Molecular and cellular analysis of G protein function

描述(由申请人提供)：杂三聚体(a？)G蛋白介导G蛋白偶联受体(GPCRs)的信号，G蛋白偶联受体在对健康和疾病重要的广泛生物学过程中发挥作用，是最常见的药物靶点。尽管GPCRs是神经和心血管疾病相当常见的药物靶点，但在炎症领域的例子很少，尽管GPCRs可以调节T细胞信号。这项提议试图确定G蛋白是否？亚基可能是这一领域有用的药物靶点。使用小干扰RNA(SiRNA)使G蛋白1亚单位沉默，还是抑制？与小分子1帆船蛋白的信号传递导致T细胞受体(TCR)刺激的细胞因子IL-2在CD4T细胞中产生增加。这种作用可能被用于增强炎症，用于疫苗开发、肿瘤免疫治疗以及治疗系统性红斑狼疮(SLE)。这项建议的总体目标是阐明通过什么机制？复合体调节T细胞白介素2(IL-2)的产生，并评估它们作为治疗异常T细胞信号的新靶点的有效性。拟议的实验将在原代人类T细胞和/或Jurkat T白血病细胞系中测试以下假设。1.G蛋白？复合体抑制依赖于TCR的IL-2的产生。为了阐明这一效应的机制，我们将检验两个假设。(A)1？复合体可能通过降低二酰甘油(DAG)和RAS/MAPK的激活水平来抑制依赖TCR的IL-2的产生。(B)可能需要沉默特定的23个复合体以增强TCR刺激的IL-2。2.作为？1？受A2AR的刺激，而不是EP4R，抑制依赖TCR的IL-2的产生。将测试以下导致这种差异的可能机制。(A)这两种GPCR可能对DAG代谢和RAS/MAPK信号有不同的影响。(B)cAMP对两个GPCR的反应的空间和/或时间特征可能不同。(C)不同于？复合体可能被两个GPCRs激活，并因其激活、解离和/或内化的S模式而发挥不同的作用。3.瞄准A2AR-Gas？1？信号可以预防和/或逆转无能。在没有第二次刺激的情况下，通过TCR激活的T细胞变得没有反应(无能)，当用抗原重新刺激时，也不会转录IL-2。SLE患者T细胞中TCR刺激的IL-2水平降低参与了疾病过程，可能是由于无能。来研究A2AR-Gas的作用？信号在无能，下面的假设将被测试。(A)Gallein和/或A2AR拮抗剂可以预防和/或逆转T细胞的无能。(B)Gallein、1siRNA和/或A2AR拮抗剂可以恢复SLE患者T细胞在TCR刺激下的正常IL-2水平。 公共卫生相关性：必须抑制炎症才能治疗脓毒症和自身免疫性疾病，而炎症增加可能对肿瘤的免疫治疗和疫苗开发有用。目前的治疗方法疗效有限，副作用也很多。拟议研究的目标是扩大初步实验，表明G蛋白？亚基可能成为诊断和治疗免疫系统疾病的有用药物靶点。

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.