Abstract
The high-affinity interaction between the urokinase-type plasminogen activator (uPA) and its glycolipid-anchored receptor (uPAR) plays a regulatory role for both extravascular fibrinolysis and uPAR-mediated adhesion and migration on vitronectin-coated surfaces. We have recently proposed that the adhesive function of uPAR is allosterically regulated via a "tightening" of its three-domain structure elicited by uPA binding. To challenge this proposition, we redesigned the uPAR structure to limit its inherent conformational flexibility by covalently tethering domains DI and DIII via a non-natural interdomain disulfide bond (uPAR(H47C-N259C)). The corresponding soluble receptor has 1) a smaller hydrodynamic volume, 2) a higher content of secondary structure, and 3) unaltered binding kinetics towards uPA. Most importantly, the purified uPAR(H47C-N259C) also displays a gain in affinity for the somatomedin B domain of vitronectin compared with uPAR(wt), thus recapitulating the improved affinity that accompanies uPA-uPAR(wt) complex formation. This functional mimicry is, intriguingly, operational also in a cellular setting, where it controls lamellipodia formation in uPAR-transfected HEK293 cells adhering to vitronectin. In this respect, the engineered constraint in uPAR(H47C-N259C) thus bypasses the regulatory role of uPA binding, resulting in a constitutively active uPAR. In conclusion, our data argue for a biological relevance of the interdomain dynamics of the glycolipid-anchored uPAR on the cell surface.
Highlights
The urokinase receptor acts as a modulator of lamellipodia formation on vitronectin-rich matrices
We have recently proposed a model for the role of urokinase-type plasminogen activator receptor (uPAR) in vitronectin-dependent cell adhesion and migration, where unoccupied uPAR predominantly exists in an open inactive conformation [24]
Inspired by this intriguing possibility, we searched the interface between uPAR DI and DIII for optimal distances and geometries, which could permit the formation of a stable disulfide bond, i.e. with an optimal distance between the C-atoms of 5.6 Å [37]
Summary
The urokinase receptor (uPAR) acts as a modulator of lamellipodia formation on vitronectin-rich matrices. Besides emphasizing the predictive power of our molecular model for uPAR function [24], this study opens new avenues for crystallizing the unoccupied receptor, the structure of which has so far proven elusive This constrained uPAR mutant maintains unaltered binding kinetics towards uPA even after cleavage of the proteasesensitive linker region between DI and DII, which offers the unique possibility of studying the functional consequences of this cleavage per se, without facing confounding effects from subsequent interdomain dissociations and release of DI as is the case for the wild-type protein [27,28,29]
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