BIOGENESIS OF EXOCRINE SECRETORY GRANULE MEMBRANES

真核细胞显示出高度的大分子组织，其中 特定的组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细胞的生物学特性。 在细胞中运作的分子机制， 蛋白质到细胞器，如分泌颗粒，用于储存 激素、酶、神经递质和其他重要分子， 在适当的刺激下释放。 外分泌颗粒是 本质上是上皮细胞的产物， 其内容物在顶端质膜。 的主要目标 本项目旨在研究外分泌的机制， 形成颗粒。 我们选择GP-2作为模型， 腺泡颗粒膜蛋白，并已分离出编码它的cDNA。 在体内，这种蛋白质具有生命周期，除了其 掺入到颗粒膜中，其获得 糖基-磷酸肌醇（GPT）连接，其运输到细胞表面 其部分分泌物呈可沉淀形式。 GP-2，表达时 从其cDNA通过转染，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 转化的垂体AtT-20细胞，它的一部分进入一个新的 AtT-20垂体细胞中的一种储存细胞器。 这些GP-2丰富的 颗粒与内源性含ACTH的分泌颗粒不同， 囊泡 相比之下，尿调蛋白/Tamm Horsfall蛋白，一种肾顶质， 与GP-2有88%同源性的膜蛋白，不具有这一点。 在AtT-20细胞中仅在细胞表面发现 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，这些分子特征是其储存在 AtT-20细胞。 这将成为隔离的前奏。 负责GP-2分选的细胞组分。 我们还将 确定分泌蛋白和膜蛋白是否共享相同的分子 针对调节途径的机制，并检查 不同组织来源的分泌细胞系支持 GP-2在颗粒中的储存。 此外，我们将使用分子生物学 鉴定推定的GP-2/尿调节蛋白的其他成员的技术 家人 预计这项研究将提供重要 不仅在外分泌中， 细胞，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.