MEMBRANE FUNCTION IN PAROTID SECRETORY MECHANISMS

本申请中提出的研究的长期目标是 为唾液蛋白分泌机制提供了新的见解。 细胞内事件耦合分泌刺激的输入， 第二信使的产生和唾液蛋白的分泌 很大程度上是未知的，但可能是由分泌的蛋白质介导的。 颗粒和质膜。 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 已经使用单克隆抗体鉴定了多肽， 第一个被报道为储存颗粒膜的常见组分 在外分泌、内分泌和神经细胞中。 它们的广泛分布表明 它们可能是蛋白质输出的远端步骤中的常见调节剂， 因此可能是分泌过程所必需的。 单克隆 抗体将被用来表征分布和膜 通过免疫细胞化学和免疫化学的抗原组织 技术，并从腮腺膜免疫纯化多肽。 多肽的一级结构将由肽推导出 从腮腺cDNA文库测序和克隆。 三种研究 计划实现建立这些功能的主要目标 颗粒膜蛋白 一、多肽的鉴定 具有相关的序列和已知的功能将通过计算机来寻找- 搜索集中的序列数据库。 第二，影响 将在培养的细胞中检查分泌，其中全长cDNA 编码这些蛋白质的基因已经被表达出来。 最后，单克隆 抗体，以及产生的多克隆抗体， 抗原，将作为蛋白质排放的预期扰动物进行检查 使用腮腺分泌颗粒胞吐作用的无细胞类似物。 这些 研究可以确定唾液蛋白的某些最终效应物 分泌物 因此，他们将对其他人产生重大兴趣。 试图澄清整合的调查人员， 转导的分泌刺激，他们可能会导致新的见解 关于流体的大分子组成的调节 口腔的阶段。

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.