Molecular and cellular analysis of G protein function

描述（申请人提供）：异源三聚体（a？）G蛋白介导来自G蛋白偶联受体（GPCR）的信号，GPCR在对健康和疾病重要的广泛生物过程中发挥作用，并且是最常见的药物靶标。虽然GPCR是神经系统和心血管疾病相当常见的药物靶标，但在炎症领域的例子较少，尽管事实上GPCR可以调节T细胞信号传导。这项建议旨在确定是否G蛋白？亚基可能是该领域有用的药物靶点。沉默G蛋白1亚基使用小干扰RNA（siRNA）或抑制？用小分子1-gallein进行信号传导，导致T细胞受体（TCR）刺激的细胞因子、CD 4 + T细胞中白细胞介素2（IL-2）的产生增加。潜在地，这种作用可用于增强炎症以用于疫苗开发、肿瘤免疫疗法，并且还用于治疗系统性狼疮性肾炎（SLE）。这项建议的总体目标是阐明机制，其中？复合物调节T细胞白细胞介素2（IL-2）的产生，并评估它们作为治疗异常T细胞信号传导的新靶点的效用。所提出的实验将在原代人T细胞和/或Jurkat T白血病细胞系中测试以下假设。1. G蛋白1？复合物抑制TCR依赖性IL-2产生。为了阐明这种效应的机制，将测试两个假设。(A)1号？复合物可通过降低二酰基甘油（DAG）和Ras/ MAPK活化水平来抑制TCR依赖性IL-2产生。(B)特异性23复合物的沉默可能是增强TCR刺激的IL-2所必需的。2. 1？受A2 AR而非EP 4 R刺激的IL-2抑制TCR依赖性IL-2的产生。将测试这种差异的以下可能机制。(A)这两种GPCR可能对DAG代谢和Ras/MAPK信号通路有不同的影响。(B)cAMP对两种GPCR的响应的空间和/或时间特征可能不同。(C)不一样吗？复合物可被两种GPCR激活，并由于其GPCR刺激的激活、解离和/或内化模式而发挥不同的作用。3.瞄准A2 AR-Gas 1？信号传导可以预防和/或逆转无反应性。在没有第二刺激的情况下，通过TCR活化的T细胞变得无反应（无反应性），并且当用抗原再刺激时不转录IL-2。SLE患者T细胞中TCR刺激的IL-2减少有助于疾病过程，可能是由于无反应性。调查A2 AR-Gas <$1的作用？在无反应性中的信号传导，将测试以下假设。(A)Gallein和/或A2 AR拮抗剂可以预防和/或逆转T细胞中的无反应性。(B)Gallein、siRNA和/或A2 AR拮抗剂可以恢复SLE患者T细胞中正常的TCR刺激的IL-2水平。 公共卫生相关性：为了治疗败血症和自身免疫性疾病，必须抑制炎症，而增加的炎症可用于肿瘤的免疫治疗和疫苗开发。目前的治疗方法疗效有限，而且有副作用。拟议研究的目标是扩大初步实验，表明G蛋白？亚基可以是用于诊断和治疗免疫系统疾病的有用的药物靶标。

DESCRIPTION (provided by applicant): Heterotrimeric (a¿?) G proteins mediate signals from G protein coupled receptors (GPCRs), which play roles in a vast range of biological processes important for health and disease, and are the most common pharmaceutical drug targets. Although GPCRs are fairly common drug targets for neurological and cardiovascular diseases, there are fewer examples in the field of inflammation despite the fact that GPCRs can modulate T cell signaling. This proposal seeks to determine whether G protein ¿? subunits might be useful drug targets in this area. Silencing the G protein ¿1 subunit using small interfering RNA (siRNA) or inhibition of ¿? signaling with the small molecule, 1 gallein, leads to increased T cell receptor (TCR)-stimulated production of the cytokine, interleukin 2 (IL-2) in CD4+ T cells. Potentially this effect could be used to enhance inflammation for vaccine development, tumor- immunotherapy, and also for treating systemic lupus erythematosis (SLE). The overall goal of this proposal is to elucidate the mechanisms by which ¿? complexes modulate T cell interleukin 2 (IL-2) production and to assess their utility as novel targets for treating aberrant T cell signaling. The proposed experiments will test the following hypotheses in primary human T cells and/or the Jurkat T leukemia cell line. 1. G protein ¿1? complexes inhibit TCR-dependent IL-2 production. To elucidate the mechanism of this effect, two hypotheses will be tested. (A) ¿1? complexes may inhibit TCR-dependent IL-2 production by decreasing levels of diacylglycerol (DAG) and Ras/ MAPK activation. (B) Silencing of specific 23 complexes may be required for potentiation of TCR-stimulated IL-2. 2. as¿1? stimulated by the A2AR, but not EP4R, inhibits TCR-dependent IL-2 production. The following possible mechanisms for this difference will be tested. (A) The two GPCRs may differentially affect DAG metabolism and Ras/MAPK signaling. (B) The spatial and/or temporal characteristics of cAMP responses to the two GPCRs may differ. (C) Different as¿? complex(es) may be activated by the two GPCRs and exert different effects as a result s of their GPCR-stimulated activation, dissociation, and/or internalization patterns. 3. Targeting A2AR-Gas¿1? signaling may prevent and/or reverse anergy. In the absence of a second stimulus, T cells activated through the TCR become unresponsive (anergic) and do not transcribe IL-2 when re-stimulated with antigen. Decreased TCR-stimulated IL-2 in T cells from SLE patients contributes to the disease process and may be due to anergy. To investigate a role for A2AR-Gas¿1? signaling in anergy, the following hypotheses will be tested. (A) Gallein and/or A2AR antagonists may prevent and/or reverse anergy in T cells. (B) Gallein, ¿1 siRNA, and/or A2AR antagonists may restore normal TCR-stimulated IL-2 levels in T cells from patients with SLE. PUBLIC HEALTH RELEVANCE: Inflammation must be inhibited in order to treat sepsis and autoimmune diseases, whereas increased inflammation can be useful for immunotherapy of tumors and vaccine development. Current treatments have limited efficacy as well as side effects. The goal of the proposed studies is to expand on preliminary experiments that suggest that G protein ¿? subunits could be useful drug targets for the diagnosis and treatment of immune system disorders.