INTERLEUKIN-8 RECEPTOR OF HUMAN NEUTROPHILS

人类中性粒细胞的 INTERLEUKIN-8 受体

• 批准号: 5210014
• 负责人: JAMES A KATANCIK
• 金额: --
• 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/5210014
• 关键词: cytokine; cytokine receptors; human tissue; interleukin 8; neutrophil; northern blottings; protein purification; protein sequence; protein structure function; radiotracer; receptor binding; tissue /cell culture; transfection

Interleukin-8 (IL-8), neutrophil-activating peptide 2 (NAP-2), and gro /melanoma growth-stimulatory activity (GRO) are structurally related human peptides that act as mediators of inflammation. Two human neutrophil IL-8 receptors have been cloned, type A (IL8RA) and type B (IL8RB). The two receptors bind IL-8 with high affinity, but IL8RB also binds GRO and NAP-2. Our immediate goal is to define the regions to which IL-8, GRO, and NAP-2 bind the IL8RB. Using the mammalian expression vector pcDNA 3 (Invitrogen) we have cloned and expressed the IL8RB into the human cell line 293 (human kidney epithelial). The sequence of the IL8RB fragment was confirmed by the dideoxy sequencing method. Stable transfection has been accomplished by selection with the antibiotic G418 and has been confirmed by fluorescence activated cell sorting (FACS) and radioactive ligand binding studies with IL8. Saturable binding is seen at approximately 2 nM IL8. Working with Dr. Ernesto DeNardin, I'm currently characterizing binding of the ligands IL-8, NAP-2 and GRO-alpha to the IL8RB. This work involves radioactive binding assays with peptide inhibition to map the binding regions of the receptor. I have synthesized eight peptides, approximately 20 amino acids in length, that correspond to the extracellular regions of the IL8RB. These peptides have been purified by HPLC and composition confirmed by amino acid analysis. Preliminary data suggests that the N-terminal region of the receptor and he first extracellular loop are critical areas for ligand binding. Peptides corresponding to these regions demonstrate approximately 50% inhibition of control binding levels. These studies will provide information on the function of the IL8RB and may allow for the future design of receptor antagonists. Receptor antagonists may provide a novel means of modulating destructive inflammatory reactions, including those seen in periodontal disease.

白细胞介素 8 (IL-8)、中性粒细胞激活肽 2 (NAP-2) 和 gro/黑色素瘤生长刺激活性 (GRO) 是 结构相关的人类肽，充当炎症介质。 两种人类中性粒细胞 IL-8 受体已被克隆，A 型 (IL8RA) 和 B 型（IL8RB）。 两种受体以高亲和力结合IL-8，但IL8RB 还结合 GRO 和 NAP-2。 我们的近期目标是定义区域 其中 IL-8、GRO 和 NAP-2 结合 IL8RB。 使用我们克隆的哺乳动物表达载体 pcDNA 3 (Invitrogen) 并将IL8RB表达到人细胞系293（人肾 上皮）。 IL8RB片段的序列由 双脱氧测序法。 稳定转染已完成 用抗生素G418进行选择并已通过荧光证实 活化细胞分选 (FACS) 和放射性配体结合研究 IL8。 在大约 2 nM IL8 时观察到饱和结合。 与...一起工作 Ernesto DeNardin 博士，我目前正在表征配体的结合 IL-8、NAP-2 和 GRO-α 至 IL8RB。 这项工作涉及放射性 通过肽抑制进行结合测定，以绘制肽的结合区域图谱 受体。 我合成了8种肽，大约20个氨基酸 长度，对应于 IL8RB 的细胞外区域。 这些肽已通过 HPLC 纯化，其成分通过 氨基酸分析。 初步数据表明 N 末端区域 受体和第一个细胞外环是关键区域 配体结合。 对应于这些区域的肽表明 对照结合水平的约50%抑制。 这些研究将提供有关 IL8RB 功能的信息 可能有助于未来受体拮抗剂的设计。 受体 拮抗剂可能提供一种调节破坏性的新方法 炎症反应，包括牙周病中常见的炎症反应。