SORTING OF PROTEINS IN REGULATED SECRETORY CELLS

调节分泌细胞中的蛋白质排序

• 批准号: 3287621
• 负责人: HSIAO-PING H MOORE
• 金额: $ 10.52万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287621
• 关键词: Golgi apparatus; adrenocorticotropic hormone; affinity chromatography; antibody formation; cell cell interaction; cell type; complementary DNA; genetic manipulation; histochemistry /cytochemistry; hormone regulation /control mechanism; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; intracellular membranes; molecular biology; molecular cloning; monoclonal antibody; mutagens; neoplastic cell culture for noncancer research; nucleic acid sequence; proinsulin; proteolysis; radioimmunoassay; secretion; somatotropin; vesicle /vacuole

The general aim of this proposal is to understand how proteins are targeted to their correct intracellular compartment after synthesis. Specifically, the tumor cell line, AtT-20, derived from the mouse anterior pituitary, will be used to study assembly of the secretory granules which are involved in regulated secretion of peptide hormones. Recent evidence suggests that cells with regulated secretory function (i.e. neural, endocrine and exocrine cells) sort their secretory and plasma membrane proteins into distinct secretory pathways for externalization. Thus these cells have a regulated pathway in which specialized secretory products are stored in dense core secretory granules. Release of these products is regulated by secretagogues. In contrast, the cells utilize a second, constitutive, pathway for transporting other plasma membrane and secreted proteins to the cell surface. Externalization from this pathway is not regulated, and does not involve storage in the secretory granules. It is the aim of this proposal to understand how proteins are sorted in these cells such that only a subset of proteins are transported to the secretory granules. First, sorting signals will be sought on proteins targeted to the regulated pathway. This will be achieved by in vitro mutagenesis of DNA sequences encoding secretory granule proteins, followed by expressing the altered DNAs in AtT-20 cells to examine their effects on targeting. We will seek to determine if a common sorting signal is shared by secretory granule proteins from the different cell types which are capable of regulated secretion. Secondly, the temporal and the spatial relationships of sorting to other posttranslational processes will be examined by DNA transfection and immunoelectron microscopy. Finally, we will attempt to identify the cellular machinery involved in sorting by affinity chromatography and monoclonal antibody techniques.

该建议的一般目的是了解蛋白质的目标 合成后它们正确的细胞内室。 具体来说， 肿瘤细胞系ATT-20，源自小鼠前垂体， 将用于研究所涉及的分泌颗粒的组装 在受调节的肽激素分泌中。 最近的证据表明 具有调节分泌功能的细胞（即神经，内分泌和 外分泌细胞）将其分泌和质膜蛋白分类为 外部化的独特分泌途径。 因此，这些细胞具有 专门分泌产品存储在中的规范途径中 密集的核心分泌颗粒。 这些产品的释放受 秘密。 相比之下，细胞利用第二个本构， 将其他质膜和分泌蛋白运输到的途径 细胞表面。 该途径的外部化不受调节，并且确实 不涉及分泌颗粒中的存储。 这是这个目的 建议了解如何在这些细胞中分类蛋白质，以便 只有一部分蛋白质被运输到分泌颗粒。 首先，将寻求针对受监管的蛋白质的分类信号 路径。 这将通过DNA序列的体外诱变来实现 编码分泌颗粒蛋白，然后表达改变 DNA中的ATT-20细胞中的DNA检查其对靶向的影响。 我们将寻求 确定分泌颗粒是否共享一个常见的排序信号 来自不同细胞类型的蛋白质，能够调节 分泌。 其次，分类的时间和空间关系 DNA转染将检查其他翻译后过程 和免疫电子显微镜。 最后，我们将尝试确定 通过亲和力色谱和分类涉及的细胞机械 单克隆抗体技术。