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

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.