Abstract
The urokinase-type plasminogen activator receptor (uPAR) has been implicated as a modulator of several biochemical processes that are active during tumor invasion and metastasis, e.g. extracellular proteolysis, cell adhesion, and cell motility. The structural basis for the high affinity interaction between the urokinase-type plasminogen activator (uPA) and uPAR, which focuses cell surface-associated plasminogen activation in vivo, is now thoroughly characterized by site-directed mutagenesis studies and x-ray crystallography. In contrast, the structural basis for the interaction between uPAR and the extracellular matrix protein vitronectin, which is involved in the regulation of cell adhesion and motility, remains to be clarified. In this study, we have identified the functional epitope on uPAR that is responsible for its interaction with the full-length, extended form of vitronectin by using a comprehensive alanine-scanning library of purified single-site uPAR mutants (244 positions tested). Interestingly, the five residues identified as "hot spots" for vitronectin binding form a contiguous epitope consisting of two exposed loops connecting the central fourstranded beta-sheet in uPAR domain I (Trp(32), Arg(58), and Ile(63)) as well as a proximal region of the flexible linker peptide connecting uPAR domains I and II (Arg(91) and Tyr(92)). This binding topology provides the molecular basis for the observation that uPAR can form a ternary complex with uPA and vitronectin. Furthermore, it raises the intriguing possibility that the canonical receptor and inhibitor for uPA (uPAR and PAI-1) may have reached a convergent solution for binding to the somatomedin B domain of vitronectin.
Highlights
The urokinase-type plasminogen activator receptor is a modular, glycolipid-anchored membrane protein (1, 2)
This method detects binding of preformed pro-uPA1⁄7uPAR complexes to immobilized vitronectin using a high affinity monoclonal anti-urokinase-type plasminogen activator receptor (uPAR) antibody (R2), which is conjugated with chelated Eu3ϩ as a time-resolved fluorescence reporter
This interaction is considered functionally important as several studies in cell culture clearly show that the level of uPAR expression regulates vitronectin-dependent cell adhesion and motility (21, 22, 25, 27, 40)
Summary
The urokinase-type plasminogen activator receptor (uPAR2/ CD87) is a modular, glycolipid-anchored membrane protein (1, 2). The binding sites mediating the interactions between uPAR and its auxiliary binding partners, e.g. vitronectin (21, 22) and integrins (23, 24), must reside outside this cavity, but the molecular mechanisms underlying these interactions are largely unknown As these interactions are dependent on or modulated by receptor occupancy with uPA (22, 25–27), uPAR may orchestrate the assembly of these ternary complexes on the cell surface and assist in the regulation of cell adhesion and migration. We find that the functional epitope for this interaction encompasses a small region located outside the central uPA-binding cavity and involves residues in two loops connecting the central four-stranded -sheet in uPAR domain I as well as residues in the linker peptide connecting uPAR domains I and II. This functional epitope on uPAR for vitronectin binding resembles the corresponding binding site on PAI-1 for the somatomedin B-like (SMB) domain of vitronectin as both interactions employ arginine as the critical hot spot residue
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