GTP-BINDING PROTEINS IN T CELL SIGNALING

蛋白酪氨酸激酶密切参与T细胞信号传导 通过TCR/CD 3分子复合物。 异三聚体G的作用 蛋白质在这个过程中没有明确的规定。 鉴于最近 有证据表明酪氨酸激酶和G蛋白调节途径 “相声”的对方，我们建议研究 G蛋白参与T细胞信号转导。 我们首先 计划研究G蛋白和 TCR/CD3。我们将利用嵌合体和突变体 CD 3xi链的胞质内拖尾，试图定义 参与物理关联的关键部位，以及特定的 代表Ga α亚基上位点的肽，以确定是否 它们会干扰这种联系。 共沉淀 和用特异性抗体的蛋白质印迹分析，和CD 3xi- GST融合蛋白将是主要利用的工具， 解决这些问题。 该分析将扩展到人类 胸腺细胞、外周T细胞和小鼠脾细胞， 更好地建立物理的生物学意义 协会. 此外，G蛋白“功能缺陷”突变体 将被用来进一步探索物理的意义， 协会. 我们接下来将利用免疫共沉淀， 用抗酪氨酸激酶抗体进行蛋白质印迹分析， 以及特异性肽竞争，以研究 G蛋白与特定蛋白酪氨酸激酶的结合。 G 蛋白质“功能缺陷”突变体将用于评估 物理关联的功能相关性。 此外，我们将 评估G蛋白在ras刺激中的参与， 随后激活MAP-激酶级联反应，通过研究 ras-GTP交换因子与G蛋白的相互作用， G蛋白“功能缺陷”突变体对这种相互作用的影响。 将通过以下方法评估上述结果的生物学意义： 决定对晚期事件如raf和MAP激酶的影响 活化以及IL 2产生和CD 69表达。 的 已经建立的系统，现有的技术专长， 现有的数据，以及所提出的实验的可行性，使 这一提议直截了当，可能会产生新的和重要的 信息. 此外，我们的研究结果将提供重要的 对未来实验的见解。

Protein tyrosine kinases are intimately involved in T cell signaling via the TCR/CD3 molecular complex. The role of heterotrimeric G proteins in this process is not clearly established. In view of recent evidence that the tyrosine kinase- and G protein-regulated pathway "cross-talk" to each other, we are proposing to study the involvement of G proteins in T cell signal transduction. We first plan to study the physical association between G proteins and TCR/CD3. We'll utilize chimeras and mutants of the intracytoplasmic trail of the CD3xi chain in an attempt to define the critical sites involved in the physical association, and specific peptides representing sites on Galpha subunits to determine whether they can interfere with the association. Co-immunoprecipitation and western blotting analyses with specific antibodies, and CD3xi- GST fusion proteins will be the primary tolls utilized in order to address these issues. The analysis will be extended to human thymocytes, peripheral T cells, and mouse splenic cells in order to better establish the biologic significance of the physical associations. Furthermore, G proteins "function-deficient" mutants will be utilized to further explore the significance of the physical associations. We will next utilize co-immunoprecipitation and western blotting analyses with anti-tyrosine kinase antibodies, as well as specific peptide competition in order to investigate the association of G proteins with specific protein tyrosine kinases. G protein "function-deficient" mutants will be utilized to assess the functional relevance of the physical associations. Further, we will assess the involvement of G proteins in the stimulation of ras and the subsequent activation of the MAP-kinase cascade, by studying the interaction of ras-GTP exchange factors with G proteins and the effects of G protein "function-deficient" mutants on this interaction. The biological significance of the above findings will be assessed by determining effects on late events such as raf and MAP-kinase activation, as well as IL2 production, and CD69 expression. The already established systems, the available technical expertise, the existing data, and the feasibility of the proposed experiments render this proposal straightforward, and likely to yield new and important information. In addition, our findings will provide important insights for future experimentation.