REGULATION OF CHEMOATTRACTANT ACTIVATION OF NEUTROPHILS

中性粒细胞趋化剂激活的调节

• 批准号: 6170222
• 负责人: ALGIRDAS JOSEPH JESAITIS
• 金额: $ 21.26万
• 依托单位: MONTANA STATE UNIVERSITY - BOZEMAN
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-01 至 2004-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6170222
• 关键词: CHO cells; Escherichia coli; G protein; actins; active sites; biological signal transduction; cell free system; chemical association; chemical substitution; chemoattractants; conformation; guanosinetriphosphatases; human tissue; laboratory rabbit; leukocyte activation /transformation; membrane proteins; neutrophil; nuclear magnetic resonance spectroscopy; phosphorylation; protein sequence; protein structure function; receptor; receptor coupling; recombinant proteins; site directed mutagenesis; synthetic peptide

The long term goal of the proposed research is to determine the molecular basis of activation and regulation of chemoattractant-induced neutrophil responses initiated by the occupancy of N-formyl chemotactic peptide receptors (FPR). FPR belong to the family of heptahelical receptors which transmits a signal through guanyl nucleotide binding proteins (G proteins). This receptor family has more than 2-3 hundred members and is therefore of central importance for a great many signal transduction pathways in cells. In addition to the interaction of FPR with G proteins, interactions of FPR with the membrane skeletal actin have been implicated in regulation of signal transduction. The research proposed here seeks to identify and structurally characterize the intracellular receptor domains which are interacting with G proteins and membrane skeletal proteins. For this purpose peptides with sequences from the predicted intracellular domains of the FPR will be synthesized and then utilized to study their effect on physical and functional coupling of FPR to signal transduction or regulatory proteins. The physical coupling will be studied with a recently developed reconstitution assay for FPR-G protein complexes which are separated from G protein-free receptors by velocity sucrose density gradients. Functional coupling will be evaluated by measuring peptide perturbation of high affinity agonist binding, receptor-mediated stimulation of U protein GTPase activity, formyl peptide-induced superoxide production and actin polymerization in electroporated cells, and in vitro actin polymerization stimulated by formyl peptides. Peptide mimetics in these studies will also be used in combination to uncover synergistic interactions of peptide pairs which might be expected because of the multi-site nature of these interactions. The three-dimensional structure of peptides identified as being part of regulatory protein- protein contacts will be studied in solution with NMR methods including COSY, TOCSY, ROESY, NOESY, and Tr-NOESY. In addition, the structure of the receptor peptides bound to G protein and regulatory proteins will be determined using Tr-NOESY NMR. The structural information obtained will then be used to identify amino acids critical to the predicted bound structures. These amino acids will then be substituted in recombinant FPR expressed in CHO cells by amino acids that would either interfere with or promote the formation of such three-dimensional conformations, thus providing functional confirmation of these structures. Such studies will be the basis for the development of synthetic peptide derivatives or even non-peptide analogs which may permit the design of new classes of drugs which can specifically inhibit signal transduction pathways important in the pathogenesis of inflammatory diseases.

拟议研究的长期目标是确定分子 趋化剂诱导的中性粒细胞激活和调节的基础 由 N-甲酰趋化肽占据引发的反应 受体（FPR）。 FPR 属于七螺旋受体家族 通过鸟苷酸结合蛋白（G 蛋白质）。该受体家族有 2-300 多个成员， 因此对于许多信号转导至关重要 细胞内的通路。除了 FPR 与 G 蛋白的相互作用外， FPR 与膜骨架肌动蛋白的相互作用已被证实 在信号转导的调节中。这里提出的研究旨在 识别和结构表征细胞内受体结构域 与 G 蛋白和膜骨骼蛋白相互作用。为了 此目的肽具有来自预测的细胞内的序列 FPR 的域将被合成，然后用于研究它们 FPR 与信号转导的物理和功能耦合的影响 或调节蛋白。物理耦合将通过 最近开发的 FPR-G 蛋白复合物重构测定法 通过速度蔗糖密度与无 G 蛋白的受体分离 梯度。通过测量肽来评估功能耦合 受体介导的高亲和力激动剂结合的扰动 甲酰肽诱导的 U 蛋白 GTP 酶活性刺激 电穿孔细胞中的超氧化物产生和肌动蛋白聚合， 以及甲酰基肽刺激的体外肌动蛋白聚合。肽 这些研究中的模拟物也将结合使用来揭示 肽对的协同相互作用可能是预期的，因为 这些交互的多站点性质。三度空间 被鉴定为调节蛋白一部分的肽的结构- 将使用 NMR 方法研究溶液中的蛋白质接触，包括 COSY、TOCSY、ROESY、NOESY 和 Tr-NOESY。此外，该结构的 与 G 蛋白和调节蛋白结合的受体肽将 使用Tr-NOESY NMR测定。获得的结构信息将 然后用于识别对预测结合至关重要的氨基酸 结构。然后这些氨基酸将在重组 FPR 中被取代 在 CHO 细胞中表达的氨基酸会干扰或 促进这种三维构象的形成，从而 提供这些结构的功能确认。此类研究将 成为开发合成肽衍生物的基础，甚至 可能允许设计新类别药物的非肽类似物 它可以特异性抑制重要的信号转导途径 炎症性疾病的发病机制。