CD45 REGULATION OF T CELL RECEPTOR SIGNAL TRANSDUCTION

T淋巴细胞在机体防御 病原体 特异性抗原对T细胞的刺激涉及活化 T细胞抗原受体复合物（TCR）的产生， 磷脂酰肌醇（PI）衍生的第二信使和激活 蛋白酪氨酸激酶（PTK）。 我们实验室的最新证据， 其他人指出，另一种T细胞表面抗原CD 45， 对T细胞活化很重要。 CD 45蛋白的表面表达 酪氨酸磷酸酶，似乎是至关重要的，以使TCR偶联 与PI和PTK第二信使途径。 的总目标 这个建议是为了了解CD 45调节信号的机制， 通过TCR进行转导。 为了探讨这个问题，CD 45缺陷突变体， 人类T细胞白血病细胞系HPB-ALL和Jurkat已经被开发出来。 本项目的具体目标是研究CD 45的结构如何影响细胞的增殖和分化。 可能与其作为酪氨酸磷酸酶的功能和作为 TCR介导的信号传导。 这将由现场完成- 鼠CD 45的定向诱变和嵌合分子的产生 然后通过转染到CD 45缺陷细胞中进行重构。 的 各种突变体恢复TCR介导的信号转导的能力 将被评估。 更详细的研究解决了CD 45如何调节 TCR介导的信号转导被提出关注于CD 45的作用 调节src样激酶fyn和lck的活性。 此外，描述了将试图识别 在我们的T细胞中通过免疫共沉淀的CD 45的生理底物 白血病细胞系和正常人T淋巴细胞。 一种遗传学方法， 利用诱变和细胞分选技术分离假 回复突变体，试图确定潜在的CD 45底物，也是 提出了 最后，我们建议评估CD 45在 通过检查非TCR相关PTK的功能， 在我们的CD 45阳性、CD 45缺陷和CD 45转染的小鼠中， 克隆

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.