INTERLEUKIN-8 RECEPTOR OF HUMAN NEUTROPHILS

人类中性粒细胞的 INTERLEUKIN-8 受体

• 批准号: 6238299
• 负责人: JAMES A KATANCIK
• 金额: $ 6.57万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-07-01 至 1998-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6238299
• 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），嗜中性粒细胞活化肽2 （NAP-2）和gro /黑素瘤生长刺激活性（GRO）是 作为炎症介质的结构相关的人肽。 已经克隆了两种人中性粒细胞IL-8受体，A型（IL 8 RA）和 B型（IL 8 RB）。 这两种受体以高亲和力结合IL-8，但IL 8 RB 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IL 8 RB。 使用哺乳动物表达载体pcDNA 3（Invitrogen），我们克隆了 并在人细胞系293（人肾）中表达IL 8 RB 上皮的）。 IL 8 RB片段的序列通过测序确认。 双脱氧测序法 稳定转染已经通过以下方法实现： 用抗生素G418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， IL 8。 在约2nM IL 8处观察到可饱和结合。 使用 博士埃内斯托·德纳丁，我正在研究 IL-8、NAP-2和GRO-α与IL 8 RB结合。 这项工作涉及放射性 用肽抑制的结合测定，以绘制 受体的 我已经合成了八种肽，大约20种氨基酸 在长度上，其对应于IL 8 RB的细胞外区域。 这些肽已经通过HPLC纯化，并且通过HPLC确认组成。 氨基酸分析 初步数据表明， 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证明 对照结合水平抑制约50%。 这些研究将提供关于IL 8 RB功能的信息， 可能允许未来设计受体拮抗剂。 受体 拮抗剂可以提供一种新的手段， 炎症反应，包括牙周病中出现的炎症反应。