PHOTOAFFINITY-LABELING OF THE HUMAN RECEPTOR FOR UROKINASE-TYPE PLASMINOGEN-ACTIVATOR USING A DECAPEPTIDE ANTAGONIST - EVIDENCE FOR A COMPOSITE LIGAND-BINDING SITE AND A SHORT INTERDOMAIN SEPARATION

Binding of urokinase-type plasminogen activator (uPA) to its cellular receptor (uPAR) renders the cell surface a favored site for plasminoge n activation. Recently, a 15-mer peptide antagonist of the uP4-uPAR in teraction, with an IC50 value of 10 nM, was identified using phage dis play technology [Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Ros enberg, S. (1994) Proc. Natl. Acad. Sci. 91, 7129-7133]. In the presen t study, the molecular aspects of the interaction between this peptide and uPAR have been investigated. We have characterized the real-time receptor binding kinetics for the antagonist using surface plasmon res onance and identified critical residues by alanine replacements. The m inimal peptide antagonist thus derived (SLNFSQYLWS) was rendered photo activatable by replacing residues important for uPAR binding with phot ochemically active derivatives of phenylalanine containing either (tri fluoromethyl)diazirine or benzophenone, These peptides incorporated co valently into purified soluble uPAR upon photoactivation, and this was inhibited by preincubation with receptor binding derivatives of uPA. The intact three-domain structure of uPAR was essential for efficient photoaffinity labeling. Proteolytic domain mapping using chymotrypsin revealed a specific labeling of both uPAR domain I and domains II + II I dependent on the position of the photoprobe in the antagonist. On th e basis of these studies, we propose the existence of a composite liga nd binding site in uPAR combined of residues located in distinct struc tural domains. According to this model, a close spatial proximity betw een uPAR domain I and either domains II or III in intact uPAR is requi red for the assembly of this composite binding site. Since the recepto r binding properties of the peptide antagonist closely mimic those of uPA itself, these two ligands presumably share a coincident binding si te in uPAR.