BIOGENESIS OF EXOCRINE SECRETORY GRANULE MEMBRANES

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。 细胞生物学领域的中心兴趣是阐明 在细胞中工作的分子机制适当地分布 蛋白质到细胞器，如用于储存的分泌颗粒 荷尔蒙、酶、神经递质和其他重要分子 在适当的刺激下释放。外分泌分泌颗粒是 本质上是上皮的细胞的产物，并专门释放 它们在顶端质膜上的含量。的主要目标是 本项目旨在研究外分泌分泌的机制。 颗粒就形成了。我们选择了GP-2作为模型，主要的胰腺 腺泡颗粒膜蛋白，并克隆了编码该蛋白的基因。 在体内，这种蛋白质有一个生命周期，除了它的 掺入颗粒膜，它获得了一种 糖基磷酸肌醇(GPT)的连接及其向细胞表面的转运 其部分分泌物以可沉淀的形式存在。GP-2在表达时 从其cdna通过转染，主要定位在细胞表面。 在缺乏分泌颗粒的Hela细胞中。然而，在永久的 转化子的垂体ATT-20细胞，它的一部分进入小说 At-20垂体细胞中储存细胞器的类型。这些富含GP-2的 颗粒不同于内源性含ACTH的分泌 水泡。相比之下，尿调蛋白/Tamm Horsfall蛋白，一种肾尖部 与GP-2有88%同源性的膜蛋白不具有这一点 能力，只在ATT-20细胞的细胞表面发现 用编码它的cDNA腺病毒载体进行转染。利用同源性 在GP-2和THP之间，我们建议使用重组DNA技术来 定义GP-2的分子特征，解释其在体内的存储 ATT-20细胞。这将是隔离的前奏 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 靶向调控途径的机制并检查 不同组织来源的分泌细胞系支持 颗粒剂中GP-2的储存。此外，我们还将使用分子生物学 识别推测的GP-2/尿调蛋白其他成员的技术 一家人。预计这项研究将提供重要的 对分泌颗粒形成的洞察不仅仅是在外分泌 单元格，但也在其他 分泌细胞类型。

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.