The long term objective of this proposal is to derive an understanding of the protein biochemistry of cell adhesion to the extracellular matrix. This will be accomplished by examining structure-function relationships of two closely related cell adhesion receptors, integrins alphaIIBbeta3 and alphavbeta3. The proposed studies relate directly to the pathological biochemistry of cancer, osteoporosis and thrombosis. One of the key functional domains of the integrin adhesion receptors is the ligand binding site. Current opinion suggests that this domain encompasses amino-terminal regions of both the alpha and beta subunits of the integrins. However, a structural basis for the remarkably broad range of ligands that the integrins bind has not been put forth. the first specific aim of this proposal is to identify the region of the alpha subunit that confers ligand selection by integrins. This will be achieved by genetically engineering and expressing recombinant """"""""hybrid"""""""" receptors in an attempt to define the region of the receptor that regulates ligand binding and selection. This analysis will focus on substituting sequences from the alphaIIB subunit for homologous sequences within the alphav subunit. The resulting hybrid will be expressed with the beta3 subunit, and the ligand binding phenotype of the hybrid receptor will be assessed to determine which changes enact a phenotypic switch in ligand recognition. The integrin adhesion receptors also contain divalent cation binding sites which regulate the receptors activation state and ligand binding specificity. Little information is available regarding a mechanism and structural basis for this critical regulatory element.
The second aim of this proposal is to determine the number od divalent cation binding sites on the integrin which must be occupied for ligand binding to occur. This will be accomplished by performing direct binding studies between purified integrins and divalent cation. The central hypothesis of the proposed studies is that the site on the integrin alpha subunit that confers ligand binding specificity is identical to the divalent cation binding site that triggers ligand binding. this could explain the profound effect divalent cations have on ligand selection by the integrins. This hypothesis will be tested by identifying the location of the critical divalent cation binding site with a series of standard biochemical approaches including ligand blotting and site-specific protein modification. Results from this analysis will be compared to results from aim #1 to determine if the ligand contact site is also a regulatory divalent cation binding site. It is anticipated that successful completion of these aims will significantly advance current understanding of cell-extracellular matrix interactions.
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