title: Internalization of the urokinase-plasminogen activator inhibitor type-1 complex is mediated by the urokinase receptor.
abstract: The role of the urokinase receptor (uPAR) in the internalization of the urokinase-plasminogen activator inhibitor type-1 (uPA.PAI-1) complex has been investigated. First, exploiting the species specificity of uPA binding, we show that mouse LB6 cells (that express a mouse uPAR) were unable to bind or degrade the human uPA.PAI-1 complex. On the other hand, LB6 clone 19 cells, which express a transfected human uPAR, degraded uPA.PAI-1 complexes with kinetics identical to the human monocytic U937 cells. We also show by immunofluorescence experiments with anti-uPA antibodies that in LB6 clone 19 cells, the uPA.PAI-1 complex is indeed internalized. While at 4 degrees C uPA fluorescence was visible at the cell surface, shift of the temperature to 37 degrees C caused a displacement of the immunoreactivity to the cytoplasmic compartment, with a pattern indicating lysosomal localization. If uPA.PAI-1 internalization/degradation is mediated by uPAR, inhibition of uPA.PAI-1 binding to uPAR should block degradation. Three different treatments, competition with the agonist amino-terminal fragment of uPA, treatment with a monoclonal antibody directed toward the binding domain of uPAR or release of uPAR from the cell surface with phosphatidylinositol-specific phospholipase C completely prevented uPA.PAI-1 degradation. The possibility that a serpin-enzyme complex receptor might be primarily or secondarily involved in the internalization process was excluded since a serpin-enzyme complex peptide failed to inhibit uPA.PAI-1 binding and degradation. Similarly, complexes of PAI-1 with low molecular mass uPA (33 kDa uPA), which lacks the uPAR binding domain, were neither bound nor degraded. Finally we also show that treatment of cells with uPA.PAI-1 complex caused a specific but partial down-regulation of uPAR. A similar result was obtained when PAI-1 was allowed to complex to uPA that had been previously bound to the receptor. The possibility therefore exists that the entire complex uPA.PAI-1-uPAR is internalized. All these data allow us to conclude that internalization of the uPA.PAI-1 complex is mediated by uPAR.
authors.affiliation: Institute of Microbiology, University of Copenhagen, Denmark.
authors.forename: D
authors.initials: D
authors.lastname: Olson
authors.forename: J
authors.initials: J
authors.lastname: Pöllänen
authors.forename: G
authors.initials: G
authors.lastname: Høyer-Hansen
authors.forename: E
authors.initials: E
authors.lastname: Rønne
authors.forename: K
authors.initials: K
authors.lastname: Sakaguchi
authors.forename: T C
authors.initials: TC
authors.lastname: Wun
authors.forename: E
authors.initials: E
authors.lastname: Appella
authors.forename: K
authors.initials: K
authors.lastname: Danø
authors.forename: F
authors.initials: F
authors.lastname: Blasi
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D015536:Down-Regulation; D005455:Fluorescent Antibody Technique; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054801:Phosphoinositide Phospholipase C; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D015849:Plasminogen Inactivators / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator; D013045:Species Specificity; D013696:Temperature; D014162:Transfection; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; C524898:PLAUR protein, human; D015849:Plasminogen Inactivators; C524899:Plaur protein, mouse; D011956:Receptors, Cell Surface; D055293:Receptors, Urokinase Plasminogen Activator; D010727:Phosphoric Diester Hydrolases; D054801:Phosphoinositide Phospholipase C; D014568:Urokinase-Type Plasminogen Activator; D043265:Phosphatidylinositol Diacylglycerol-Lyase