Characterization of intestinal adenosine A2b receptor

肠腺苷 A2b 受体的表征

• 批准号: 6669814
• 负责人: SHANTHI Vasudevan SITARAMAN
• 金额: $ 7.65万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-20 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6669814
• 关键词: adenosine; apical membrane; basolateral membrane; biological signal transduction; cell line; clinical research; cytoskeletal proteins; gastrointestinal epithelium; gene deletion mutation; human subject; immunofluorescence technique; inflammation; interleukin 6; neuroimmunomodulation; neutrophil; point mutation; protein protein interaction; protein structure function; purinergic receptor; site directed mutagenesis

DESCRIPTION (provided by applicant): Acute flares of inflammatory diseases of the intestine are characterized by the migration of neutrophils across the intestinal epithelium into the lumen to form 'crypt abscess'. Adenosine is generated during active intestinal inflammation by the conversion of neutrophil-derived 5'AMP into adenosine by the epithelial 5'ectonucleotidase. Adenosine, acting via the A2b receptor, not only mediates electrogenic chloride secretion but also induces an apically polarized secretion of the pro-inflammatory cytokine, interleukin-6 (IL-6). IL-6, in turn, induces a calcium flux in neutrophils, thus providing a paracrine signal to neutrophils. In this R03 application, we propose to investigate the hypothesis that the A2b receptors play a central role in orchestrating both the diarrheal and inflammatory components of the intestinal inflammatory response. If so, pharmacological manipulation of the A2b receptor may be therapeutic for intestinal inflammation. The overall goals of this proposal are to characterize the intestinal A2b receptor, to examine the regulation of adenosine-induced IL-6 secretion, and to determine the effect of IL-6 on neutrophil function. Specific aim 1 : We will use model intestinal cell line, T84, expressing native A2b receptor and Caco2-BBE (which do not express A2b receptor) overexpressing GFP-A2b to study: i) the recruitment of the A2b receptor to apical vs basolateral membrane using domain-specific biotinylation and immunoflourescence ii) the molecular mechanism of interaction of A2b receptor with E3KARP will be studied using deletion constructs and site-directed mutagenesis iii) the role of A2b receptor recruitment in receptor signaling and function and desensitization. Specific aim 2: We will investigate: i) the mechanism of adenosine-mediated IL-6 induction using IL-6 promoter constructs with deletion and point mutations of the various nuclear factor binding sites,.Specific aim 3: We will study: i) the effect of IL-6 on degranulation of neutrophils.

描述（由申请人提供）： 肠道炎症性疾病的急性发作的特征是中性粒细胞穿过肠上皮迁移到管腔中形成“隐窝脓肿”。腺苷是在活动性肠道炎症期间通过上皮 5' 核酸外切酶将中性粒细胞衍生的 5'AMP 转化为腺苷而产生的。腺苷通过 A2b 受体发挥作用，不仅介导氯化物分泌，还诱导促炎细胞因子白细胞介素 6 (IL-6) 的顶端极化分泌。 IL-6 反过来诱导中性粒细胞中的钙流，从而向中性粒细胞提供旁分泌信号。在此 R03 应用中，我们建议研究 A2b 受体在协调肠道炎症反应的腹泻和炎症成分中发挥核心作用的假设。如果是这样，A2b 受体的药理学操作可能可以治疗肠道炎症。该提案的总体目标是表征肠道 A2b 受体，检查腺苷诱导的 IL-6 分泌的调节，并确定 IL-6 对中性粒细胞功能的影响。具体目标 1：我们将使用表达天然 A2b 受体的模型肠细胞系 T84 和过表达 GFP-A2b 的 Caco2-BBE（不表达 A2b 受体）来研究：i) 使用域特异性生物素化和免疫荧光将 A2b 受体招募到顶膜和基底膜 ii) A2b 相互作用的分子机制 将使用缺失构建体和定点诱变来研究具有 E3KARP 的受体 iii) A2b 受体募集在受体信号转导和功能以及脱敏中的作用。具体目标 2：我们将研究：i) 使用具有各种核因子结合位点缺失和点突变的 IL-6 启动子构建体，腺苷介导的 IL-6 诱导机制。具体目标 3：我们将研究：i) IL-6 对中性粒细胞脱颗粒的影响。