TYROSINE PHOSPHORYLATION IN MUSCARINIC SIGNALLING

此应用程序中提出的研究的目的是测试 假设酪氨酸基团的磷酸化状态在 颌下腺腺泡细胞的蛋白质参与了 当M受体激动剂与 这些细胞膜上的胆碱能受体。为了测试 这一假说认为，细胞蛋白质中的酪氨酸磷酸含量 在静息和激活的细胞中被确定。磷酸酪氨酸含量 将使用免疫印迹和特异性抗体进行测量 磷酸酪氨酸残基和~(32)P标记的免疫沉淀 用相同的抗体提取细胞，然后变性 聚丙烯酰胺凝胶电泳法和放射自显影技术。两国关系 不同蛋白质的激动剂剂量和磷酸酪氨酸水平之间的关系 将被检查，以及将磷酸化和/或 去磷酸化事件。这些变化是不是 参与信号转导机制的问题将通过 将这些剂量反应和时间进程数据与其他 细胞功能的变化，包括肌醇的产生 三磷酸，从细胞内存储和通过 从介质进入，并通过依赖于钙离子的氯(-)开放 频道。酪氨酸激酶和酪氨酸磷酸酶活性的抑制剂 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.