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.

这项提案的总体目标是了解蛋白质是如何靶向的 在合成后回到正确的细胞内区室 具体地说， 肿瘤细胞系AtT-20，来源于小鼠垂体前叶， 将被用来研究组装的分泌颗粒， 调节肽类激素的分泌。 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）将它们的分泌蛋白和质膜蛋白分类为 不同的分泌途径外化。 因此，这些细胞具有 调节途径，其中专门的分泌产物储存在 致密的核心分泌颗粒。 这些产品的放行受以下法规的监管： 促分泌素 相比之下，细胞利用第二种组成性的， 转运其他质膜和分泌蛋白质的途径 细胞表面 这一途径的外化不受调节， 不涉及在分泌颗粒中的储存。 这就是目的 了解蛋白质如何在这些细胞中分类的建议， 只有一部分蛋白质被转运到分泌颗粒。 首先，分选信号将在靶向受调控的蛋白质上寻找。 通路 这将通过DNA序列的体外诱变来实现 编码分泌颗粒蛋白，然后表达改变的 AtT-20细胞中的DNA，以检查它们对靶向的影响。 我们将寻求 以确定分泌颗粒是否共享共同的分选信号 来自不同细胞类型的蛋白质， 分泌物 其次，时间和空间关系的排序 其他翻译后过程将通过DNA转染进行检查 和免疫电镜。 最后，我们将尝试识别 参与亲和层析分选的细胞机制， 单克隆抗体技术。