The mechanism by which single-chain urokinase (scuPA) binds to its receptor (uPAR) is incompletely understood. We report that a fragment comprising the first domain of recombinant soluble uPAR (sDI) as well as a fragment comprising the remaining domains (sDII-DIII) competes with the binding of recombinant fulllength soluble uPAR (suPAR) to scuPA with an IC 50 ‫؍‬ 253 nM and an IC 50 ‫؍‬ 1569, respectively. sDII-III binds directly to scuPA with K d ‫؍‬ 238 nM. Binding of scuPA to each fragment

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... so induces the expression of plasminogen activator activity. sDI and sDII-DIII (200 nM each) induced activity equal to 66 and 36% of the maximum activity induced by full-length suPAR (5 nM), respectively. Each fragment also stimulates the binding of scuPA to cells lacking endogenous uPAR. Although scuPA binds to sDI and to sDII-DIII through its aminoterminal fragment, the fragments act synergistically to inhibit the binding of suPAR and to stimulate plasminogen activator activity. Furthermore, sDII-DIII retards the velocity and alters the pattern of cleavage of sDI by chymotrypsin. These results suggest that binding of scuPA to more than one epitope in suPAR is required for its optimal activation and association with cell membranes. Binding of urokinase (uPA) 1 to its receptor may play an important role in inflammation (1, 2) and the development of tumor metastases (3) , among other processes. The urokinase receptor (uPAR) is a single-chain glycoprotein that is attached to cell membranes by a glycosylphosphatidylinositol-linked receptor (3, 4). Binding of single chain urokinase (scuPA) to uPAR stimulates its enzymatic activity (5), provides relative protection from plasmin and plasminogen activator type 1 (6),