FMLF BINDING SITE ON FORMYL PEPTIDE RECEPTOR

甲酰肽受体上的 FMLF 结合位点

• 批准号: 2672811
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 10.19万
• 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/2672811
• 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依赖于 识别和结合细菌 N-甲酰化肽的能力 （例如 fMLF。）与细菌肽结合后，N-甲酰基 肽受体 (FPR) 激活鸟苷酸结合蛋白 (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。除了普通主机外 防御功能，中性粒细胞在慢性疾病中也发挥着核心作用 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引起的大部分组织损伤可能 被阻止。了解甲酰肽的分子机制- 与 FPR 的结合以及 FPR-G 蛋白相互作用可能会促进 设计可用于控制慢性病的受体拮抗剂 炎症性疾病。总体目标是开发站点定向 光亲和扫描与质谱分析相结合 用于膜蛋白研究的普遍适用的工具。此外 将开发方法来阐明可能引起的结构变化 通过使用噬菌体展示库的位点特异性诱变及其影响 光活性激动剂标记。我们提出了一个工作假设 N- 甲酰基-Met-Leu-Phe (fMLF) 与甲酰基肽受体 (FPR) 结合 基于所提出的 G 蛋白偶联受体的结构模型 Baldwin 提出，已知的 fMLF 3D 结构与特定的 免疫球蛋白，以及视网膜和 fMLF 之间的结构相似性。我们 提议使用站点定向来测试和完善这个工作假设 光亲和标记与定点诱变相结合。我们有 先前光亲和标记甲酰肽受体（FPR）和 已经使用这种标记受体来监测其与 G- 蛋白质和肌动蛋白。目前的研究将大大扩展 通过制备更紧凑的光亲和力的光亲和力激动剂方法 可用于绘制 FPR 激动剂结合位点图谱的类似物。我们 将使用各种光亲和类似物来充分探测 激动剂位点并对光亲和交联位点进行测序以提供 有关受体跨膜组织的结构信息。 此外，使用定点诱变，我们将确定那些 对激动剂结合和蛋白质折叠很重要的残基。 这 工作应提供有关 FPR 结构和 吞噬细胞趋化作用的主要事件。它也应该作为一个 其他七螺旋受体研究的概念框架。