PILOT STUDY--ROLE OF LEUKOTRIENE B4 RECEPTOR ACTIVATION IN RHEUMATOID ARTHRITIS

试点研究--白三烯 B4 受体激活在类风湿性关节炎中的作用

• 批准号: 3747754
• 负责人: DAVID T HUNG
• 金额: --
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 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/3747754
• 关键词: G protein; RNase protection assay; Xenopus oocyte; biological signal transduction; clone cells; complementary DNA; gene expression; immunoprecipitation; in situ hybridization; leukocyte activation /transformation; leukotrienes; messenger RNA; molecular cloning; neutrophil; northern blottings; polymerase chain reaction; protein structure function; receptor binding; receptor coupling; receptor expression; rheumatoid arthritis; second messengers; site directed mutagenesis

LTB4 is the most potent of all the arachidonic acid-derived mediators of neutrophil chemotaxis and degranulation. LTB4 also activates other leukocytes (eosinophils, lymphocytes, and monocytes) and in addition, exerts profound effects on vascular endothelial cells, pulmonary smooth muscle cells and bronchoepithelium, and skin. Ample medical evidence implicates LTB4-mediated activation of leukocytes and other cells in the pathogenesis of medical disorders as diverse as rheumatoid arthritis, ulcerative colitis, myocardial infraction, asthma, and psoriasis. All of these cell activation events appear to be mediated by the interaction of LTB4 with a specific cell surface molecule, the LTB4 "receptor". The LTB4 receptor remains unidentified. The identification and characterization of the LTB4 receptor is critical to the understanding of LTB4 signaling and the rational design of LTB4 inhibitors. The specific aims of this proposal are: Specific Aim 1) To obtain a cDNA clone for the LTB4 receptor. Two complementary approaches to clone the LTB4 receptor will be taken. a) Expression cloning in Xenopus oocytes. An assay for the mRNA encoding a functional LTB4 receptor has been developed using mRNA expression in Xenopus oocytes. Established screening strategies will utilize this assay to obtain the LTB4 receptor cDNA. b) Cloning by PCR and screening by expression in Xenopus oocytes. Considerable evidence suggests that the LTB4 receptor is a seven- transmembrane domain receptor. cDNA from LTB4- responsive cells will be subjected to PCR amplification using oligonucleotide primers based on consensus sequences of seven-transmembrane domain receptors and products will be used to screen cDNA libraries for full length clones. Pools of candidate clones will be screened for the ability to LTB4 responsiveness to microinjected oocytes, and if positive, will be progressively subdivided and rescreened until a single clone conferring LTB4 responsiveness can be identified. Specific Aim 2) To define the mechanism of LTB4 receptor activation and signaling. The LTB4 receptor cDNA clone will be used to address the following questions: Which structural domains in the LTB4 receptor mediate LTB4 binding? What is the relationship of the cloned receptor to the high and low affinity neutrophil LTB4 receptors and which neutrophil functions does it mediate? Where is the receptor expressed? Is the LTB4 receptor related to the receptors for leukotrienes C4, D4, and E4 or other arachidonic acid metabolites? Which G-proteins and second messenger pathways is the LTB4 receptor coupled to? Can structure-function studies with the LTB4 receptor clone be used to design receptor antagonists superior to the empirically-derived antagonists currently existing?

LTB4是所有蛛网膜酸衍生的介体中最有效的 中性粒细胞趋化性和脱粒。 LTB4还激活其他 白细胞（嗜酸性粒细胞，淋巴细胞和单核细胞），此外， 对血管内皮细胞产生深远的影响，肺部光滑 肌肉细胞和支气管上皮和皮肤。 充足的医学证据 暗示LTB4介导的白细胞和其他细胞激活 医学疾病的发病机理，如类风湿关节炎， 溃疡性结肠炎，心肌违规，哮喘和牛皮癣。 全部 这些细胞激活事件似乎是由相互作用介导的 具有特定细胞表面分子的LTB4 LTB4“受体”的LTB4。 这 LTB4受体保持不明。 标识和 LTB4受体的表征对于理解至关重要 LTB4信号传导和LTB4抑制剂的合理设计。 这 该提案的具体目的是： 具体目标1）获得LTB4受体的cDNA克隆。 二 将采用克隆LTB4受体的互补方法。 a）在异武卵母细胞中表达克隆。 编码mRNA的测定 在使用mRNA表达中开发了功能性LTB4受体 爪蟾卵母细胞。 既定的筛选策略将利用这一点 测定以获得LTB4受体cDNA。 b）通过PCR克隆并通过表达在异武卵母细胞中进行筛选。 大量证据表明，LTB4受体是7- 跨膜结构域受体。 来自LTB4响应细胞的cDNA将是 使用基于寡核苷酸引物进行PCR扩增 七跨膜域受体和产物的共识序列 将用于筛选cDNA库的全长克隆。 池 候选克隆将被筛选，以获得LTB4响应能力的能力 为了显微注射的卵母细胞，如果阳性，将逐渐为 细分并重新分组，直到单个克隆授予LTB4 可以确定响应能力。 具体目的2）定义LTB4受体激活的机制和 信号。 LTB4受体cDNA克隆将用于解决 以下问题：LTB4受体中的哪些结构域 介导LTB4结合？克隆受体与 高和低亲和力中性粒细胞LTB4受体和中性粒细胞 功能可以调节吗？受体在哪里表达？是LTB4 与白细胞C4，D4和E4的受体有关的受体 其他花生四烯酸代谢产物？哪个G蛋白和第二信使 LTB4受体是否耦合到途径？可以结构功能研究 使用LTB4受体克隆来设计受体拮抗剂 优于当前存在的经验衍生的对手？