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Assembly of urokinase receptor-mediated plasminogen activation complexes involves direct, non-active-site interactions between urokinase and plasminogen.

Ellis, V; Whawell, SA; Werner, F; Deadman, JJ; (1999) Assembly of urokinase receptor-mediated plasminogen activation complexes involves direct, non-active-site interactions between urokinase and plasminogen. Biochemistry , 38 (2) pp. 651-659. 10.1021/bi981714d.

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Abstract

The binding of the zymogenic form of urokinase-type plasminogen activator (pro-uPA) to its specific cellular receptor, uPAR, leads to a large potentiation of plasmin generation. This is dependent on the concurrent cellular binding of plasminogen, and is completely abrogated by the plasminogen lysine-binding site ligand, 6-aminohexanoic acid. Previous data have provided circumstantial evidence for the formation of specific complexes to mediate the kinetically favorable reciprocal interactions between the protease and zymogen components [Ellis, V., and Dano, K. (1993) J. Biol. Chem. 268, 4806-4813]. To further investigate the formation of these putative complexes, we have studied the effect of various lysine-binding site ligands on the binding and activation of plasminogen on U937 cells. Lysine-binding site ligands resembling internal lysine residues, such as Nalpha-acetyl-L-lysine methyl ester, were found to specifically inhibit uPAR-mediated cell-surface plasminogen activation at concentrations up to 40-fold lower than those inhibiting the cellular binding of 125I-labeled plasminogen (IC50s 300 microM vs 8.5 mM). By contrast, 6-aminohexanoic acid, resembling a C-terminal lysine residue, did not display this disparity (IC50s 25 vs 30 microM). These lysine analogues were also found to compete a non-active-site interaction between uPA and plasminogen, detected by surface plasmon resonance (Kd 50 nM), at concentrations correlating with their effect on cell-surface plasminogen activation, suggesting that this interaction is part of the kinetic mechanism. Consistent with this, synthetic peptides corresponding to the sequence uPA149-158 (GQKTLRPRFK) and uPA149-157 (GQKTLRPRF) specifically abolished the amplification of cell-surface plasminogen activation. These data demonstrate that a novel non-active-site interaction between uPA and plasminogen is necessary for the assembly and efficiency of cell-surface plasminogen activation complexes.

Type: Article
Title: Assembly of urokinase receptor-mediated plasminogen activation complexes involves direct, non-active-site interactions between urokinase and plasminogen.
Location: United States
DOI: 10.1021/bi981714d
Keywords: Aminocaproic Acid, Binding Sites, Biosensing Techniques, Cell Membrane, Enzyme Activation, Enzyme Inhibitors, Humans, Lysine, Macromolecular Substances, Oligopeptides, Peptide Fragments, Plasminogen, Plasminogen Activators, Receptors, Cell Surface, Receptors, Urokinase Plasminogen Activator, U937 Cells, Urokinase-Type Plasminogen Activator
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences > Structural and Molecular Biology
URI: http://discovery.ucl.ac.uk/id/eprint/151184
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