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

目前所述研究的长期目标 建议阐明Rab蛋白以及辅助蛋白的作用。 调节Rab蛋白功能的蛋白质，在调节胞吐中 在腮腺里。这项建议的具体目的是：1) 检测Rab3和Rab-GDP的表达和定位 解离抑制物(RAB-GDI)亚型；以及2)检测激动剂- 这些异构体在大鼠腮腺中的亚细胞再分布 腺体。唾液腺执行许多重要的功能，这些功能影响 口腔和胃肠道环境，包括蛋白质分泌。 因此，了解调节唾液的基本机制 腺体分泌是必不可少的。尽管Rab蛋白参与了 囊泡运输和分泌的不同阶段，表达和 这些重要蛋白质在唾液腺中的功能尚未得到证实。 学习。四种Rab3亚型(A、B、C和D)和至少两种Rab-GDI 已经在哺乳动物细胞中发现了异构体。Rab3亚型 将用RT-PCR方法检测腮腺中Rab3- 特定的引子。获得的聚合酶链式反应产物将被克隆和 测序鉴定该组织中表达的Rab3亚型。拉布3 Northern和Western还将检测Rab-GDI的表达 使用异构体特异的DNA探针和抗体进行印迹， 分别进行了分析。这个实验室的初步研究表明 表达至少一种Rab3亚型、Rab3D和一种Rab-GDI亚型 在大鼠腮腺中。Rab3与Rab-GDI异构体的关联 在腮腺以不同的亚细胞组分表达， 包括分泌颗粒，将通过细胞学检查 分级分离和Western blotting。精确的亚细胞定位 这些蛋白质中的一部分也将通过免疫荧光和 免疫金电子显微镜。刺激剂的作用 淀粉酶分泌对Rab3和Rab-GDI亚细胞定位的影响 异构体将通过定量Western blotting进行检测。这些研究 本建议书中概述的内容应指明Rab3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.