SIGNAL TRANSDUCTION IN T CELLS

这项建议的目的是研究GTP结合的作用 人类T细胞活化中的蛋白质。GTP结合蛋白，由 三个亚基(Alpha、Beta、Gamma)已被证明与配体偶联- 受体与细胞内效应器的相互作用。没有这样的蛋白质 到目前为止在免疫系统的细胞中已被发现。 我们积累的初步证据有力地表明 这样的蛋白质确实存在于人类T淋巴细胞中。我们已经演示了 人T细胞膜GTP结合蛋白α亚基的存在 细胞通过使用~(32)P-NAD和利用 细菌毒素(霍乱和百日咳毒素)对ADP- 以放射性NAD为靶点体外核糖化α亚基 ADP-核糖的供体。此外，这些毒素可以调节 通过TCR/CD3复合体诱导T细胞活化。例如, 皮摩尔浓度的霍乱毒素抑制抗CD3+IL2介导的抗体 人T细胞的增殖。相反，这种毒素不会抑制 离子霉素+PMA在10倍以上的浓度下仍有增殖作用。 更多数据表明，霍乱毒素介导的抑制是 可能是在抗CD3抗体诱导的阶段，而不是在控制的阶段 被IL2。我们还鉴定并分离了GTP结合蛋白α 人Gamma gt11亚基cDNA文库的筛选 合成寡核苷酸诱导的白血病T细胞系Jurkat。一个 其中一个cdna克隆的部分核苷酸序列也被测得。 获得。最后，针对合成肽的抗体可以代表 Alpha亚基的保守区已经准备好作为探针 研究T细胞中这些蛋白质的特性。 我们建议对分离的a亚单位cdna克隆进行鉴定。 将它们的序列与已经克隆的Alpha亚基的序列进行比较。vbl.使用 DNA-RNA杂交分析我们将确定是否 分离的cDNA克隆在淋巴样细胞中唯一表达 血统。此外，我们将比较这些基因在 静息和激活的T细胞，以及处于不同阶段的T细胞 其分化(胸腺细胞)。此外，我们将确定 GTP结合蛋白与特异性T细胞的局部结合 表面受体通过使用双官能团的交联剂化学。最后， 我们将利用合成的多肽来代表 α亚基及其反应性抗体，以确定 GTP结合蛋白参与T细胞的激活 TCR/CD3分子复合体。我们将测量这些毒剂对 IP3和Ca++积累使用渗透性细胞系统和 电穿孔。

The purpose of this proposal is to investigate the role of GTP binding proteins in human T cell activitation. GTP binding proteins, composed of three subunits (Alpha,Beta,Gamma), have been shown to couple ligand- receptor interactions to intracellular effectors. No such proteins have been identified as yet in cells of the immune system. We have accumulated preliminary evidence which strongly suggests that such proteins do exist in human T lymphocytes. We have demonstrated the presence of GTP-binding protein Alpha subunits in membranes of human T cells by using 32p-NAD and by taking advantage of the ability of bacterial toxins (cholera and pertussis toxin) to specifically ADP- ribosylate the Alpha subunits in vitro using the radioactive NAD as a donor of the ADP-ribose. Furthermore, these toxins can modulate activation of T cells induced via the TCR/CD3 complex. For example, cholera toxin at picomolar concentrations inhibits anti-CD3+IL2 mediated proliferation of human T cells. In contrast, this toxin does not inhibit ionomycin+PMA proliferation even at 10-fold higher concentration. Additional data suggest that the cholera toxin mediated inhibition is probably at the step induced by anti-CD3 and not at the step controlled by IL2. We have also identified and isolated GTP-binding protein alpha subunit cDNA clones by screening a Gamma gt11 cDNA library from the human leukemic T cell line Jurkat with synthetic oligodeoxynucleotides. A partial nucleotide sequence of one of the cDNA clones has been also obtained. Finally, antibodies to synthetic peptides that represent conserved regions of Alpha subunits have been prepared as probes for characterizing these proteins in T cells. We proposed to characterize the isolated a subunit cDNA clones and compare their sequences to those of already cloned Alpha subunits. Using DNA-RNA hybridization analysis we shall determine whether or not the isolated cDNA clones are uniquely expressed in cells of the lymphoid lineage. Furthermore, we shall compare the expression of these genes in resting and activated T cells, as well as, in T cells at various stages of their differentiation (thymocytes). Furthermore, we'll determine the topographic association of GTP-binding proteins to specific T cell surface receptors by using bifunctional crosslinking chemistry. Finally, we'll utilize synthetic peptides that represent conserved portions of the alpha subunits and their reactive antibodies in order to determine the involvement of GTP-binding proteins in the triggering of T cells via the TCR/CD3 molecular complex. We'll measure the effects of these agents on IP3 and Ca++ accumulation using a permeabilized cell system and electroporation.