MODULATION OF TCR /CD3 SIGNAL TRANSDUCTION BY C-AMP /PROTEIN KINASE A

C-AMP /蛋白激酶 A 对 TCR /CD3 信号转导的调节

• 批准号: 3804897
• 负责人: M A ALAVA
• 金额: --
• 依托单位: CENTER BIOLOGICS EVALUATION RESEARCH HEMATOLOGY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3804897
• 关键词: CD3 molecule; T cell receptor; T lymphocyte; adenylate cyclase; biological signal transduction; cyclic AMP; enzyme induction /repression; enzyme inhibitors; enzyme mechanism; forskolin; guanosine triphosphate; hydrolysis; inositol; isozymes; leukocyte activation /transformation; lymphocyte proliferation; lymphokines; membrane proteins; nucleotide analog; phospholipase C; phospholipids; phosphorylation; prostaglandin E; protein kinase; secretion; transport proteins

To study the mechanism of activation of the hydrolysis of inositol phospholipids (InsPL) in response to perturbation of the murine T lymphocyte Ag receptor (TCR/CD3), and its regulation by activators of the adenylate cyclase (AC) system. The hydrolysis of InsPL, one of the most rapid responses observed in T cells, is deemed to have a role as a transducer of the signal initiated at the TCR/CD3. This metabolic pathway is centered on the activation of an enzyme, an InsPL-specific phospholipase C (PLC). A second signal transduction mechanism of relevance in T lymphocyte activation is represented by the AC/cAMP pathway. AC activation in Th cells has been clearly documented in response to either pharmacological treatment (i.e. FSK, CTx) of lymphocytes or to autocoids (i.e.: prostaglandin E2). Increased levels of cAMP are associated with down regulation of several T cell responses, including lymphokine secretion and proliferation. The mechanism by which increased levels of cAMP depress lymphocyte function is not defined, but our preliminary data indicates that it is associated with a decrease in InsPL hydrolysis. We propose to investigate the effect of AC activators and cAMP on the regulation of InsPL hydrolysis in response to TCR/CD3 perturbation in intact or permeabilized cells. This approach will allow us to characterize the site of action of cAMP in inhibiting TCR/CD3-mediated InsPL hydrolysis. We have been able to induce InsPL hydrolysis in permeabilized T cells by the addition of non-hydrolyzable analogs of GTP (such as GTPgammaS), which are otherwise non-permeable to intact cells. GTPgammaS activates a G-protein that regulates PLC activity, thus bypassing the TCR/CD3 (or other cell surface structure). Activation of the cAMP/PKA pathway results in inhibition of InsPL hydrolysis triggered via TCR/CD3 perturbation, but does not affect GTPgammaS-induced PLC activation. Therefore the two pathways may be distinct. Although cAMP-mediated inhibition does not occur after permeabilization, addition of purified PKA reconstituted the inhibitory effect, which was associated with a specific pattern of phosphorylation of membrane proteins, and included a PLC isozyme, PLC-gamma1. Attempts will be made to understand the functional relationship between PLC- gamma1 phosphorylation by PKA and CD3-mediated activation.

肌醇水解酶的活化机理研究 磷脂(InsPL)对小鼠T细胞扰动的反应 淋巴细胞抗原受体(TCR/CD3)及其激活剂的调节 腺苷环化酶(AC)系统。InsPL的水解是一种 在T细胞中观察到的大多数快速反应被认为具有 在TCR/CD3启动的信号传感器。这种新陈代谢 途径以一种酶的激活为中心，这种酶是InsPL特有的 磷脂酶C(PLC)。第二种信号转导机制 AC/cAMP与T淋巴细胞活化的相关性 路径。Th细胞中AC的激活已清楚地记录在 对药物治疗(如FSK、CTX)的反应 淋巴细胞或自体激素(如：前列腺素E_2)。增加的级别 CAMP的表达与几种T细胞反应的下调有关， 包括淋巴因子的分泌和增殖。这个机制是通过 CAMP水平升高抑制淋巴细胞功能不是 已定义，但我们的初步数据表明它与 InsPL水解率降低。我们建议调查一下 AC激活剂和cAMP对InsPL水解酶的调节作用 对完整或通透性细胞中TCR/CD3的扰动。这种方法 将使我们能够确定cAMP抑制的作用部位 TCR/CD3介导的InsPL水解酶。我们已经能够诱导InsPL 添加非水解物对通透性T细胞的水解作用 GTP的类似物(如GTP GammaS)，否则对 完好无损的细胞。GTPGammaS激活一种调节PLC的G蛋白 活性，从而绕过TCR/CD3(或其他细胞表面结构)。 CAMP/PKA通路激活导致InsPL抑制 通过TCR/CD3微扰触发的水解酶，但不影响 GTP-GammaS诱导PLC激活。因此，这两条路径可能是 截然不同。尽管cAMP介导的抑制不会在 通透性，加入纯化的PKA重建了抑制作用 效应，这与特定的磷酸化模式有关 包括一个PLC同工酶PLC-Gamma1。尝试 将使理解PLC之间的功能关系- 通过PKA和CD3介导的激活使Gamma1磷酸化。