SORTING OF PROTEINS IN REGULATED SECRETORY CELLS

调节分泌细胞中的蛋白质排序

• 批准号: 3287624
• 负责人: HSIAO-PING H MOORE
• 金额: $ 22.62万
• 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 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3287624
• 关键词: Golgi apparatus; adrenocorticotropic hormone; affinity chromatography; antibody formation; binding proteins; cell cell interaction; cell type; complementary DNA; endocrine gland /system; genetic manipulation; histochemistry /cytochemistry; hormone regulation /control mechanism; immunoelectron microscopy; immunofluorescence technique; immunoprecipitation; intracellular membranes; laboratory mouse; laboratory rabbit; membrane proteins; molecular biology; molecular cloning; monoclonal antibody; mutagens; neoplastic cell culture for noncancer research; nucleic acid hybridization; nucleic acid sequence; proinsulin; protein biosynthesis; protein transport; proteolysis; radioimmunoassay; secretion; somatotropin; vesicle /vacuole; virus protein

The general aim of this proposal is to understand how proteins are targeted to their correct intracellular compartments after synthesis. Specifically, we will use the pituitary cell line, AtT- 2O, to study molecular events underlying the assembly of secretory granules. Recent evidence suggests that neuroendocrine cells possess a constitutive and a regulated pathway for secretion, and that proteins destined for the cell surface are selectively sorted into different secretory vesicles for externalization. How proteins select the proper organelles, and how the machinery of these two types of secretory vesicles differs from each other are not understood. In this proposal, we will try to identify the sorting signals that direct peptide hormones into regulated granules by in vitro mutagenesis and DNA transfection. We will also purify the sorting compartment and characterize them biochemically. To elucidate the cellular sorting mechanisms, we will isolate specific sorting proteins from the sorting compartment and study their interactions with the transported proteins. We will ask if different sorting proteins are used by different types of cells with regulated secretory functions, or whether these cells share a common sorting machinery. To identify other components involved in sorting, we will set up an in vitro system to assay sorting in permeabilized cells. This system will be used to assess the role of clathrin in regulated secretion. Finally, we will devise systems to dissect functional components of the granule that are involved in the temporal and spatial regulation of secretory activities.

这项建议的总体目标是了解蛋白质是如何 靶向其正确的细胞内隔室后， 合成. 具体来说，我们将使用垂体细胞系AtT- 2 O，以研究分泌的组装的分子事件， 颗粒。 最近的证据表明神经内分泌细胞 具有组成性和调节性分泌途径，和 蛋白质会被选择性地分选到细胞表面 分泌到不同的分泌囊泡中进行外化。 如何 蛋白质选择适当的细胞器，以及如何机器的 这两种类型的分泌囊泡彼此不同， 不理解。在本建议中，我们将尝试确定 将信号分类，将肽激素引导到受调节的 颗粒通过体外诱变和DNA转染。 我们将 还净化分选室并表征它们 生物化学 为了阐明细胞分选机制，我们 将从分选室中分离出特定的分选蛋白 并研究它们与转运蛋白的相互作用。 我们 我会问不同的类型是否使用不同的分选蛋白 具有调节分泌功能的细胞，或者这些细胞 共用一台分拣机械。 识别其他组件 参与分选，我们将建立一个体外系统， 在透化细胞中分选。 该系统将用于评估 网格蛋白在调节分泌中的作用。 最后我们将 设计系统来剖析颗粒的功能成分， 参与分泌的时间和空间调节 活动