SIGNAL TRANSDUCTION VIA THE T CELL RECEPTOR /CD3 COMPLEX

通过 T 细胞受体 /CD3 复合物进行信号转导

• 批准号: 3804896
• 负责人: E BONVINI
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH HEMATOLOGY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804896
• 关键词: CD3 molecule; G protein; T cell receptor; biological signal transduction; guanosine triphosphate; helper T lymphocyte; immunologic assay /test; immunoprecipitation; inositol phosphates; leukocyte activation /transformation; membrane permeability; monoclonal antibody; nucleotide analog; oncogenes; phosphatase inhibitor; phospholipase C; protein kinase; receptor coupling; tissue /cell culture

The objective of this study is to determine the coupling mechanism between the T cell receptor (TCR) of T helper (Th) lymphocytes and phospholipase C (PLC), the key enzyme in the inositol phospholipid (InsPL) hydrolysis pathway. An understanding of the mechanism of lymphocyte activation represents the base for designing immunomodulatory strategies targeted to critical elements of the signal transduction pathway, based upon the information acquired. Preliminary findings: Effect of quanine nucleotide analogs. Exposure of murine Th cells permeabilized with streptolysin O (SLO) or tetanolysin (TL) to the non- hydrolyzable guanosine triphosphate (GTP) analog, guanosine-5'-0-(3- - thiotriphosphate) (GTPgammaS), resulted in inositol phosphate (InsP) generation. Similarly, perturbation of the TCR/CD3/ with the MoAb 145.2C11 (directed against the CD3 epsilon chain) resulted in InsPL hydrolysis by permeabilized cells. A role for a G-protein in TCR/CD3/ PLC coupling was further indicated by the inhibition of TCR/CD3-mediated InsPL hydrolysis by GDP, a quanine nucleotide analog that competes for GTP. Microelements and nucleotide requirement. InsP generation induced by either TCR/CD3 perturbation or GTPgammaS treatment showed similar Ca2+ dependence. An indication for a role of kinases in PLC activation was supported by the observation that ATP was strictly required for TCR/CD3-mediated InsPL hydrolysis, while only potentiated GTPgammaS- induced InsP generation. Other nucleotides (CTP, GDP, GTP, ITP) did not affect the response. Attempts will be made in the future to gain information on the nature of both the putative G-protein and tyrosine kinase involved in modulating PLC activity. A strategy of coprecipitation using anti-PLC Ab (PLC- gamma1 and PLC-beta) and/or anti-src and src-related oncogene products (fyn, lck, yes, etc.) will be used to this end. By taking advantage of permeabilized cells, transient, "weak" interaction could be stabilized by using homobifunctional chemical cross-linker followed by immunoprecipitation. Preliminary efforts using this techniques have been satisfactory. By using inhibitors of protein tyrosine phosphate phosphatase (sodium orthovanadate or molybdate) in intact and permeabilized cells, attention will be paid to the role of these enzymes, including the CD45 phosphatase, in modulating InsPL hydrolysis.

本研究的目的是确定耦合机制 辅助性T淋巴细胞（Th）的T细胞受体（TCR）与 磷脂酶C（PLC）是肌醇磷脂合成的关键酶 （InsPL）水解途径。 了解其机制， 淋巴细胞活化是设计免疫调节剂的基础。 针对信号转导关键要素的策略 路径，根据所获得的信息。 初步调查结果： 鸟嘌呤核苷酸类似物的作用。 鼠Th细胞的暴露 用链球菌溶血素O（SLO）或破伤风溶血素（TL）透化至非- 可水解鸟苷三磷酸（GTP）类似物，鸟苷-5 '-0-（3- 硫代三磷酸）（GTP γ S），导致肌醇磷酸（InsP） 一代 类似地，用MoAb干扰TCR/CD 3/ 145.2C11（针对CD 3 β链）导致InsPL 通过透化细胞水解。 G蛋白在TCR/CD 3/T细胞中的作用 PLC偶联进一步通过抑制TCR/CD 3介导的细胞凋亡来指示。 InsPL被GDP水解，GDP是一种竞争InsPL的鸟嘌呤核苷酸类似物。 GTP 微量元素和核苷酸需要量。 InsP生成诱导 通过TCR/CD 3扰动或GTP γ S处理显示类似的 钙依赖。 激酶在PLC激活中的作用的指示 支持这一观点的是， TCR/CD 3介导的InsPL水解，而仅增强GTP γ S- 诱导InsP生成。 其他核苷酸（CTP，GDP，GTP，ITP）不 会影响反应。 今后将努力获得关于下列问题性质的资料： 假定的G蛋白和酪氨酸激酶都参与调节 PLC活动 用抗PLC抗体（PLC-1）共沉淀的策略， γ 1和PLC-β）和/或抗src和src相关癌基因产物 (fyn，lck，yes，etc.）将被用于这一目的。 通过利用 透化细胞，瞬时，“弱”相互作用可以稳定 通过使用同双功能化学交联剂， 免疫沉淀。 使用这种技术的初步努力已经 是满意的。 通过使用蛋白酪氨酸磷酸抑制剂 磷酸酶（原钒酸钠或磷酸钠）在完整和 透化细胞，注意将支付这些作用 酶，包括CD 45磷酸酶，在调节InsPL水解。