Identification of specific sites involved in ligand binding by photoaffinity labeling of the receptor for the urokinase-type plasminogen activator. Residues located at equivalent positions in uPAR domains I and III participate in the assembly of a composite ligand-binding site.
Plasminogen activation by the urokinase-type plasminogen activator (uPA) is facilitated in the presence of cells expressing the glycolipid-anchored high-affinity receptor for uPA (denoted uPAR). Structures involved in the interaction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinity labeling [Ploug, M., Ostergaard, S., Hansen, L. B. L., Holm, A., and Dano, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe4 and Trp9 with either benzophenone or (trifluoromethyl)aryldiazirine rendered this peptide antagonist photoactivatable, and as a consequence, it incorporated covalently upon photolysis into either uPAR domain I or domain III depending on the actual position of the photophore in the sequence. The residues of uPAR specifically targeted by photoaffinity labeling were identified by matrix-assisted laser desorption mass spectrometry, NH2-terminal sequence analysis, and amino acid composition analysis after enzymatic fragmentation and HPLC purification. According to these data, the formation of the receptor-ligand complex positions Phe4 of the peptide antagonist very close to Arg53 and Leu66 in uPAR domain I and Trp9 of the antagonist in the vicinity of His251 in uPAR domain III. The gross molecular arrangement of the deduced receptor-ligand interface provides a rational structural basis for the observed requirement for the intact multidomain state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPAR domains I and III. In addition, these data suggest that the assembly of the composite ligand binding site in uPAR may resemble the homophilic interdomain dimerization of kappa-bungarotoxin, a structural homologue of the Ly-6/uPAR domain family.
Ploug
M
M
Finsen Laboratory, Rigshospitalet, Copenhagen, Denmark. m-ploug@finsenlab.dk
D000595:Amino Acid Sequence; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001389:Azirines / Q000378:metabolism; D001577:Benzophenones / Q000378:metabolism; D001665:Binding Sites; D016466:CHO Cells; D006224:Cricetinae; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000378:metabolism; D008024:Ligands; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D010649:Phenylalanine / Q000378:metabolism; D019920:Photoaffinity Labels / Q000378:metabolism*; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D014364:Tryptophan / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors / Q000378:metabolism*
D001389:Azirines; D001577:Benzophenones; D008024:Ligands; D009842:Oligopeptides; C524898:PLAUR protein, human; D019920:Photoaffinity Labels; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; C116195:peptide antagonist AE68; D010649:Phenylalanine; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; D010960:Plasminogen Activators; D014568:Urokinase-Type Plasminogen Activator; D007930:Leucine