文摘

Binding of urokinase-type plasminogen activator (uPA) to itscellular receptor (uPAR) rendersthe cell surface a favored site for plasminogen activation.Recently, a 15-mer peptide antagonist of theuPA-uPAR interaction, with an IC₅₀ value of 10 nM, wasidentified using phage display technology[Goodson, R. J., Doyle, M. V., Kaufman, S. E., and Rosenberg, S.(1994) Proc. Natl. Acad. Sci.91,7129-7133]. In the present study, the molecular aspects of theinteraction between this peptide anduPAR have been investigated. We have characterized the real-timereceptor binding kinetics for theantagonist using surface plasmon resonance and identified criticalresidues by alanine replacements. Theminimal peptide antagonist thus derived (SLNFSQYLWS) was renderedphotoactivatable by replacingresidues important for uPAR binding with photochemically activederivatives of phenylalanine containingeither (trifluoromethyl)diazirine or benzophenone. Thesepeptides incorporated covalently into purifiedsoluble uPAR upon photoactivation, and this was inhibited bypreincubation with receptor bindingderivatives of uPA. The intact three-domain structure of uPAR wasessential for efficient photoaffinitylabeling. Proteolytic domain mapping using chymotrypsin revealed aspecific labeling of both uPARdomain I and domains II + III dependent on the position of thephotoprobe in the antagonist. On thebasis of these studies, we propose the existence of a composite ligandbinding site in uPAR combined ofresidues located in distinct structural domains. According to thismodel, a close spatial proximity betweenuPAR domain I and either domains II or III in intact uPAR is requiredfor the assembly of this compositebinding site. Since the receptor binding properties of the peptideantagonist closely mimic those of uPAitself, these two ligands presumably share a coincident binding site inuPAR.