BIOGENESIS OF EXOCRINE SECRETORY GRANULE MEMBRANES

外分泌分泌颗粒膜的生物发生

• 批准号: 3245775
• 负责人: MICHAEL J. RINDLER
• 金额: $ 19.96万
• 依托单位: NEW YORK UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1994-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3245775
• 关键词: adrenal glands; animal tissue; brain cell; cell type; exocytosis; fusion gene; genetic recombination; granule; immunocytochemistry; laboratory rabbit; membrane proteins; neurotransmitters; pancreas; protein biosynthesis; protein transport; site directed mutagenesis; transfection

Eukaryotic cells show a high degree of macromolecular organization in which specific components are selectively segregated into specialized organelles. Of central interest in the field of cell biology is the elucidation of the molecular mechanisms operating in the cell to appropriately distribute proteins to organelles such as the secretory granules that serve to store hormones, enzymes, neurotransmitters, and other important molecules for release upon appropriate stimulus. Exocrine secretory granules are the product of cells that are epithelial in nature and specifically release their contents at the apical plasma membrane. The principal objective of this project is to study the mechanisms by which exocrine secretory granules are formed. We have chosen as a model GP-2, the major pancreatic acinar granule membrane protein, and have isolated the cDNA encoding it. In vivo, this protein has a life cycle which includes, in addition to its incorporation into the granule membrane, its acquisition of a glycosyl-phosphoinositol (GPT) linkage, its transport to the cell surface and its partial secretion in a sedimentable form. GP-2, when expressed from its cDNA by transfection, is primarily localized on the cell surface in Hela cells, which lack secretory granules. However, in permanent transformants of pituitary AtT-20 cells, a portion of it enters a novel type of storage organelle in AtT-20 pituitary cells. These GP-2-rich granules are distinct from the endogenous ACTH-containing secretory vesicles. By contrast, uromodulin/Tamm Horsfall protein, a renal apical membrane protein that is 88% homologous to GP-2, does not share this ability and is found exclusively on the cell surface in AtT-20 cells transfected with the cDNA encoding it. Taking advantage of the homology between GP-2 and THP, we propose to use recombinant DNA techniques to define the molecular features of GP-2 that are account for its storage in AtT-20 cells. This will serve as a prelude to the isolation of the cellular components responsible for the sorting of GP-2. We will also determine whether secretory and membrane proteins share the same molecular mechanisms for targetting to the regulated pathway and examine the capability of secretory cell lines of diverse tissue origin to support the storage of GP-2 in granules. In addition, we will use molecular biological techniques to identify other members of the putative GP-2/uromodulin family. It is anticipated that this research will provide important insights into the formation of secretory granules not only in exocrine cells but also in other secretory cell types.

真核细胞表现出高度的大分子组织，其中 特定的成分被选择性地分离到专门的细胞器中。 细胞生物学领域的核心兴趣是阐明 细胞内运作的分子机制以适当分布 蛋白质到细胞器，例如用于储存的分泌颗粒 激素、酶、神经递质和其他重要分子 适当刺激后释放。 外分泌颗粒是 本质上是上皮细胞的产物，并且专门释放 它们在顶端质膜上的内容物。 主要目标 该项目旨在研究外分泌分泌的机制 形成颗粒。 我们选择 GP-2 作为模型，主要的胰腺 腺泡颗粒膜蛋白，并分离出编码它的cDNA。 在体内，这种蛋白质有一个生命周期，除了它的 结合到颗粒膜中，它获得了 糖基-磷酸肌醇 (GPT) 连接，其转运至细胞表面 及其以可沉淀形式的部分分泌。 GP-2，当表达时 通过转染从其 cDNA 中提取，主要定位于细胞表面 在缺乏分泌颗粒的 Hela 细胞中。 然而，在永久 垂体AtT-20细胞的转化体，其一部分进入新的 AtT-20 垂体细胞中的储存细胞器类型。 这些富含GP-2的 颗粒与内源性含 ACTH 的分泌物不同 囊泡。 相比之下，尿调节蛋白/Tamm Horsfall 蛋白（一种肾尖 与 GP-2 88% 同源的膜蛋白，不具有这一点 能力，仅存在于 AtT-20 细胞的细胞表面 用编码它的cDNA转染。 利用同源性 在 GP-2 和 THP 之间，我们建议使用重组 DNA 技术 定义 GP-2 的分子特征，这些特征决定了其存储在 AtT-20 细胞。 这将成为隔离的前奏 负责 GP-2 分选的细胞成分。 我们还将 确定分泌蛋白和膜蛋白是否具有相同的分子 靶向调节途径的机制并检查 不同组织来源的分泌细胞系的能力，以支持 GP-2以颗粒形式储存。 此外，我们将利用分子生物学 鉴定假定的 GP-2/尿调节素其他成员的技术 家庭。 预计这项研究将提供重要的 不仅在外分泌中，还深入了解分泌颗粒的形成 细胞也存在于其他细胞中 分泌细胞类型。