Abstract
The urokinase-type plasminogen activator receptor (uPAR) provides a rendezvous between proteolytic degradation of the extracellular matrix and integrin-mediated adhesion to vitronectin. These processes are, however, tightly linked because the high affinity binding of urokinase regulates the binding of uPAR to matrix-embedded vitronectin. Although crystal structures exist to define the corresponding static bi- and trimolecular receptor complexes, it is evident that the dynamic property of uPAR plays a decisive role in its function. In the present study, we combine small angle x-ray scattering, hydrogen-deuterium exchange, and surface plasmon resonance to develop a structural model describing the allosteric regulation of uPAR. We show that the flexibility of its N-terminal domain provides the key for understanding this allosteric mechanism. Importantly, our model has direct implications for understanding uPAR-assisted cell adhesion and migration as well as for translational research, including targeted intervention therapy and non-invasive tumor imaging in vivo.
Highlights
The urokinase receptor is a modular receptor containing three LU domains
Structural Studies Using SAXS—To study the structure of urokinase-type plasminogen activator receptor (uPAR) in solution, small angle x-ray scattering data were collected for soluble uPARwt in complex with two different antagonist peptides, AE105 [31] and AE234 [32], a receptor-binding fragment of the high affinity protease ligand uPA (ATF), and for the unoccupied receptor alone
This discrepancy between the predicted molecular mass and that estimated from the scattering data could theoretically indicate the presence of a small proportion of oligomers; the shape parameters Rg and Dmax remain constant with increasing concentration, 34306 JOURNAL OF BIOLOGICAL CHEMISTRY
Summary
The urokinase receptor (uPAR) is a modular receptor containing three LU domains. Results: Ligand-free uPAR is inherently flexible with a detached N-terminal domain (DI). One complicating factor in targeting uPAR is the dual function this receptor exerts on both degradation of and adhesion to the extracellular matrix [16] These distinct functional properties are, interrelated because the low affinity binding between uPAR and the somatomedin B domain (SMB) of vitronectin [17] is regulated by uPAR occupancy with its high affinity protease ligand uPA [18, 19]. To better understand the structural transition(s) regulating the interaction between uPAR and vitronectin, we in this study embark on a more dynamic approach by probing the conformation(s) of the ligand-free state of uPAR with small angle x-ray scattering (SAXS) and hydrogen-deuterium exchange (HDX) These data are integrated with functional data obtained by surface plasmon resonance. Trapping uPAR in the open state provides a new avenue for therapeutic inhibition of the proteolytic activity of the plasminogen activation system without stimulating cellular migration
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