FMLF BINDING SITE ON FORMYL PEPTIDE RECEPTOR

甲酰肽受体上的 FMLF 结合位点

• 批准号: 2887247
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 10.59万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2887247
• 关键词: G protein; affinity labeling; bactericidal immunity; calcium flux; chemoattractants; crosslink; flow cytometry; immunoglobulin structure; mass spectrometry; method development; neutrophil; oxidative stress; peptides; photochemistry; protein folding; protein structure function; receptor; receptor binding; receptor coupling; site directed mutagenesis; tissue /cell culture

The migration of neutrophils towards invading bacteria is partly dependent on their ability to recognize and bind bacterial N-formylated peptides (such as fMLF.) Upon binding of the bacterial peptide, the N-formyl peptide receptor (FPR) activates a guanyl nucleotide binding protein (G protein) which transduces the signal to intracellular effector molecules causing a cascade of cellular events including chemotaxis, lysosomal enzyme secretion and production of superoxide. In addition to normal host defense functions, neutrophils also play a central role in chronic inflammatory processes. By regulating the behavior of the neutrophils, much of the tissue damage caused by superoxide and its metabolites could be prevented. Understanding the molecular mechanism of formyl peptide- binding to FPR, as well as FPR-G protein interaction might facilitate the design of receptor antagonists that could be useful in controlling chronic inflammatory diseases. The broad goal is to develop site directed photoaffinity scanning in conjunction with mass spectral analysis as a generally applicable tool for studies of membrane proteins. In addition methods will be developed to elucidate possible structural changes caused by site specific mutagenesis using phage display libraries and affects of photoactive agonist labeling. We have developed a working hypothesis of N- formyl-Met-Leu-Phe (fMLF) binding to the formyl peptide receptor (FPR) based upon a structural model of G-protein coupled receptors as proposed by Baldwin, the known 3D structure of fMLF bound to a specific immunoglobulin, and the structural similarity between retinal and fMLF. We propose to test and refine this working hypothesis using site directed photoaffinity labeling in concert with site directed mutagenesis. We have previously photoaffinity labeled the formyl peptide receptor (FPR) and have used this labeled receptor to monitor its interaction with both G- protein and actin. The present studies will greatly extend the photoaffinity agonist approach by preparing more compact photoaffinity analogues which can be used to map the agonist binding site of FPR. We will use a wide variety of photoaffinity analogues that fully probe the agonist site and sequence the photoaffinity crosslinked sites to provide structural information on the transmembrane organization of the receptor. In addition, using site directed mutagenesis, we will determine those residues which are important in agonist binding and protein folding. This work should provide useful information about the structure of FPR and the primary events in the chemotaxis of phagocytes. It should also serve as a conceptual framework for the study of other heptahelical receptors.

中性粒细胞向入侵细菌的迁移部分取决于 关于它们识别和结合细菌N-甲基化肽的能力 （例如fmlf。）细菌肽的结合后，N-纤维基 肽受体（FPR）激活鸟苷核苷酸结合蛋白（G 蛋白质），将信号转导至细胞内效应分子 引起一系列细胞事件，包括趋化性，溶酶体 酶的分泌和超氧化物的产生。除了正常的主机 国防功能，中性粒细胞在慢性中也起着核心作用 炎症过程。通过调节中性粒细胞的行为， 超氧化物及其代谢物造成的许多组织损伤可能 被阻止。了解甲基肽的分子机制 与FPR结合以及FPR-G蛋白相互作用可能有助于 受体拮抗剂的设计可能有助于控制慢性 炎症性疾病。广泛的目标是开发定向网站 与质谱分析一起作为一个光谱扫描作为一个 通常适用于膜蛋白研究的工具。此外 将开发方法来阐明可能导致的结构变化 使用现场特定诱变，使用噬菌体显示库和影响 光性激动剂标签。我们已经提出了n-的工作假设 与甲基肽受体（FPR）结合的甲基 - 米尔-LEU-PHE（FMLF） 根据提出的基于G蛋白偶联受体的结构模型 由鲍德温（Baldwin），已知的FMLF的已知3D结构与特定 免疫球蛋白以及视网膜和FMLF之间的结构相似性。我们 建议使用定向网站测试和完善此工作假设 与网站定向诱变共同标记。我们有 以前的照片亲和力标记了甲基肽受体（FPR）和 已经使用此标记的受体来监测其与两个G-的相互作用 蛋白质和肌动蛋白。目前的研究将大大扩展 通过准备更紧凑的光恋性，光性激动剂方法 可用于绘制FPR的激动剂结合位点的类似物。我们 将使用各种各样的光性类似物来完全探测 激动剂站点并对光附上的交联位点进行序列以提供 有关受体跨膜组织的结构信息。 此外，使用站点定向诱变，我们将确定 在激动剂结合和蛋白质折叠中很重要的残基。 这 工作应提供有关FPR结构的有用信息 吞噬细胞趋化性的主要事件。它也应该用作 研究其他七值受体的概念框架。