Urokinase binding to laminin‐nidogen

Recently we have shown that heparin and related sulfated polyanions are low‐affinity ligands of the kringle domain in the amino‐terminal region (ATF) of human urokinase (u‐PA), and proposed that this may facilitate loading of u‐PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin‐nidogen) for u‐PA binding, and found that laminin‐nidogen is also a ligand of the u‐PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin‐nidogen showed that there are u‐PA binding sites in fragment E4 of laminin as well as in nidogen. The long‐arm terminal domain of laminin (fragment E3), which contains a heparin‐binding site, competed for binding of u‐PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u‐PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy‐terminal domain. Direct binding assays confirmed that u‐PA binds to nidogen through a site in the u‐PA ATF. We conclude that u‐PA binds to laminin‐nidogen by interactions involving the ATF region of u‐PA, the E4 domain of laminin and the rod or amino‐terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u‐PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.