title: Specific binding of urinary-type plasminogen activator (u-PA) to vitronectin and its role in mediating u-PA-dependent adhesion of U937 cells.
abstract: The present paper described interactions of urinary-type plasminogen activator (u-PA) with isolated protein components of the extracellular matrix (ECM) using kinetic and ligand-blotting analyses, as well as adhesion studies with u-PA-saturated U937 monocytic cells. Kinetic analyses showed that fibronectin and laminin were moderately effective at decreasing activation of plasminogen by u-PA (3-4-fold decrease in kcat/Km), while activation was stimulated slightly by collagen types I and IV (2-4-fold increase in kcat/Km). Ligand-blotting experiments using intact immobilized ECM proteins demonstrated that u-PA binds predominantly to vitronectin. This was supported by ELISA studies, which showed concentration dependent, saturable, reversible binding of u-PA to vitronectin (Kd,app. of 97 nM). Limited proteolysis of vitronectin followed by ligand-blotting analysis demonstrated u-PA binding to a specific vitronectin fragment (M(r) 49,000), and binding was shown to occur through the N-terminal fragment of u-PA. N-terminal sequence analysis indicated that this binding fragment of vitronectin originates with Thr-122 and comprises the hemopexin domain, including the heparin-binding region of the vitronectin molecule. Plasminogen activator inhibitor type I did not compete with u-PA for binding to vitronectin, suggesting both molecules may co-localize on vitronectin. In contrast, binding of u-PA to vitronectin was significantly inhibited by plasminogen, suggesting these molecules share a common binding site on vitronectin. In addition to in vitro studies, experiments were performed to assess the contribution of direct binding of u-PA to vitronectin on the adhesive behaviour of U937 cells. Binding of u-PA-saturated U937 cells to vitronectin was inhibited 66% by excess vitronectin, suggesting that direct binding of u-PA to vitronectin is the mechanism by which u-PA-dependent adhesion of U937 cells to vitronectin is mediated.
authors.affiliation: Department of Pathology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: T L
authors.initials: TL
authors.lastname: Moser
authors.forename: J J
authors.initials: JJ
authors.lastname: Enghild
authors.forename: S V
authors.initials: SV
authors.lastname: Pizzo
authors.forename: M S
authors.initials: MS
authors.lastname: Stack
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002448:Cell Adhesion / Q000502:physiology; D004797:Enzyme-Linked Immunosorbent Assay; D016326:Extracellular Matrix Proteins / Q000378:metabolism / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D007948:Leukemia, Monocytic, Acute / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000494:pharmacology; D011485:Protein Binding; D011956:Receptors, Cell Surface / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D019096:Vitronectin
chemical_list: D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; C524898:PLAUR protein, human; D010446:Peptide Fragments; D017395:Plasminogen Activator Inhibitor 1; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D019096:Vitronectin; D014568:Urokinase-Type Plasminogen Activator