Recognition of Fibrinogen by Leukocyte Integrins

描述（申请人提供）：白细胞整合素aM <$2（CD 11b/CD 18，Mac-1）在正常保护性炎症反应和病理性炎症中起关键作用。这种受体具有惊人的粘附和信号传导能力，使其成为宿主防御中的首要主力。它也是许多疾病的潜在治疗靶点，其中炎症起着重要作用，包括心血管疾病。aM <$2的不同功能和活性源于其结合多种结构不同配体的能力。然而，允许aM <$2表现出广泛的配体识别的机制仍然知之甚少。我们以前的研究与原型aM <$2配体纤维蛋白原提供了初步的洞察机制，通过该受体的aM-域识别其配体。在过去的资助期内，我们已经解决了共识的aMI结构域识别基序，我们称之为p53。一个关键的特点是一个小的核心组成的特定组合的碱性和疏水氨基酸残基普遍存在于许多aM <$2配体。这些特征与aM <$2识别各种不相关序列的能力一致，从而形成了aM <$2配体结合混杂的分子基础。特异性目的1是进一步表征aM <$2的广泛识别特异性的机制。将使用组合肽库和突变分析来阐明ESTs的结构特征。质谱法将用于确定炎症相关蛋白质修饰对炎症蛋白功能的影响。我们的初步研究表明，中性粒细胞分泌产物富含IRMs，这使得它们被预测为一类新的aM？2配体。我们已经发现，其中之一，人嗜中性粒细胞cathelicidin肽LL-37，有效地结合aMb 2，并诱导一个有效的aM 2依赖性迁移反应。基于这一发现，我们提出LL-37和其他嗜中性粒细胞衍生的蛋白质/肽通过结合单核细胞/巨噬细胞上的aM？2发挥其有效的免疫调节作用。具体目标2是通过表征LL-37引起的α M2依赖性单核细胞应答来检验该假设。LL-37对aM <$2的信号传导和迁移功能的影响将使用aM <$2表达和aM <$2缺陷细胞并在体内动物模型中测定。在过去的资助期内，研究将整合素aD <$2鉴定为具有与aM <$2相似的特异性的多配体受体，并揭示其对炎性巨噬细胞的上调抑制其迁移。具体目标3是表征α M <$2和α D <$2（巨噬细胞上两种最丰富和粘附的整合素）在炎症消退期间这些细胞从炎症部位迁移中的作用。将在野生型和整合素缺陷小鼠中研究巨噬细胞通过引流淋巴细胞的流出。总的来说，这些研究将导致对aM <$2的配体识别原理的进一步理解，将对aM <$2和aD <$2的生物学提供新的见解，并可能有助于设计新的治疗策略。 公共卫生相关性：炎症与许多疾病的发病机制密切相关，包括心血管疾病。整合素α M <$2（Mac-1）是白细胞上最通用的受体，并且在炎症反应期间介导这些细胞的许多反应。Mac-1所表现出的功能多样性取决于它结合无数不同蛋白质的能力。了解Mac-1极端粘性的分子基础和受体广泛识别的生物学意义可能会导致治疗炎症发挥作用的疾病的新方法。

DESCRIPTION (provided by applicant): Leukocyte integrin aM¿2 (CD11b/CD18, Mac-1) plays a pivotal role in normal protective inflammatory response and pathological inflammation. This receptor has prodigious adhesive and signaling capabilities which allowed it to become the premier workhorse in host defense. It is also a potential therapeutic target in many diseases in which inflammation plays an essential role, including cardiovascular diseases. The diverse functions and activities ascribed to aM¿2 arise from its ability to bind a multitude of structurally diverse ligands. However, the mechanisms which allow aM¿2 to exhibit broad ligand recognition are still poorly understood. Our previous studies with a prototype a aM¿2 ligand fibrinogen provided initial insight into the mechanism by which the aMI-domain of the receptor recognizes its ligands. In the past funding period we have solved the consensus aMI-domain recognition motif, we termed IRM. A key feature of IRM is a small core consisting of specific combinations of basic and hydrophobic amino acid residues ubiquitous in many aM¿2 ligands. The characteristics of IRM are consistent with the capacity of aM¿2 to recognize a wide variety of unrelated sequences and, thus, form a molecular basis for aM¿2 ligand binding promiscuity. Specific Aim1 is to further characterize the mechanism underlying broad recognition specificity of aM¿2. Combinatorial peptide libraries and mutational analyses will be used to clarify the structural features of IRM. Mass spectrometry will be used to determine the effect of inflammation-associated protein modifications on the function of IRM. Our preliminary studies revealed that neutrophil secretion products are enriched in IRMs which allowed their prediction as a new class of aM¿2 ligands. We have found that one of them, human neutrophil cathelicidin peptide LL-37, effectively binds aMb2 and induces a potent aM¿2- dependent migratory response. Based on this finding we propose that LL-37 and other neutrophil-derived proteins/peptides exert their potent immunomodulatory effects by binding aM¿2 on monocyte/macrophages. Specific Aim 2 is to test this hypothesis by characterizing aM¿2-dependent monocyte responses elicited by LL-37. The effect of LL-37 on signaling and migratory functions of aM¿2 will be determined using aM¿2- expressing and aM¿2-deficient cells and in the in vivo animal model. Studies over the past funding period identified integrin aD¿2 as a multi-ligand receptor with specificity similar to that of aM¿2 and revealed that its upregulation on inflammatory macrophages inhibits their migration. Specific Aim 3 is to characterize the role of aM¿2 and aD¿2, two most abundant and adhesive integrins on macrophages, in emigration of these cells from the inflammatory site during the resolution of inflammation. The efflux of macrophages by draining lymphatics will be investigated in wild-type and integrin-deficient mice. Overall, these studies will lead to an increased understanding of the principles which govern ligand recognition by aM¿2, will give new insights into the biology of aM¿2 and aD¿2 and may be useful in the design of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: Inflammation is critically involved in the pathogenesis of many disorders, including cardiovascular disease. Integrin aM¿2 (Mac-1) is the most versatile receptor on leukocytes and mediates numerous responses of these cells during the inflammatory response. The multiplicity of functions exhibited by Mac-1 depends on its ability to bind a myriad of diverse proteins. Understanding the molecular basis for the extreme stickiness of Mac-1 and the biological significance of receptor's broad recognition could lead to new methods of treatment of disorders in which inflammation plays a role.