title: Discrimination of different forms of the murine urokinase plasminogen activator receptor on the cell surface using monoclonal antibodies.
abstract: The urokinase plasminogen activator receptor (uPAR) is a versatile three-domain GPI-anchored protein, which binds urokinase plasminogen activator (uPA) and thereby focalises plasminogen activation on the cell surface. Generation of a proteolytic potential is essential in both normal physiological and pathological extracellular tissue remodelling processes. uPA can also cleave uPAR, resulting in liberation of the amino-terminal domain I, which encompasses binding sites for both uPA and the adhesion molecule, vitronectin. In order to localise the different uPAR forms on the plasma membrane of murine monocyte macrophage-like P388D.1 cells, we have now generated and characterised two high-affinity murine mAbs, mR3 and mR4, raised against murine uPAR. mR3 was found to recognise an epitope located in domain I of uPAR. Surface plasmon resonance analyses and cell binding studies revealed that this mAb was able to bind preformed complexes of murine pro-uPA and murine uPAR. In contrast, mR4 recognises domains II-III in uPAR and does not bind preformed pro-uPA-uPAR complexes in similar analyses. Immunofluorescence microscopy of P388D.1 cells revealed that mR3 stained the cells equally well in the presence or absence of saturation with the amino-terminal fragment of uPA, ATF. However, the signal intensity obtained using another uPAR domain I specific mAb, mR1, was significantly reduced upon ATF saturation. Furthermore, when adding ATF, mR4 selectively stained the cleaved receptor. Applying these newly generated mAbs, we additionally demonstrated that cleaved and intact uPAR was evenly distributed on the surface of these cells.
authors.affiliation: Finsen Laboratory, Rigshospitalet section 3735, Copenhagen Biocenter, Copenhagen N, Denmark.
authors.forename: Morten G
authors.initials: MG
authors.lastname: Rasch
authors.forename: Jesper
authors.initials: J
authors.lastname: Pass
authors.forename: Martin
authors.initials: M
authors.lastname: Illemann
authors.forename: Gunilla
authors.initials: G
authors.lastname: Høyer-Hansen
authors.forename: Ida K
authors.initials: IK
authors.lastname: Lund