Molecular and cellular analysis of G protein function

G 蛋白功能的分子和细胞分析

• 批准号: 7890012
• 负责人: CATHERINE H BERLOT
• 金额: $ 34.22万
• 依托单位: GEISINGER CLINIC
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-01-01 至 2014-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7890012
• 关键词: 1,2-diacylglycerol; Adenosine A2A Receptor; Adverse effects; Affect; Alprostadil; Antigens; Area; Autoimmune Diseases; Biological Process; CD4 Positive T Lymphocytes; Cancer Vaccines; Cardiovascular Diseases; Cell Line; Characteristics; Chemosensitization; Complex; Cyclic AMP; Data; Development; Diagnosis; Diagnostic; Diglycerides; Disease; Dissociation; Drug Delivery Systems; Exhibits; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Goals; Health; Human; Immune System Diseases; Immunotherapy; Inflammation; Interleukin-2; MAP Kinase Gene; Mediating; Metabolism; Molecular; Neurologic; Patients; Pattern; Pharmacologic Substance; Play; Process; Production; Proteins; Reagent; Role; Sepsis; Signal Transduction; Small Interfering RNA; Stimulus; Systemic Lupus Erythematosus; T-Cell Receptor; T-Lymphocyte; Testing; Therapeutic; anergy; base; cytokine; gallein; inhibitor/antagonist; leukemia; novel; novel strategies; prevent; protein function; public health relevance; receptor; receptor coupling; research study; response; small molecule; tumor; vaccine development

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.

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，其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点。尽管 GPCR 是神经系统和心血管疾病相当常见的药物靶点，但尽管 GPCR 可以调节 T 细胞信号传导，但在炎症领域的例子却较少。该提案旨在确定 G 蛋白是否 ¿？亚基可能是该领域有用的药物靶标。使用小干扰 RNA (siRNA) 沉默 G 蛋白 ¿1 亚基或抑制 ¿小分子 1 加仑蛋白的信号传导导致 CD4+ T 细胞中 T 细胞受体 (TCR) 刺激的细胞因子白细胞介素 2 (IL-2) 的产生增加。这种效应可能可用于增强疫苗开发、肿瘤免疫治疗的炎症，也可用于治疗系统性红斑狼疮 (SLE)。该提案的总体目标是阐明 ??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标的效用。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¿1？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，将测试两个假设。 (A) ¿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) TCR 刺激的 IL-2 的增强可能需要特定 23 个复合物的沉默。 2.as¿1？受 A2AR（而非 EP4R）刺激，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) 两种 GPCR 可能对 DAG 代谢和 Ras/MAPK 信号传导产生不同的影响。 (B) cAMP 对两种 GPCR 的反应的空间和/或时间特征可能不同。 (C) 不同为??复合物可以被两个 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A2AR-Gas¿1？信号传导可以防止和/或逆转无反应性。在没有第二次刺激的情况下，通过 TCR 激活的 T 细胞变得无反应（无反应性），并且在用抗原重新刺激时不会转录 IL-2。 SLE 患者 T 细胞中 TCR 刺激的 IL-2 减少有助于疾病进程，可能是由于无反应所致。研究 A2AR-Gas¿1 的作用？无能信号传导，将测试以下假设。 (A) Gallein 和/或 A2AR 拮抗剂可以预防和/或逆转 T 细胞的无反应性。 (B) Gallein、1 siRNA 和/或 A2AR 拮抗剂可以恢复 SLE 患者 T 细胞中 TCR 刺激的正常 IL-2 水平。 公共卫生相关性：必须抑制炎症才能治疗败血症和自身免疫性疾病，而炎症的增加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且有副作用。拟议研究的目标是扩展初步实验，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。