REGIONS INVOLVED IN BINDING OF UROKINASE-TYPE-1 INHIBITOR COMPLEX ANDPROUROKINASE TO THE ENDOCYTIC ALPHA(2)-MACROGLOBULIN RECEPTOR LOW-DENSITY-LIPOPROTEIN RECEPTOR-RELATED PROTEIN - EVIDENCE THAT THE UROKINASE RECEPTOR PROTECTS PROUROKINASE AGAINST BINDING TO THE ENDOCYTIC RECEPTOR

The alpha(2)-macroglobulin receptor/low density lipoprotein receptor-r elated protein (alpha(2)MR/LRP) binds several ligands, including compl ex between the two chain urokinase-type plasminogen activator (uPA) an d type-1 plasminogen activator inhibitor (PAI-1), and the single chain zymogen pro-urokinase (pro-uPA). We have used truncated variants of u PA and PAI-1 as well as Fab fragments of monoclonal antibodies with kn own epitopes to identify regions in the uPA PAI-1 complex and in pro-u PA involved in binding to alpha(2)MR/LRP. uPA PAI-1 complex bound with high affinity (EC(50) about 0.4 nM) via contacts in the PAI-1 moiety as well as the uPA serine proteinase domain and the uPAA chain. Pro-uP A bound with lower affinity (EC(50) about 10 nM), and efficient bindin g to alpha(2)MR/LRP was dependent on contact with both the A chain and the serine proteinase domain. We analyzed the effect of complex forma tion with the urokinase receptor since this is the primary target for binding of uPA.PAI-1 and pro-uPA at the cell surface, and since it has been demonstrated that urokinase receptor-bound uPA PAI-1 complex is internalized following interaction with alpha(2)MR/LRP (Nykjaer, A., P etersen, C. M., Moller, B., Jensen, P. H., Moestrup, S. K., Holtet, T. L., Etzerodt, M., Thogersen, H. C., Munch, M., Andreasen, P. A., and Gliemann, J. (1992) J. Biol. Chem. 267, 14543-14546). Soluble recombin ant urokinase receptor blocked the binding of pro-uPA to alpha(2)MR/LR P but caused only a slight reduction in the affinity for binding of uP A PAI-1. Moreover, pro-uPA bound to the urokinase receptor at the cell surface was not internalized and degraded unless activated to uPA and complexed with PAI-1. We conclude that pro-uPA is protected against d egradation via alpha(2)MR/LRP when bound to uPAR due to shielding of a binding contact in the A chain, whereas the affinity of uPAR-bound uP A PAI-1 complex for binding to alpha(2)MR/LRP remains sufficient to al low rapid internalization and degradation.