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

本研究的目的是确定两者之间的耦合机制。 辅助性T细胞(Th)T细胞受体与磷脂酶C 肌醇磷脂(InsPL)水解的关键酶(PLC) 路径。对淋巴细胞活化机制的认识 表示设计针对以下目标的免疫调节策略的基础 信号转导途径的关键要素，基于 获取的信息。初步研究结果：奎宁核苷酸的作用 类比。链球菌溶血素O对小鼠Th细胞的暴露 (SLO)或破伤素(TL)对非水解性鸟苷三磷酸的作用 (GTP)类似物，鸟苷-5‘-O-(3-硫代三磷酸)(GTP-伽马-S) 在INSP生成中。同样，TCR对摩押的摄动 145.2Cll(针对TCR CD3 epsilon链)导致InsPL 渗透性细胞的水解液。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 GDPBS，一种与GTP竞争的鸟嘌呤核苷酸类似物。 微量元素和核苷酸需求。由任何一种引起的InSP产生 TcR扰动和GTP-γ-S处理表现出相似的钙依赖关系。 TCR介导的INSPL水解严格需要ATP，并且 增强GTP-γ-S诱导的INSP生成。其他核苷酸(CTP， GDP、GTP、ITP)不影响反应。细菌毒素对人体的影响 共价修饰G蛋白。百日咳毒素(PTX)是一种细菌毒素 通过ADP-核糖化修饰许多G蛋白 亚单位。这种修饰防止了G蛋白的解离 并阻止其发挥正常的受体偶联作用 功能。PTX处理不影响对INSP产生的刺激 通过在完整或通透性细胞中Tcr或Gtp-γ-S的扰动， 分别进行了分析。在未来，我们将尝试对此进行初步鉴定 蛋白质(S)与氨基酸抗血清的交叉反应 已知的G蛋白序列。我们将特别关注 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.