Recognition of fibrinogen by leukocyte integrins

白细胞整合素对纤维蛋白原的识别

• 批准号: 6917095
• 负责人: Tatiana P Ugarova
• 金额: $ 30.6万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6917095
• 关键词: binding sites; cell adhesion; cell migration; clinical research; extracellular matrix; fibrinogen; genetically modified animals; human subject; inflammation; integrins; intermolecular interaction; laboratory mouse; leukocytes; ligands; neutrophil; phage display; protein binding; protein sequence; site directed mutagenesis

DESCRIPTION (provided by applicant): Integrin-alpha-M-beta-2 (CD11b/CD18, Mac-l) plays a pivotal role in the inflammatory response and host defense. By mediating critical adhesive reactions of neutrophils it participates in regulating neutrophil influx to the sites of inflammation and initiates numerous neutrophil responses. This receptor is also a potential therapeutic target in many diseases in which the inflammatory component plays an essential role. Alpha-M-beta-2 exerts its functions by binding a multitude of biologically diverse molecules. However, the mechanism which allows alpha-Mbeta-2 to exhibit broad specificity and the biological value of alpha-M-beta-2 ligand promiscuity are not known. Our long term goal is to understand the molecular basis for ligand recognition by alpha-M-beta-2, and to determine how alpha-M-beta-2 ligand promiscuity affects its adhesive functions. Studies over the past funding period have mapped the alpha-M-beta-2-binding site in the physiologically important ligand fibrinogen, and identified the recognition peptide that effectively disrupted alpha-M-beta-2-mediated cell adhesion not only to fibrinogen but also to many other ligands. Based on these findings, we have hypothesized that the fibrinogen recognition motif contains critical structural information required for alpha-M-beta-2 binding and that it may serve as a prototype adhesive signal in many alpha-M-beta-2 ligands. We will test this hypothesis by analyzing alpha-M-beta-2-binding sites in fibrinogen and in other physiological ligands using combinatorial peptide libraries and mutational analyses of fibrinogen domains. We will also use phage display to delineate the general principles that control alpha--beta-2 ligand recognition. Our data further indicated that a discrete region in the alpha-M-I-domain of alpha-M-beta-2 participates in binding of numerous ligands which has led us to propose the hypothesis that the unique region in the alpha-M-I-domain constitutes the consensus binding site for recognition of many ligands. Through the use of chimeric and point mutations between alpha-M-beta-2 and alpha-L-beta-2, the related beta-2 integrin with a different ligand specificity, we will determine the structural features required for the interaction of the consensus binding site with multiple ligands. Finally, the impact of alpha-M-beta-2's multi-ligand binding potential on its adhesive functions will be studied. We hypothesize that the alpha-M-beta-2 ability to be upregulated to high density on the surface of neutrophils and its capacity to engage numerous ligands in the extracellular matrix may modulate general cell adhesiveness and, thus, stop neutrophil migration at the site of inflammation. We will test this hypothesis by manipulating alpha-M-beta-2 expression on the surface of neutrophils and will also use neutrophils with alpha-M-beta-2 deficiency. Together, these studies will provide important insights into the structural basis of alpha-m-beta-2 ligand binding and define the mechanism by which alpha-M-beta-2 binding promiscuity controls leukocyte adhesive functions. Thus, these studies may lead to an increased understanding of the principles that govern ligand recognition by integrins and will be useful in the design of novel therapeutic agents to specifically disrupt alpha-M-beta-2-ligand interactions

描述（由申请人提供）：整联蛋白-Alpha-M-Beta-2（CD11b/CD18，MAC-L）在炎症反应和宿主防御中起关键作用。通过介导中性粒细胞的临界粘附反应，它参与调节嗜中性粒细胞向炎症部位的涌入，并引发许多中性粒细胞反应。在许多疾病中，炎症成分起着至关重要的作用，该受体也是潜在的治疗靶标。 Alpha-M-Beta-2通过结合多种生物学上不同的分子来发挥其功能。但是，允许α-MBETA-2表现出广泛特异性的机制和α-M-Beta-2配体滥交的生物学价值。我们的长期目标是了解Alpha-M-Beta-2识别配体识别的分子基础，并确定alpha-M-Beta-2配体滥交如何影响其粘附功能。在过去的资金期间，研究在生理上重要的配体纤维蛋白原中绘制了α-M-Beta-2结合位点，并确定了有效破坏α-M-Beta-2-2介导的细胞粘附的识别肽不仅对纤维蛋白原，而且还也对许多其他配体。基于这些发现，我们假设纤维蛋白原识别基序包含α-M-Beta-2结合所需的关键结构信息，并且可以用作许多Alpha-M-Beta-2配体中的原型粘合信号。我们将通过分析纤维蛋白原和其他生理配体中的α-M-Beta-2结合位点，使用组合肽库和纤维蛋白原域的突变分析来检验该假设。我们还将使用噬菌体显示来描述控制alpha-beta-2配体识别的一般原则。我们的数据进一步表明，Alpha-M-Beta-2α-M-I域中的一个离散区域参与了众多配体的结合，这使我们提出了以下假设：Alpha-M-I-I-ragain中的独特区域构成共识结合位点，以识别许多配体。通过使用alpha-M-Beta-2和alpha-l-beta-2之间的嵌合和点突变，具有不同配体特异性的相关β-2整合蛋白，我们将确定共识相互作用所需的结构特征具有多个配体的结合位点。最后，将研究Alpha-M-Beta-2多配体结合电位对其粘合剂功能的影响。我们假设α-M-Beta-2能力将中性粒细胞表面上调至高密度及其在细胞外基质中接合大量配体的能力可能调节一般细胞的粘附性，从而阻止中性粒细胞迁移在炎。我们将通过在中性粒细胞表面操纵α-M-Beta-2表达来检验这一假设，并且还将使用具有α-M-Beta-2缺乏症的中性粒细胞。总之，这些研究将为α-M-Beta-2配体结合的结构基础提供重要的见解，并定义了Alpha-M-Beta-2结合杂交控制白细胞粘附功能的机制。因此，这些研究可能会导致人们对控制配体识别的原理的更多了解，并将在新型治疗剂的设计中有用，以特别破坏Alpha-M-Beta-2-2-rigand相互作用