GTP-BINDING PROTEINS IN T CELL SIGNALING

蛋白酪氨酸激酶与T细胞信号转导密切相关 通过TCR/CD3分子复合体。杂三聚体G的作用 蛋白质在这一过程中的作用尚不清楚。鉴于最近 酪氨酸激酶和G蛋白调节通路的证据 相互“相声”，我们提议研究 G蛋白参与T细胞信号转导。我们首先 计划研究G蛋白和G蛋白之间的物理联系 TCR/CD3。我们将利用嵌合体和突变体 CD3xi链的胞质内踪迹，试图定义 物理关联中涉及的关键站点，以及特定 代表Galpha亚基上的位点的多肽 他们可能会干扰这种联系。免疫共沉淀 用特异性抗体进行免疫印迹分析，以及CD3xi- GST融合蛋白将是主要的通行费，以便 解决这些问题。这一分析将扩展到人类 胸腺细胞、外周T细胞和小鼠脾细胞 更好地确立生物体的生物学意义 联想。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 联想。接下来我们将利用免疫共沉淀和 用抗酪氨酸激酶抗体进行免疫印迹分析 以及特定的多肽竞争，以便研究 G蛋白与特异性蛋白酪氨酸激酶的相关性。G 蛋白质“功能缺陷”突变体将被用来评估 物理关联的功能相关性。此外，我们还将 评估G蛋白在ras和ras刺激中的作用 通过研究MAP-激酶级联反应的后续激活 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。 上述发现的生物学意义将通过以下方式进行评估 确定对晚期事件的影响，如RAF和MAP-K 活化，以及IL-2的产生和CD69的表达。这个 已经建立的系统、可用的技术专长、 现有数据，以及拟议实验的可行性 这一建议直截了当，可能会产生新的重要成果 信息。此外，我们的发现将提供重要的 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.