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
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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.
Original language | English |
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Journal | Biochemistry |
Volume | 37 |
Issue number | 47 |
Pages (from-to) | 16494-505 |
Number of pages | 12 |
ISSN | 0006-2960 |
DOIs | |
Publication status | Published - 24 Nov 1998 |
Externally published | Yes |
- Amino Acid Sequence, Animals, Arginine, Azirines, Benzophenones, Binding Sites, CHO Cells, Cricetinae, Histidine, Humans, Hydrogen-Ion Concentration, Leucine, Ligands, Molecular Sequence Data, Oligopeptides, Phenylalanine, Photoaffinity Labels, Plasminogen Activators, Protein Structure, Tertiary, Receptors, Cell Surface, Receptors, Urokinase Plasminogen Activator, Tryptophan, Urokinase-Type Plasminogen Activator, Journal Article, Research Support, Non-U.S. Gov't
Research areas
ID: 178215761