PAROTID RAB3/RAB-GDI ISOFORMS--EXPRESSION/LOCALIZATION

本报告所述研究的长期目标是 建议是阐明Rab蛋白的作用，以及辅助 调节Rab蛋白功能的蛋白质，在调节的胞吐作用中 在腮腺里这项建议的具体目标是：1） 检测Rab 3和Rab-GDP的表达和定位 解离抑制剂（rab-GDI）同种型;和2）检查激动剂- 诱导这些亚型在大鼠腮腺的亚细胞再分布 腺。唾液腺执行许多重要的功能， 口腔和胃肠道环境，包括蛋白质分泌。 因此，了解调节唾液分泌的基本机制， 腺体分泌是必需的。尽管Rab蛋白参与了 囊泡运输和分泌的各个阶段，表达和 唾液腺中这些重要蛋白质的功能尚未被 研究了四种Rab 3同种型（A、B、C和D）和至少两种rab-GDI 已经在哺乳动物细胞中鉴定了同种型。Rab 3亚型 腮腺中的表达将通过RT-PCR使用Rab 3- 特异引物获得的PcR产物将被克隆， 测序以鉴定在该组织中表达的Rab 3同种型。Rab3 和rab-GDI表达也将通过北方和西方免疫组织化学方法检测。 使用同种型特异性DNA探针和抗体进行印迹， 分别该实验室的初步研究表明， 表达至少一种Rab 3同种型、Rab 3D和一种rab-GDI同种型， 在大鼠腮腺中。Rab 3和rab-GDI同种型的关联 在腮腺中以各种亚细胞组分表达， 包括分泌颗粒，将通过细胞 分级分离和Western印迹。精确亚细胞定位 这些蛋白质也将通过免疫荧光进行研究， 免疫金电镜刺激剂的作用 淀粉酶分泌对Rab 3和rab-GDI亚细胞定位的影响 通过定量Western印迹检测同种型。研究 在这个建议中所概述的应该指出Rab 3和Rab-GDI的作用 调节腮腺分泌的蛋白质。

The long-term objective of the studies described in the present proposal is to elucidate the role of Rab proteins, as well as accessory proteins which regulate Rab protein function, in regulated exocytosis in the parotid gland. The specific aims of this proposal are to: 1) examine the expression and localization of Rab3 and rab-GDP dissociation inhibitor (rab-GDI) isoforms; and 2) examine agonist- induced subcellular redistribution of these isoforms in rat parotid gland. The salivary glands perform many important functions that affect the oral and gastrointestinal environment including protein secretion. Hence, an understanding of the basic mechanisms that regulate salivary gland secretion is essential. Although Rab proteins are involved in various stages of vesicular transport and secretion, the expression and function of these important proteins in salivary glands have not been studied. Four Rab3 isoforms (A, B, C and D) and at least two rab-GDI isoforms have been identified in mammalian cells. Rab3 isoform expression in the parotid gland will be examined by RT-PCR using Rab3- specific primers. The PcR products obtained will be cloned and sequenced to identify the Rab3 isoforms expressed in this tissue. Rab3 and rab-GDI expression will also be examined by Northern and Western blotting using isoform-specific DNA probes and antibodies, respectively. Preliminary studies from this laboratory indicate that at least one Rab3 isoform, Rab3D, and one rab-GDI isoform are expressed in rat parotid gland. The association of Rab3 and rab-GDI isoforms expressed in the parotid gland with various subcellular fractions, including secretory granules, will be examined by cellular fractionation and Western blotting. Precise subcellular localization of these proteins will also be studied by immunofluorescence and immunogold electron microscopy. The effects of agents which stimulate amylase secretion on the subcellular localization of Rab3 and rab-GDI isoforms will be examined by quantitative Western blotting. The studies outlined in this proposal should indicate a role for Rab3 and rab-GDI proteins in mediating regulated secretion in the parotid gland.