CD45 REGULATION OF T CELL RECEPTOR SIGNAL TRANSDUCTION

CD45 对 T 细胞受体信号转导的调节

• 批准号: 3200529
• 负责人: GARY A. KORETZKY
• 金额: $ 6.83万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-05-01 至 1995-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3200529
• 关键词: CD antigens; T cell receptor; biological signal transduction; calcium flux; chimeric proteins; clone cells; electroporation; flow cytometry; human tissue; immunoprecipitation; insulin receptor; interleukin 2; laboratory mouse; laboratory rabbit; phosphoprotein phosphatase; phosphorylation; protein structure function; protein tyrosine kinase; site directed mutagenesis; transfection

T lymphocytes play an essential role in the body's defense against pathogens. Stimulation of T cells by specific antigens involves activation of the T cell antigen receptor complex (TCR) resulting in the generation of phosphatidylinositol (PI)-derived second messengers and the activation of a protein tyrosine kinase (PTK). Recent evidence from our laboratory and others has indicated that another T cell surface antigen, CD45, is important in T cell activation. The surface expression of CD45, a protein tyrosine phosphatase, appears to be critical in order for the TCR to couple with both the PI and PTK second messenger pathways. The overall goal of this proposal is to understand the mechanism by which CD45 regulates signal transduction via the TCR. To pursue this question, CD45-deficient mutants of the human T cell leukemic lines, HPB-ALL and Jurkat have been developed. The specific aims of this project are to study how the structure of CD45 may relate to its function as a tyrosine phosphatase and as a regulator of TCR mediated signal transduction. This will be accomplished by site- directed mutagenesis of murine CD45 and the creation of chimeric molecules before reconstitution by transfection into the CD45-deficient cells. The ability of the various mutants to restore TCR-mediated signal transduction will be evaluated. More detailed studies addressing how CD45 regulates TCR-mediated signal transduction are proposed focusing on the role of CD45 in regulating the activity of the src-like kinases, fyn and lck. Additionally, experiments are described which will attempt to identify physiological substrates of CD45 by co-immunoprecipitation in our T cell leukemic lines and normal human T lymphocytes. A genetic approach, utilizing mutagenesis and cell-sorting techniques to isolate pseudo- revertants, to attempt to identify potential CD45 substrates is also proposed. Finally, we propose to evaluate the role of CD45 in the regulation of non TCR-associated PTK's by examining the function of the insulin receptor in our CD45-positive, CD45-deficient, and CD45-transfected clones.

T淋巴细胞在机体防御中发挥重要作用 病原体。 特定抗原对 T 细胞的刺激涉及激活 T 细胞抗原受体复合物 (TCR) 的产生 磷脂酰肌醇 (PI) 衍生的第二信使及其激活 蛋白酪氨酸激酶（PTK）。 我们实验室的最新证据和 其他人指出另一种 T 细胞表面抗原 CD45 对 T 细胞激活很重要。 CD45（一种蛋白质）的表面表达 酪氨酸磷酸酶，对于 TCR 偶联似乎至关重要 PI 和 PTK 第二信使途径。 总体目标为 这个提议是为了了解CD45调节信号的机制 通过 TCR 进行转导。 为了解决这个问题，CD45缺陷突变体 已开发出人类 T 细胞白血病系 HPB-ALL 和 Jurkat。 该项目的具体目的是研究CD45的结构如何 可能与其作为酪氨酸磷酸酶和作为调节剂的功能有关 TCR 介导的信号转导。 这将通过站点来完成- 鼠 CD45 的定向诱变和嵌合分子的产生 在通过转染到 CD45 缺陷细胞中进行重建之前。 这 各种突变体恢复TCR介导的信号转导的能力 将被评估。 关于 CD45 如何调节的更详细的研究 提出 TCR 介导的信号转导，重点关注 CD45 的作用 调节 src 样激酶 fyn 和 lck 的活性。 此外，还描述了尝试识别的实验 通过 T 细胞中的免疫共沉淀研究 CD45 的生理底物 白血病细胞系和正常人 T 淋巴细胞。 遗传方法， 利用诱变和细胞分选技术来分离假 回复体，尝试识别潜在的 CD45 底物也是 建议的。 最后，我们建议评估 CD45 在 通过检查非 TCR 相关 PTK 的功能来调节 CD45 阳性、CD45 缺陷和 CD45 转染中的胰岛素受体 克隆。