Blood cells such as platelets, lymphocytes and neutrophils exert their physiological function through adhesive interactions with the extracellular matrix or with other cells. The integrins, a family of transmembrane alpha, beta heterodimers which mediate the adhesion of many of these cell types, are notable for their dynamic regulation. In response to external stimuli, integrins upregulate ligand binding affinity and alter cellular adhesiveness. Preliminary data suggest that integrin """"""""activation"""""""" or affinity modulation involves cell- specific signalling through integrin cytoplasmic domains. In this proposal, the PI will elucidate this physiological pathway. First, he will identify alpha and beta subunit cytoplasmic residues which are involved. Using extracellular alphaIIb, beta3 as a reporter group, he will generate variant cytoplasmic domains, transfect these constructs into heterologous cells and analyze their ability to bind antibody or protein ligands. Second, he will examine the effect of overexpression of integrin cytoplasmic domains on physiological activation mechanisms. Chimera consisting of the extracellular and transmembrane regions of the IL-2 receptor joined to specific integrin cytoplasmic domains have been obtained. These constructs will be co-transfected with constitutively active integrins and the inhibitory effect on binding analyzed. Finally, he will attempt to identify hypothetical cytosolic factors which interact with integrin cytoplasmic domains. Novel factors will be isolated and identified in crosslinking studies utilizing the inhibitory chimera identified above, or by regenerating the GAL4 transcription factor in a two hybrid system. Defining the mechanisms of affinity modulation is crucial to understanding the role of integrins in the physiology and pathology of blood cells.
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