Molecular and cellular analysis of G protein function

描述（由申请人提供）：异三聚体（a¿？）G蛋白介导来自G蛋白偶联受体（gpcr）的信号，在一系列对健康和疾病重要的生物过程中发挥作用，是最常见的药物靶点。尽管gpcr是神经系统和心血管疾病的常见药物靶点，但在炎症领域的例子较少，尽管gpcr可以调节T细胞信号传导。这一提议旨在确定G蛋白¿？亚基可能是这一领域有用的药物靶点。使用小干扰RNA （siRNA）沉默G蛋白¿1亚基或抑制¿1 ？小分子1 gallein的信号传导导致CD4+ T细胞中T细胞受体（TCR）刺激的细胞因子白介素2 （IL-2）的产生增加。这种效应可能用于疫苗开发、肿瘤免疫治疗以及系统性红斑狼疮（SLE）的治疗。本提案的总体目标是阐明下列机制：复合物调节T细胞白细胞介素2 （IL-2）的产生，并评估其作为治疗异常T细胞信号传导的新靶点的效用。拟议的实验将在原代人T细胞和/或Jurkat T白血病细胞系中测试以下假设。1. G蛋白？复合物抑制tcr依赖性IL-2的产生。为了阐明这种效应的机制，我们将检验两个假设。(一)1 ?复合物可能通过降低二酰基甘油（DAG）水平和Ras/ MAPK激活来抑制tcr依赖性IL-2的产生。(B)为了增强tcr刺激的IL-2，可能需要沉默特定的23个复合物。2. 是1 ?由A2AR刺激，而不是EP4R，抑制tcr依赖性IL-2的产生。将测试造成这种差异的以下可能机制。(A)两种gpcr可能对DAG代谢和Ras/MAPK信号传导有不同的影响。(B) cAMP对两种gpcr反应的空间和/或时间特征可能不同。(C)不同于？复合物（es）可能被两种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. 针对A2AR-Gas 1 ?信号可以阻止和/或逆转能量。在没有第二种刺激的情况下，通过TCR激活的T细胞在再次受到抗原刺激时变得无反应（无能）并且不转录IL-2。SLE患者的T细胞中tcr刺激的IL-2减少有助于疾病进程，可能是由于能量。调查A2AR-Gas的作用？能量信号，以下假设将被验证。(A) Gallein和/或A2AR拮抗剂可以阻止和/或逆转T细胞的能量。(B) Gallein，¿1 siRNA和/或A2AR拮抗剂可以恢复SLE患者tcr刺激的T细胞中正常的IL-2水平。

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.