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 hi gh-affinity receptor for uPA (denoted uPAR). Structures involved in the int eraction between human uPAR and a decamer peptide antagonist of uPA binding (SLNFSQYLWS) were previously tagged by specific site-directed photoaffinit y labeling [Ploug, M., Olstergaard, S., Hansen, L. B. L., Helm, A., and Dan o, K. (1998) Biochemistry 37, 3612-3622]. Replacement of the key functional residues Phe(4) and Trp(9) with either benzophenone or (trifluoromethyl)-a ryldiazirine rendered this peptide antagonist photoactivatable, and as a co nsequence, it incorporated covalently upon photolysis into either uPAR doma in I or domain III depending on the actual position of the photophore in th e sequence. The residues of uPAR specifically targeted by photoaffinity lab eling 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, th e formation of the receptor-ligand complex positions Phe4 of the peptide an tagonist very close to Arg(53) and Leu(66) in uPAR domain I and Trp(9) of t he antagonist in the vicinity of His(251) in uPAR domain HI. The gross mole cular arrangement of the deduced receptor-ligand interface provides a ratio nal structural basis for the observed requirement for the intact multidomai n state of uPAR for achieving high-affinity ligand binding, since according to this model ligand binding must rely on a close spatial proximity of uPA R domains I and III. In addition, these data suggest that the assembly of t he composite ligand binding site in uPAR may resemble the hemophilic interd omain dimerization of kappa-bungarotoxin, a structural homologue of the Ly- 6/uPAR domain family.