMOGRANIN A

内分泌和神经内分泌细胞储存多肽、激素和神经肽 在调节分泌途径的分泌颗粒中 构成分泌蛋白是直接分泌的，不需要 细胞内存储。受调控的多肽前体的分选 分泌途径是生物活性物质被刺激释放的关键 多肽，但分选机制在很大程度上仍不清楚。钙和 低pH诱导的聚集和膜结合被认为起到了作用 在不同调节的分泌蛋白的分类中起作用。然而， 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 用于分类受调节的分泌蛋白的聚集。嗜铬麦角菌素A (CGA)与多肽激素共同储存在大多数 内分泌和神经内分泌细胞。我们最近发现了不同的 CGA中可能发挥膜结合和聚集功能的结构域， 分别进行了分析。因此，CGA提供了一个独特的机会来确定 膜结合和聚集对分选的相对贡献 一种单独的蛋白质。基于这些观察，我们建议分开 膜结合和聚集区作为冗余的排序结构域 用于将CGA分选到内分泌细胞调节的分泌途径。 为了验证这一假说，本文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 并对调节的分泌途径进行分选；2.确定 CGA的C-末端结构域在聚集和分选中起作用 神经内分泌细胞的调节分泌途径。要解决这些问题 问题，我们将删除推定的膜结合和聚集 结构域，并确定对分类、膜结合和 CGA的聚集。将构建转移嵌合体来测试这些 结构域足以进行分类、膜结合和聚集 内分泌细胞。拟议研究将提供一个框架，以 了解内分泌细胞如何传递的生理和病理 生物活性多肽进入血液循环。

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.