Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor.
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.
Finsen Laboratory, Rigshospitalet & Biotech Research and Innovation Centre, Copenhagen Biocenter, Ole Maaløes Vej 5, DK-2200 Copenhagen N, Denmark.
Henrik
H
Gårdsvoll
Magnus
M
Kjaergaard
Benedikte
B
Jacobsen
Mette C
MC
Kriegbaum
Mingdong
M
Huang
Michael
M
Ploug
D000818:Animals; D002460:Cell Line; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004330:Drosophila; D006801:Humans; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011554:Pseudopodia / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000737:chemistry* / Q000378:metabolism*; D013002:Somatomedins / Q000737:chemistry / Q000378:metabolism; D020349:Surface Plasmon Resonance; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*; D019096:Vitronectin / Q000737:chemistry / Q000378:metabolism
D055293:Receptors, Urokinase Plasminogen Activator; D013002:Somatomedins; D019096:Vitronectin; C016756:somatomedin B; D014568:Urokinase-Type Plasminogen Activator