TYROSINE PHOSPHORYLATION IN MUSCARINIC SIGNALLING

本申请中提出的研究目的是测试 这一假说认为，酪氨酸基团的磷酸化状态的变化， 下颌下腺腺泡细胞的蛋白质参与了 当毒蕈碱激动剂与 这些细胞膜上的胆碱能受体。 为了测试 根据这一假设，细胞蛋白质的酪氨酸磷酸含量将 在静止和激活的细胞中确定。 磷酸酪氨酸含量 将使用免疫印迹法和特异性抗体进行测量， 磷酸酪氨酸残基，并通过免疫沉淀的32 P-标记 细胞提取物与相同的抗体，然后变性 聚丙烯酰胺凝胶电泳和放射自显影。 的关系 激动剂剂量和磷酸酪氨酸水平之间的差异 将被检查，因为将磷酸化和/或磷酸化的时间过程， 去磷酸化事件。 这些变化是否 涉及的信号转导机制将解决的 将这些剂量反应和时间过程数据与其他剂量反应和时间过程数据进行比较。 细胞功能的变化，包括肌醇的产生 三磷酸盐，从细胞内储存和通过 进入，从介质中，并通过，Ca 2+依赖性开放的Cl（-） 渠道 酪氨酸激酶和酪氨酸磷酸酶活性抑制剂 也将被用来研究这些反应在信号中的作用 转导过程

The purpose of the research proposed in this application is to test the hypothesis that changes in the phosphorylation state of tyrosine groups in proteins of the submandibular gland acinar cells are involved in the biochemical cascade that occurs when muscarinic agonists interact with cholinergic receptors in the membranes of these cells. In order to test this hypothesis, the tyrosine phosphate content of cellular proteins will be determined in resting and activated cells. The phosphotyrosine content will be measured using both immunoblotting with an antibody specific for phosphotyrosine residues, and by immunoprecipitation of 32P-labelled cellular extracts with the same antibody, followed by denaturing polyacrylamide gel electrophoresis and autoradiography. The relationship between agonist dose and phosphotyrosine levels for the various proteins will be examined, as will the time course of the phosphorylation and/or dephosphorylation events. The question of whether these changes are involved in the signal transduction mechanism will be addressed by comparing these dose response and time course data to those of other changes in cell function, including the production of inositol trisphosphate, mobilization of Ca2+ from intracellular stores and through entry, from the medium, and by, the Ca2+- dependent opening of Cl(-) channels. Inhibitors of tyrosine kinase and tyrosine phosphatase activity will also be used to investigate the role of these reactions in the signal transduction process.