This renewal application proposes to continue ongoing studies of the structure and function of integrin (cytoadhesin) adhesion receptors. Integrins, cell-surface heterodimeric molecules, mediate a very broad range of cell-cell and cell-matrix interactions and, thereby, control a diverse set of cellular functions and responses. Two integrins, alpha4/beta1, and alphaM/beta2, have been implicated in many physiological events, including the development of the immune response and hematopoiesis, and pathophysiologic events, including inflammation, reprefusion injury and atherosclerosis. The overall objective of this proposal is to establish the molecular basis for ligand binding to these two integrins as a means of understanding their function. It is further anticipated that insights derived from studies of these two integrins will be broadly applicable and contribute to a general understanding of the ligand binding functions of integrin receptors. Small peptide ligands for alpha4/beta1 and alphaM/beta2 have been identified, and these will be used to define the precise recognition specificities of these two receptors. Covalent crosslinking strategies, similar to those which we have successfully employed to identify ligand recognition sites in alphaIIb/beta3, will then be used to localize sequences within these integrins that mediate ligand binding. Synthetic peptides, monoclonal antibody and mutational strategies will then be brought to bear to definitively establish the role of identified regions in receptor function. Independent strategies, cobalt oxidation to evaluate the role of the metal binding domains and hydropathic complementarity, will also be applied to define the ligand binding mechanisms of alpha4/beta1 and alphaM/beta2. In addition to these studies, a novel hypothesis that fibrinogen functions as a bridging molecule for linking leukocytes to endothelium will be tested. Fibrinogen markedly enhances alphaM/beta2-dependent leukocyte adhesion to endothelial cells, and the molecular and cellular specificity of this effect will be assessed under both static and flowing conditions. By defining the molecular basis for ligand binding functions of alphaM/beta2 and alpha4/beta1 and other integrins and by providing insights into the basis for leukocyte : endothelial cell interactions, which are central to many physiological and pathophysiological processes, our studies have the potential to lead to new approaches to treat inflammation, atherosclerosis and other vascular disorders and diseases.
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