A region in domain II of the urokinase receptor required for urokinase binding

The urokinase receptor is composed of three homologous domains based on dis ulfide spacing. The contribution of each domain to the binding and activati on of single chain urokinase (scuPA) remains poorly understood. In the pres ent paper we examined the role of domain II (DII) in these processes. Repos itioning DII to the amino or carboxyl terminus of the molecule abolished bi nding of scuPA as did deleting the domain entirely. By using alanine-scanni ng mutagenesis, we identified a g-amino acid continuous sequence in DII (Ar g(137)-Arg(145)) required for both activities. Competition-inhibition and s urface plasmon resonance studies demonstrated that mutation of Lys(139) and His(143) to alanine in soluble receptor (suPAR) reduced the affinity for s cuPA similar to 5-fold due to an increase in the "off rate." Mutation of Ar g(137), Arg(142), and Arg(145), each to alanine, leads to an similar to 100 -fold decrease in affinity attributable to a 10-fold decrease in the appare nt "on rate" and a 6-fold increase in off rate. These differences were conf irmed on cells expressing variant urokinase receptor. suPAR-K139A/H143A dis played a 50% reduction in scuPA-mediated plasminogen activation activity, w hereas the 3-arginine variant was unable to stimulate scuPA activity at all . Mutation of the three arginines did not affect binding of a decamer pepti de antagonist of scuPA known to interact with DI and DIII. However, this mu tation abolished both the binding of soluble DI to DII-III in the presence of scuPA and the synergistic activation of scuPA mediated by DI and wild ty pe DII-DIII. These data show that DII is required for high affinity binding of scuPA and its activation. DII does not serve merely as a spacer functio n but appears to be required for interdomain cooperativity.