CHARACTERIZATION OF THE HUMAN INTERLEUKIN 8 TYPE B RECEPTOR

人白细胞介素 8 B 型受体的表征

• 批准号: 3732391
• 负责人: JAMES A KATANCIK
• 金额: --
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 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/3732391
• 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

Characterization of the Human Interleukin-8 Type B Receptor 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. These chemokines elicit activity subseuent to binding specific receptor molecules on the surface of responsive cells - including neutrophils. Two human neutrophil L-8 receptors have been cloned, type A (L-8RA) and type B (IL-8RB). These receptors belong to the family of G-protein-coupled, seven-transmembrane receptors. The two receptors show 32% similarity in their N-termini and 83% similarity over the remainder of the protein. Both receptors bind L-8 with high affinity, but IL 8RB also binds GRO and NAP-2, both of which can compete with IL-8 for binding. The long term objective of this study is to determine the purpose of the second IL8 receptor that also binds GRO and NAP-2. We hypothesize that the binding of different chemokines may have a regulatory function on neutrophil inflammatory responses. Our immediate goal is to define the mechanism by which IL-8. GRO. and N-.2 bind to the IL-8RB. Our first specific aim is to determine the regions of the IL-8RB necessary for binding IL-8, GRO and NAP-2. We have obtained the p3 cDNA (1.9 kb) that includes the human IL 8RB ORF from Philip Murphy at the NIH. Utilizing PCR we then isolated the 1.1 kb DNA fragment corresponding to the IL8RB ORF. We have transformed E. coli cells (XL Blue) with the plasmid l-Gl8R (Pharmacia) containing our IL8RB insert by electroporation. Restriction digestion and analysis by agarose gel electrophoresis confirmed our cloned sequence in transformed E. coli cells. Next we cloned the purified 1.1 kb IL8RB sequence into the mammalian expression vector pcDNA 3 (Invitrogen). The fragment will be sequenced to confirm that no errors were introduced by the PCR. The pcDNA 3 vector will then be used to transform human 293 cells, which will express the IL-8RB on the cell surface. Expression will be confirmed by Northern analysis, FACS analysis, and by saturable binding of radiolabeled IL8. I have synthesized eight peptides of approximately 20 amino acids each corresponding to the N-terminal and three extracellular loops of the IL8RB The peptides have all been cleaved from the synthesis resin and deprotected. The next step will be to solubilize the peptides, purify by HPLC and confirm amino acid content. These peptides will be used in competitive binding assays to determine key binding domains of the IL-8RB.

人白细胞介素-8 B型受体的鉴定 白细胞介素-8（IL-8）、嗜中性粒细胞活化肽2（NAP-2）和 GRO/黑色素瘤生长刺激活性（GRO）在结构上相关 作为炎症介质的人类肽。 这些趋化因子 引发活性，其从属于结合细胞表面上的特异性受体分子， 反应细胞的表面-包括中性粒细胞。 两个人中性粒细胞 L-8受体已被克隆，A型（L-8 RA）和B型（IL-8 RB）。 这些 受体属于G蛋白偶联的七跨膜受体家族， 受体。 两种受体的N-末端有32%的相似性， 蛋白质的其余部分的相似性。 两种受体都与L-8结合， 高亲和力，但IL 8 RB也结合GRO和NAP-2，两者都可以 与IL-8竞争结合。 本研究的长期目标是 确定也结合GRO的第二IL 8受体的目的， NAP-2。 我们假设不同趋化因子的结合可能具有 对中性粒细胞炎症反应的调节功能。 我们眼前的 目的是确定IL-8. GRO。 和N-.2结合到 IL-8RB。 我们的第一个具体目标是确定IL-8 RB的必要区域， 用于结合IL-8、GRO和NAP-2。 我们已经获得了p3 cDNA（1.9kb）， 包括来自NIH的Philip Murphy的人IL 8 RB ORF。 利用PCR 然后我们分离了对应于IL 8 RB ORF的1.1kb DNA片段。 我们 改造了E.大肠杆菌细胞（XL Blue）与质粒I-Gl8 R（Pharmacia） 含有我们的IL 8 RB插入物的细胞。 限制性消化和 琼脂糖凝胶电泳分析证实了我们的克隆序列， 转化的大肠大肠杆菌细胞。 接下来，我们克隆了纯化的1.1kb IL 8 RB 序列导入哺乳动物表达载体pcDNA 3（Invitrogen）中。 的 将对片段进行测序，以确认没有错误被引入。 PCR法 然后将pcDNA 3载体用于转化人293细胞， 其将在细胞表面上表达IL-8 RB。 表达将 通过北方分析、FACS分析和通过以下的可饱和结合证实： 放射性标记的IL 8。 我已经合成了8种多肽， 每个氨基酸对应于N-末端和三个细胞外 IL 8 RB的环肽都已从合成中裂解 树脂并脱保护。 下一步是溶解肽， 经HPLC纯化，确定氨基酸含量。 这些肽将用于 在竞争性结合测定中， IL-8RB。