ROLE OF G-PROTEINS IN THE CONTROL OF INOSITOL PHOSPHOLIPID HYDROLYSIS IN T-CELLS

本研究的目的是确定之间的耦合机制 辅助性T淋巴细胞（Th）T细胞受体（TCR）与磷脂酶C (PLC)是肌醇磷脂（InsPL）水解的关键酶 通路对淋巴细胞活化机制的认识 代表了设计免疫调节策略的基础， 信号转导途径的关键要素，基于 获得的信息。初步研究结果：喹核苷酸的作用 类似物用链球菌溶血素O透化的小鼠Th细胞的暴露 (SLO)或破伤风溶素（TL）转化为不可水解的三磷酸鸟苷 (GTP)类似物，鸟苷-5'-O-（3-硫代三磷酸）（GTP-γ-S），得到 在InsP世代。类似地，用MoAb干扰TCR 145.2 Cll（针对TCR CD3 β链）导致InsPL 通过透化细胞水解。G蛋白在TCR/PLC中的作用 TCR介导的InsPL的抑制进一步表明偶联 通过GDPBS水解，GDPBS是一种竞争GTP的鸟嘌呤核苷酸类似物。 微量元素和核苷酸需要量。InsP生成由以下任一项诱导 TCR扰动或GTP-γ-S处理显示类似的Ca（2+）依赖性。 TCR介导的INSPL水解严格需要ATP， 增强GTP-γ-S诱导的InsP产生。其他核苷酸（CTP， GDP、GTP、ITP）对反应没有影响。细菌毒素的影响， 共价修饰G蛋白。百日咳毒素是一种细菌毒素 通过ADP-核糖基化G蛋白的α， 亚单位这种修饰阻止了G蛋白的解离 异源三聚体并阻断其发挥正常受体偶联 功能PTx处理不影响InsP的产生 通过干扰完整或透化细胞中的TCR或GTP-γ-S， 分别在未来，我们将尝试初步确定这一点， 通过测试其与抗氨基酸的抗血清的交叉反应性， 已知的G蛋白序列。将特别注意 保守序列（即GTP结合位点）和氨基末端或 羧基末端序列。

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'-O- (3-thiotriphosphate) (GTP-gamma-S) , resulted in InsP generation. Similarly, perturbation of the TCR with the MoAb 145.2Cll (directed against the TCR CD3 epsilon chain) resulted in InsPL hydrolysis by permeabilized cells. A role for a G-protein in TCR/PLC coupling was further indicated by the inhibition of TCR-mediated InsPL hydrolysis by GDPBS, a guanine nucleotide analog that competes for GTP. Microelements and nucleotide requirement. InsP generation induced by either TCR perturbation or GTP-gamma-S treatment showed similar Ca(2+) dependence. ATP was strictly required for TCR-mediated INSPL hydrolysis, and potentiated GTP-gamma-S-induced InsP generation. Other nucleotides (CTP, GDP, GTP, ITP) did not affect the response. Effect of bacterial toxins that covalently modify G-proteins. Pertussis toxin (PTx) is a bacterial toxins that covalently modifies many G-proteins by ADP-ribosylating their alpha subunit. This modification prevents dissociation of the G-protein heterotrimer and blocks it from exerting its normal receptor coupling function. PTx treatment did not affect the stimulation of InsP production by perturbation of the TCR or GTP-gamma-S in intact or permeabilized cells, respectively. In the future we will attempt initial identification of this protein(s) by testing its cross-reactivity with antisera against amino acid sequences of known G-proteins. Particular attention will be dedicated to conserved sequences (i.e. GTP-binding site) and amino-terminal or carboxy-terminal sequences.