G PROTEINS AND PAROTID SECRETORY GRANULE

G蛋白和腮腺分泌颗粒

• 批准号: 6379756
• 负责人: EILEEN L WATSON
• 金额: $ 28.83万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6379756
• 关键词: ADP ribosylation; G protein; acinar cell; amylases; cell membrane; chloride channels; cytoplasm; electrical conductance; electron microscopy; exocrine glands; exocytosis; granule; immunoelectron microscopy; immunofluorescence technique; laboratory rat; light microscopy; membrane permeability; membrane transport proteins; parotid gland; phosphorylation; protein structure function; radiotracer; secretion; tissue /cell culture; western blottings

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.

拟议研究的总体目标是了解 GTP 结合蛋白在唾液外分泌细胞中的功能/作用。 从我们实验室获得的证据表明，异源三聚体 单体G蛋白位于大鼠腮腺腺泡细胞上 分泌颗粒以及质膜上。假设为 该提案测试的是 G 蛋白，位于分泌颗粒上 膜的功能是调节离子通道的门控和胞吐作用 在大鼠腮腺中。具体目标是： l) 异源三聚体和单体G的鉴定和定位 蛋白质。 G 蛋白将在各种细胞组分中被鉴定 包括质膜、细胞质膜和分泌颗粒膜 使用 [32P] ADP-核糖基化进行二维凝胶电泳， 放射性标记的 GTP 结合和特异性 G 蛋白的免疫印迹分析 抗血清。 G 蛋白在腮腺组织切片和 腺泡细胞将通过免疫组织化学/免疫细胞化学（光 和电子显微镜）。 2) 确定 G 蛋白对 Cl-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-核糖基化因子（ARF）、特异性G蛋白 抗血清和影响单体 G 蛋白释放/激活的药物 将被利用。 G蛋白调节的Cl-电导的调节 磷酸化/去磷酸化机制也将在 通过使用放射性标记分离颗粒和透化细胞 分别为[32P]ATP和[32P]正磷酸盐。 3) 检查G蛋白在调节分泌中的作用 （胞吐作用）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 透化细胞。 G蛋白从分泌状态的易位 颗粒到其他细胞区室将在腺泡细胞中确定 细胞刺激后；易位将由以下人员检查 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。获得的结果将与淀粉酶相关 发布。 拟议的研究应提供有关以下方面的重要信息： G 蛋白的身份及其功能的重要见解 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相关的细胞生化缺陷 唾液功能障碍。