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 potentia tion of plasmin generation. This is dependent on the concurrent cellular bi nding of plasminogen, and is completely abrogated by the plasminogen lysine -binding site ligand, 6-aminohexanoic acid. Previous data have provided cir cumstantial evidence for the formation of specific complexes to mediate the kinetically favorable reciprocal interactions between the protease and zym ogen components [Ellis, V., and Dano, K. (1993) J. Biol. Chem. 268, 4806-48 13]. To further investigate the formation of these putative complexes, we h ave studied the effect of various lysine-binding site ligands on the bindin g and activation of plasminogen on U937 cells. Lysine-binding site ligands resembling internal lysine residues, such as Na-acetyl-L-lysine methyl este r, were found to specifically inhibit uPAR-mediated cell-surface plasminoge n activation at concentrations up to 40-fold lower than those inhibiting th e cellular binding of I-125-labeled plasminogen (IC(50)s 300 mu M vs 8.5 mM ). By contrast, 6-aminohexanoic acid, resembling a C-terminal lysine residu e, did not display this disparity (IC(50)s 25 vs 30 mu M). These lysine ana logues were also found to compete a non-active-site interaction between uPA and plasminogen, detected by surface plasmon resonance (K-d 50 nM), at con centrations correlating with their effect on cell-surface plasminogen activ ation, suggesting that this interaction is part of the kinetic mechanism. C onsistent with this, synthetic peptides corresponding to the sequence uPA(1 49-158) (GQKTLRPRFK) and UpA(149-157) (GQKTLRPRF) specifically abolished th e amplification of cell-surface plasminogen activation. These data demonstr ate that a novel non-active-site interaction between uPA and plasminogen is necessary for the assembly and efficiency of cell-surface plasminogen acti vation complexes.