Vitronectin, also known as serum spreading factor, is a major cell adhesion molecule present in the plasma and in the subendothelial matrix. At least three distinct cell surface receptors for vitronectin (alphaVb3, alphaIIbbeta3 and alphaVbeta5) belonging to the integrin superfamily, have been identified. The ligand-receptor interaction is mediated by the RGD sequence in vitronectin. Despite the fact that all the ligands have the RGD sequence, the receptors are able to distinguish various RGD-containing ligands. Thus, the fibronectin receptor does not interact with vitronectin. Among the three vitronectin receptors, only alphaVbeta5 does not interact with fibrinogen and von Willebrand protein, and only alphaIIbbeta3 recognize, in addition the RGD, the sequence HLGGAKQAGDV in fibrinogen. The mechanism of receptor specificity is not clear. One hypothesis suggests that amino acid sequences adjacent to the RGD site determine its conformation thereby imparting specificity while other hypothesis suggest additional receptor-specific binding site(s). We have expressed human recombinant wild-type vitronectin in Baby Hamster Kidney cells. The isolated wild-type vitronectin promotes cell adhesion similar to plasma derived vitronectin. Three mutants, VN(Arg45 to Leu 45), VN(Gly46 to Ala46) and VN(Asp47 to Glu47) were also expressed and isolated from BHK cells. Panc-1 cells containing alphaVbeta5, showed markedly decreased adhesion (more than 50-100-fold) to these mutants. In contrast, the interactions of alphaIIbbeta3 and alphaVbeta3 were inhibited less dramatically (2-10-fold). Thus, these receptors display varying degrees of specificity to the RGD site, aVb5 being most specific and least tolerating substitutions at the RGD site while alphaIIbbeta3 and alphaVb3 display relaxed specificity tolerating substitutions even at the RGD site. The primary aim of this proposal is to further define the amino acid residues, in addition to RGD, that are necessary for recognition by the vitronectin receptors aVb3, aVb5 and alphaIIbbeta3, using site-directed and deletion mutations of vitronectin. Vitronectin also forms a complex with plasminogen activator inhibitor-1 both in the plasma and in the subendothelium, stabilizing it from rapid inactivation. This interaction may modulate plasmin-mediated degradation of subendothelium, a critical event in neoangiogenesis, wound healing and metastasis. Additional aims of this proposal are to define the amino acids involved in PAI-I binding and to delineate the role of glycosaminoglycan binding site in the adhesion, spreading and stress fiber formation by the endothelial cells.
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