Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

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Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor. / Gårdsvoll, Henrik; Kjærgaard, Magnus; Jacobsen, Benedikte; Kriegbaum, Mette Camilla; Huang, Mingdong; Ploug, Michael.

I: Journal of Biological Chemistry, Bind 286, Nr. 50, 16.12.2011, s. 43515-26.

Publikation: Bidrag til tidsskrift/Konferencebidrag i tidsskrift /Bidrag til avisTidsskriftartikelForskningpeer review

Harvard

Gårdsvoll, H, Kjærgaard, M, Jacobsen, B, Kriegbaum, MC, Huang, M & Ploug, M 2011, 'Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor', Journal of Biological Chemistry, bind 286, nr. 50, s. 43515-26. https://doi.org/10.1074/jbc.M111.300020

APA

Gårdsvoll, H., Kjærgaard, M., Jacobsen, B., Kriegbaum, M. C., Huang, M., & Ploug, M. (2011). Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor. Journal of Biological Chemistry, 286(50), 43515-26. https://doi.org/10.1074/jbc.M111.300020

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Author

Gårdsvoll, Henrik ; Kjærgaard, Magnus ; Jacobsen, Benedikte ; Kriegbaum, Mette Camilla ; Huang, Mingdong ; Ploug, Michael. / Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor. I: Journal of Biological Chemistry. 2011 ; Bind 286, Nr. 50. s. 43515-26.

Bibtex

@article{51f7465d8fa24fd68de3e9b76d1d66b7,
title = "Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor",
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.",
keywords = "Animals, Cell Line, Chromatography, Gel, Circular Dichroism, Drosophila, Humans, Protein Structure, Secondary, Protein Structure, Tertiary, Pseudopodia, Receptors, Urokinase Plasminogen Activator, Somatomedins, Surface Plasmon Resonance, Urokinase-Type Plasminogen Activator, Vitronectin",
author = "Henrik G{\aa}rdsvoll and Magnus Kj{\ae}rgaard and Benedikte Jacobsen and Kriegbaum, {Mette Camilla} and Mingdong Huang and Michael Ploug",
year = "2011",
month = dec,
day = "16",
doi = "10.1074/jbc.M111.300020",
language = "English",
volume = "286",
pages = "43515--26",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology, Inc.",
number = "50",

}

RIS

TY - JOUR

T1 - Mimicry of the regulatory role of urokinase in lamellipodia formation by introduction of a non-native interdomain disulfide bond in its receptor

AU - Gårdsvoll, Henrik

AU - Kjærgaard, Magnus

AU - Jacobsen, Benedikte

AU - Kriegbaum, Mette Camilla

AU - Huang, Mingdong

AU - Ploug, Michael

PY - 2011/12/16

Y1 - 2011/12/16

N2 - 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.

AB - 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.

KW - Animals

KW - Cell Line

KW - Chromatography, Gel

KW - Circular Dichroism

KW - Drosophila

KW - Humans

KW - Protein Structure, Secondary

KW - Protein Structure, Tertiary

KW - Pseudopodia

KW - Receptors, Urokinase Plasminogen Activator

KW - Somatomedins

KW - Surface Plasmon Resonance

KW - Urokinase-Type Plasminogen Activator

KW - Vitronectin

U2 - 10.1074/jbc.M111.300020

DO - 10.1074/jbc.M111.300020

M3 - Journal article

C2 - 22025616

VL - 286

SP - 43515

EP - 43526

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 50

ER -