Abstract
The receptor for urokinase-type plasminogen activator (uPAR) plays important roles in a number of physiological and pathological processes by virtue of its interactions with urokinase-type plasminogen activator (uPA), vitronectin (Vn), and several other proteins. The uPA binding site spans all three domains (D1 to D3) of uPAR. However, the nature of the Vn binding site within uPAR is still not clear. In this study, we conducted homolog-scanning mutagenesis on uPAR by switching 14 individual segments of 4-8 residues to their counterpart sequences of a uPAR homolog CD59. All 14 mutants were well expressed, reacted with a panel of monoclonal antibodies, and exhibited correct molecular weights. Of these 14 mutants, six mutants were defective in both uPA and Vn binding. Most importantly, we found two unique mutants uPAR(Asn172-Lys175) and uPAR(Glu183-Asn186) within the D2 domain, which displayed differential ligand binding activity: both had high affinity uPA binding, but completely lost Vn binding, indicating that these two sequences constitute a novel Vn binding site. Indeed, two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound uPA but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA. Altogether, our data demonstrated that uPAR D2 contains two distinct ligand binding sites for uPA and Vn. Such information will help us better understand the complex roles of uPAR in cell adhesion, migration, and tumor metastasis.
Highlights
Pockets within D2, respectively, behaved like bona fide ligand binding sites: peptide P1 bound urokinase-type plasminogen activator (uPA) but not Vn, whereas peptide P2 bound Vn and inhibited uPAR-mediated cell adhesion, but did not interact with uPA
Two peptides, P1 (153CPGSNGFHNNDTFHFLKC) and P2 (171CNTTKCNEGPILELENLPQ), derived from the sequences of the identified uPA and Vn binding ated with acute injury and tissue repair, simultaneous binding of uPA and Vn to uPAR concentrates the proteolytic activity of uPA and its activation product, plasmin, on the cell surface as well as close to the provisional matrix [6]. uPAR/Vn interaction can be enhanced by uPA and attenuated by the plasminogen activator inhibitor-1, which binds to the somatomedin B domain of Vn [7]
Our results demonstrate that two novel sequences (172NTTK and 183ELEN) within the uPAR D2 domain are critical for Vn binding but dispensable for uPA binding
Summary
Materials—Human kidney 293 cells were obtained from Dr F. FACS Analysis—A total of 106 cells expressing wild-type or mutant uPAR in Hanks’ balanced salt solution (HBSS) were incubated with 1 g of mAb for 30 min at 4 °C. A total of 106 cells expressing wild-type or mutant uPAR in HBSS were incubated with different concentrations of uPA (0 to 100 nM) for 30 min at room temperature. After blocking with 300 l of 1% BSA in DPBS, a total of 2 ϫ 106 cells in HBSS in the presence or absence of different concentrations of synthetic peptides or 20 g/ml mAb VIM5 (against uPAR) were added to each well and incubated at 37 °C for 20 min. A total of 5 ϫ 106 cells expressing the wild-type or mutant uPAR were lysed, analyzed on 10% SDS-PAGE, and after transfer, visualized using a uPAR-specific mAb VIM5 and an horseradish peroxidase conjugate of goat anti-mouse IgG. Bound peptides was detected with a alkaline phosphatase-avidin conjugate, measuring absorption at 405 nm
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