G PROTEINS AND PAROTID SECRETORY GRANULE

拟议研究的总体目标是了解 唾液外分泌细胞中GTP结合蛋白的功能/作用 从我们实验室获得的证据表明，异三聚体 单体G蛋白定位于大鼠腮腺腺泡细胞 分泌颗粒和质膜上也是如此。假设是 在这个方案中测试的是位于分泌颗粒上的G蛋白 膜的功能是调节离子通道的门控和胞吐。 在大鼠腮腺中。具体目标是： L)异三聚体和单体G的鉴定和定位 蛋白质。G蛋白将在不同的细胞组分中被鉴定 包括质膜、胞浆和分泌颗粒膜。 [32P]ADP-核糖化双向凝胶电泳法， 放射性标记GTP与特异性G蛋白的结合及免疫印迹分析 抗血清。G蛋白在腮腺组织切片和腮腺组织中的定位 腺泡细胞将通过免疫组织化学/免疫细胞化学(LIGH 和电子显微镜)。 2)确定G蛋白对氯离子电导的作用 分离的分泌颗粒。对于这些研究，GTP、GTP类似物、A1F4、 细菌毒素，ADP-核糖化因子(ARF)，特异性G蛋白 抗血清和影响单体G蛋白释放/激活的试剂 将会被利用。G蛋白对氯离子电导的调节作用 磷酸化/去磷酸化机制也将在 用放射性标记技术分离颗粒和通透性细胞 [32P]ATP和[32P]正磷酸盐。 3)检测G蛋白在调节分泌中的作用 (胞吐)来自腮腺腺泡细胞。GTP和GTP类似物的作用 在分泌的后期，即淀粉酶的释放将在 通透性细胞。分泌物中G蛋白的易位 其他细胞隔间的颗粒将在腺泡细胞中确定。 在细胞刺激后；移位将通过 免疫荧光/免疫电子显微镜、免疫印迹分析和 放射性标记的GTP结合。所获得的结果将与淀粉酶相关联 放手。 拟议的研究应提供有关 G蛋白的特性以及对其功能的重大洞察 在唾液外分泌细胞中。这些研究还将提供一个框架 用于检查与以下疾病有关的细胞生化缺陷 唾液功能障碍。

The overall goal of the proposed research is to understand the function/role of GTP-binding proteins in salivary exocrine cells. Evidence obtained from Our laboratory suggests that both heterotrimeric and monomeric G proteins are located on the rat parotid acinar cell secretory granule as well as on the plasma membranes. The hypothesis to be tested in this proposal is that G proteins, located on secretory granule membranes function to modulate the gating of ion channels and exocytosis in the rat parotid gland. Specific aims are: l) Identification and localization of heterotrimeric and monomeric G proteins. G proteins will be identified in various cellular fractions including the plasma membrane, cytosol and secretory granule membranes by two-dimensional gel electrophoresis using [32P] ADP-ribosylation, radiolabeled GTP binding and immunoblot analysis with specific G protein antisera. Localization of G proteins in parotid tissue sections and in acinar cells will be by immunohistochemistry/immunocytochemistry (light and electron microscopy). 2) Determination of a role for G proteins on the Cl- conductance of isolated secretory granules. For these studies GTP, GTP analogs, A1F4, bacterial toxins, ADP-ribosylating factor (ARF), specific G protein antisera, and agents that affect monomeric G protein release/activation will be utilized. Modulation of G protein-regulated Cl- conductance by phosphorylation/dephosphorylation mechanisms will also be examined in isolated granules and in permeabilized cells by using radiolabeled [32P]ATP and [32P]orthophosphate, respectively. 3) Examination of the role of G proteins in regulating secretion (exocytosis) from parotid acinar cells. The effects of GTP and GTP analogs on the late stage of secretion, i.e. amylase release will be determined in permeabilized cells. The translocation of G proteins from secretory granules to other cellular compartments will be determined in acinar cells following cell stimulation; translocation will be examined by immunofluorescence/immunoelectron microscopy, immunoblot analysis and radiolabeled GTP binding. Results obtained will be correlated with amylase release. The proposed research should provide significant information on the identity of G proteins as well as significant insight as to their function in salivary exocrine cells. These studies will also provide a framework for examining cellular biochemical defects in diseases relating to salivary dysfunction.