T CELL ACTIVATION--G PROTEIN-TYROSINE KINASE INTERACTION

描述（改编自研究者摘要）：T细胞活化 淋巴细胞和随后的信号转导事件介导的， TCR/CD 3分子复合物是免疫应答中的重要事件 抗肿瘤和自身免疫性疾病。 蛋白酪氨酸激酶是 通过TCR/CD 3密切参与T细胞信号传导。 然而，作用 异源三聚体G蛋白在这一过程中的作用尚不清楚。 因此，在本申请中，我们提出研究 G蛋白参与T细胞信号转导。 我们已经观察到TCR/CD 3连接诱导G 蛋白质和CD 3链。 我们建议通过使用 嵌合体和突变体的胞质内尾的CD 3 链，试图定义这个物理过程中涉及的关键站点 协会 我们还将使用合成肽代表特定的 G α亚基上的位点，以及G α蛋白的点突变体， 确定它们是否能干扰G α-CD 3受体， 协会 免疫共沉淀和蛋白质印迹分析， 特异性抗体和CD 3 ε-GST融合蛋白将是额外的 为了解决这个问题而使用的工具。 我们还观察到，在TCR/CD 3扰动后，G蛋白与 与酪氨酸激酶相互作用，并调节其功能。 我们建议 通过关注特定的酪氨酸激酶EMT来研究这一过程， 家庭成员 我们将研究TCR/CD 3介导的物理结合， 以及EMT-G β和EMT-CD 3 β的功能后果， 交互. 我们将利用EMT的截短突变体， 确定对这些相互作用重要的位点。 协会 src激酶fyn和lck与EMT-G β-CD 3 β复合物的结合将是 以及这种关联对src激酶活性的影响 将被审查。 G蛋白功能缺陷突变体对 还将确定上述过程。 上述分析将 扩展到人胸腺细胞和外周T细胞，以便更好地 确定这些事件的生物学相关性。

DESCRIPTION (Adapted from Investigator's abstract): Activation of T lymphocytes and the subsequent signal transduction events mediated through the TCR/CD3 molecule complex are important events in the immune response against tumors and in autoimmune diseases. Protein tyrosine kinases are intimately involved in T cell signaling through TCR/CD3. However, the role of heterotrimeric G proteins in this process is not clearly established. Therefore, in the present application we are proposing to study the involvement of G proteins in T cell signal transduction. We have observed that TCR/CD3 ligation induces the interaction between G proteins and the CD3 chain. We propose to study this interaction by the use of chimeras and mutants of the intracytoplasmic tail of the CD3 epsilon chain in an attempt to define the critical sites involved in this physical association. We will also use synthetic peptides representing specific sites on G alpha subunits, as well as point mutants of G alpha proteins to determine whether they can interfere with the G alpha-CD3 epsilon association. Co-immunoprecipitation and western blotting analyses with specific antibodies, and CD3 epsilon-GST fusion proteins will be additional tools utilized in order to address this issue. We have also observed that upon TCR/CD3 perturbation, G proteins associate with tyrosine kinases and they regulate their function. We proposed to study this process by focusing on the specific tyrosine kinase EMT, a Tec family member. We will examine the TCR/CD3-mediated physical association and the functional consequences of EMT - G beta and EMT-CD3 epsilon interactions. We will utilize truncation mutants of EMT in order to determine sites that are important for these interactions. The association of src kinases fyn and lck with the EMT-G beta-CD3 epsilon complex will be assessed, and the consequences of this association on src kinase activity will be examined. The effects of G protein function-deficient mutants on the above processes will be also determined. The above analyses will be extended to human thymocytes and peripheral T cells in order to better establish the biological relevance of these events.