MECHANISMS FOR SORTING OF CHROMOGRANIN A

嗜铬蛋白 A 的分选机制

• 批准号: 6142443
• 负责人: SVEN-ULRIK GORR
• 金额: $ 1.16万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-06-01 至 2000-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6142443
• 关键词: calcium ion; chemical aggregate; chimeric proteins; chromogranins; crosslink; intracellular transport; membrane activity; membrane structure; molecular cloning; neuroendocrine system; nucleic acid sequence; peptide hormone; posttranslational modifications; protein binding; protein sequence; protein structure; protein structure function; protein transport; secretion; secretory protein; site directed mutagenesis; tissue /cell culture

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.

内分泌和神经内分泌细胞储存肽激素和神经肽 在调节的分泌途径中的分泌颗粒中，同时 组成型分泌蛋白直接分泌，无需 细胞内储存。将肽前体分类为受监管的 分泌途径对于生物活性的刺激释放至关重要 肽，但分选机制仍然很大程度上未知。钙和 低 pH 诱导的聚集和膜结合已被认为发挥了作用 在不同调节分泌蛋白的分类中发挥作用。然而， 它们对排序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。嗜铬素A (CgA) 与肽激素共同储存在大多数分泌颗粒中 内分泌细胞和神经内分泌细胞。我们最近发现了不同的 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来确定 膜结合和聚集对分选的相对贡献 单个蛋白质。基于这些观察，我们建议单独 膜结合和聚集位点充当冗余分选域 用于将 CgA 分类到内分泌细胞的调节分泌途径。 为了检验这一假设，提出以下具体目标： 1. 确定 CgA 的 N 末端结构域是否在膜结合中发挥作用 并分选至受调节的分泌途径； 2. 确定哪一部分 CgA的C端结构域起着聚集和排序的作用 神经内分泌细胞中受调节的分泌途径。为了解决这些 问题，我们将删除假定的膜结合和聚集 域并确定对排序、膜结合和 CgA 的聚集。将构建转移嵌合体来测试这些是否 结构域足以进行排序、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 生物活性肽进入循环。