MEMBRANE FUNCTION IN PAROTID SECRETORY MECHANISMS

本申请中提出的研究的长期目标是 为唾液蛋白质分泌的机制提供了新的见解。 细胞内事件耦合了分泌刺激的输入和 分泌唾液蛋白的第二代信使是 很大程度上未知，但很可能是由分泌物的蛋白质介导的 颗粒膜和质膜。这些研究的重点是 某些多肽的结构和功能，这些多肽是 腮腺腺泡细胞分泌颗粒的膜。这些 多肽已经使用单抗进行了鉴定，它们是 首次报道为储藏颗粒膜的常见成分 在外分泌、内分泌和神经细胞中。它们的广泛分布表明 它们可能是蛋白质出口远端步骤中的共同点 因此对分泌过程可能是必不可少的。单克隆化 抗体将被用来表征分布和膜 免疫细胞化学和免疫化学对抗原的组织 并对腮腺膜多肽进行免疫纯化。 多肽的一级结构将由多肽推导出来。 腮腺cdna文库的测序和克隆。三种研究 计划朝着建立这些功能的主要目标 颗粒膜蛋白。首先，多肽的鉴定 具有相关序列和已知功能的计算机将搜索- 集中序列数据库的检索。第二，对 将在培养的细胞中检测分泌，在其中全长cDNA 编码这些蛋白质的基因已经表达。最后，克隆人 抗体，以及免疫纯化后产生的多克隆抗体 抗原，将作为蛋白质排出的预期扰动进行检查 使用一种无细胞模拟的腮腺分泌物颗粒胞吐。这些 研究可能会确定唾液蛋白的一些最终效应者 分泌物。因此，它们将引起其他人的极大兴趣 调查人员正试图澄清整合和 分泌刺激的传导，它们可能会带来新的洞察力 关于流体的大分子组成的调节 口腔期。

The long-term objective of the research proposed in this application is to provide new insights into the mechanisms of salivary protein secretion. The intracellular events that couple the input of secretory stimuli and generation of second messengers to the discharge of salivary proteins are largely unknown but are likely to be mediated by proteins of the secretion granule and plasma membranes. The focus of these studies is on the structure and function of certain polypeptides that are components of the membranes of the secretion granules of parotid acinar cells. These polypeptides have been identified using monoclonal antibodies and are among the first to be reported as common components of storage granule membranes in exocrine, endocrine and neural cells. Their broad distribution suggests that they may be common denominators in the distal steps of protein export and thus may be essential to the secretory process. The monoclonal antibodies will be used to characterize the distribution and membrane organization of the antigens by immunocytochemical and immunochemical techniques and to immunopurify the polypeptides from parotid membranes. The primary structures of the polypeptides will be deduced by peptide sequencing and cloning from a parotid cDNA library. Three kinds of studies are planned toward the major goal of establishing the function of these granule membrane proteins. First, the identification of polypeptides having related sequences and known functions will be sought by computer- searching of centralized sequence data banks. Second, the effects on secretion will be examined in cultured cells in which full-length cDNAs encoding the proteins have been expressed. Finally, the monoclonal antibodies, as well as polyclonal antibodies generated to immunopurified antigens, will be examined as prospective perturbants of protein discharge using a cell-free analog of parotid secretion granule exocytosis. These studies may define some of the final effectors of salivary protein secretion. As such, they will be of substantial interest to other investigators who are attempting to clarify the integration and transduction of secretory stimuli, and they may lead to new insight concerning the regulation of the macromolecular composition of the fluid phase of the oral cavity.