A key mechanism for modulating enzyme activity involves inhibition of proteinases by plasma and cell derived proteinase inhibitors and removal of the proteinase-inhibitor complexes via binding to cell surface receptors. Alpha 2M is a large molecular weight plasma proteinase inhibitor that exhibits an extremely broad specificity, reacting with serine, cysteine, aspartic and metallo proteinases. Studies have documented that alpha 2M is an effective inhibitor of plasmin, collagenase kallikrein, Factor Xa, thrombin and leukocyte elastase. The reaction of a proteinase with alpha 2M is associated with conformational changes occurring in the inhibitor that have two consequences In the molecule. First of all,these alterations lead to entrapment or inhibition of the proteinase, and secondly they generate new determinants on the complex that are recognized by cell surface receptors. The removal of alpha 2M-proteinase complexes by receptor mediated endocytosis is especially important, since the complex still retains some proteolytic activity, and if this complex were to remain in the circulation, it would be a stable source of proteinase activity. The overall objectives of this proposal are to (a) deduce the structure of the alpha 2M receptor, (b) to identify important functional regions of the receptor, and (e) to elucidate the function of this receptor system and establish its relationship with other receptor systems. Anti- receptor antibodies or limited sequence information derived from the purified receptor will be utilized to identify alpha 2M receptor cDNA. Sequencing of the cDNA will allow an analysis of the deduced amino acid sequence of this receptor and a comparison of its structure with that of other receptors. The receptor cDNA will be utilized to screen a library in order to obtain the human alpha 2M receptor gene which will allow a comparison of the organization of the alpha 2M receptor gene with the gene of other coated pit receptors. Identification of functional domains of the receptor that are involved with ligand binding will be accomplished by immunological approaches, deletion studies, and site-directed mutagenesis. The role of this receptor system in macrophages will be investigated by examining the changes in gene expression that occur during monocyte activation.
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