SORTING OF PROTEINS IN REGULATED SECRETORY CELLS

调节分泌细胞中的蛋白质排序

• 批准号: 3287620
• 负责人: HSIAO-PING H MOORE
• 金额: $ 9.66万
• 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/3287620
• 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.

这项提议的总体目标是了解蛋白质是如何被靶向的。 在合成后到达正确的细胞内隔间。具体来说， 该肿瘤细胞系ATT-20来源于小鼠垂体前叶， 将用于研究所涉及的分泌颗粒的组装 调节多肽荷尔蒙的分泌。最近的证据表明 具有受调节的分泌功能的细胞(即神经、内分泌和 外分泌细胞)将其分泌和质膜蛋白分类为 不同的外化分泌途径。因此，这些细胞具有 专门化分泌产物储存的调控途径 致密的核心分泌颗粒。这些产品的发布受以下方面的监管 分泌者。相反，细胞利用第二个，构成的， 将其他质膜和分泌的蛋白质运输到 细胞表面。这条途径的外化不受调控，而且 不涉及储存在分泌颗粒中。这就是我们的目的 关于了解蛋白质在这些细胞中是如何分类的建议 只有一小部分蛋白质被输送到分泌颗粒。 首先，将寻找针对受调控分子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，然后表达改变的 检测它们对靶向的影响。我们将寻求 以确定共同的分选信号是否由分泌颗粒共享 来自不同细胞类型的能够调节的蛋白质 分泌物。第二，分类的时间关系和空间关系 对其他翻译后过程的影响将通过DNA转染进行检测 和免疫电子显微镜。最后，我们将尝试确定 参与亲和层析分选的细胞机械和 单抗技术。