The N-terminal domain of human urokinase receptor contains two distinct regions critical for ligand recognition.
The high-affinity receptor that binds human urokinase-type plasminogen activator (hu-PAR) is a glycosyl-phosphatidylinositol (GPI)-anchored cell-surface glycoprotein that belongs to the Ly-6 superfamily of T-cell-activating receptors. Binding of urokinase (u-PA) to u-PAR is species-specific, since neither murine (mu-PAR) nor hu-PAR binds u-PA from the other species. I designed and analyzed a series of exchanges between hu-PAR and mu-PAR in the N-terminal first domain to which ligand-binding function had been independently mapped. Introduction of as few as 13 murine residues (six of 13 variables) into the N-terminal region of hu-PAR abrogated binding to recombinant human pro-u-PA, whereas the opposite chimera, a mu-PAR carrying six of 13 human residues, was positive for binding. Within this region, the mu-PAR domain 1 could be minimally humanized to bind human pro-u-PA by a substitution of as few as four of the six nonconserved residues, thereby identifying the residues arginine-2, lysine-7, threonine-8, and glycine-10 as important in determining binding specificity. By alanine-scanning mutagenesis, a second recognition site within domain 1 was discovered between residues 47 and 53, a segment that is fully conserved between the human and the mouse receptors. Taken together, these results provide identification of two confined subregions within the N-terminal domain of hu-PAR critically involved in pro-u-PA recognition.
Pöllänen
J J
JJ
National Biotechnology Programmes, Center for Molecular Cell Biology, Copenhagen, Denmark.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism
C524898:PLAUR protein, human; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D011993:Recombinant Fusion Proteins; D014568:Urokinase-Type Plasminogen Activator