Identification and characterization of the murine cell surface receptor for the urokinase‐type plasminogen activator

Cell‐binding experiments have indicated that murine cells on their surface have specific binding sites for mouse urokinase‐type plasminogen activator (u‐PA). In contrast to the human system, chemical cross‐linking studies with an iodinated ligand did not yield any covalent adducts in the murine system, but in ligand‐blotting analysis, two mouse u‐PA‐binding proteins could be visualized. To confirm that these proteins are the murine counterpart of the human u‐PA receptor (u‐PAR), a peptide was derived from the murine cDNA clone assigned to represent the murine u‐PAR due to cross‐hybridization and pronounced sequence similarity with human u‐PAR cDNA [Kristensen, P., Eriksen, J., Blasi, F. & Danø, K. (1991) J. Cell Biol. 115 , 1763–1771]. A rabbit antiserum raised against this peptide specifically recognized two polypeptide bands with electrophoretic mobilities identical to those identified by ligand‐blotting analysis. Binding of mouse u‐PA to its receptor showed species specificity in ligand‐blotting analysis, since mouse u‐PA did not bind to human u‐PAR and human u‐PA did not bind to mouse u‐PAR. The apparent M r of mouse u‐PAR varied between different mouse cell lines and ranged over M r 45000–60000. In four of the cell lines, mouse u‐PA bound to two mouse u‐PAR variant proteins, whereas in the other two cell lines studied, there was only one mouse u‐PA‐binding protein. In the monocyte macrophage cell line P388D.1, trypsin‐treatment of intact cells could remove only the large mouse u‐PAR variant ( M r 60000) indicating that only this type was a cell‐surface‐exposed molecule. The smaller mouse u‐PAR variant ( M r 45000), was deglycosylated by the enzyme endo‐β‐ N ‐acetylglucosaminidase H and is probably an intracellular precursor form carrying only high‐mannose carbohydrate. Deglycosylation of this variant yielded a polypeptide with an apparent M r of about 30000, which corresponds to the M r calculated from the cDNA derived protein sequence of mouse u‐PAR. Receptor‐bound mouse u‐PA could be released by phosphatidylinositol‐specific phospholipase C treatment, indicating that mouse u‐PAR is attached to the cell surface by glycosylphosphatidylinositol. Purification of the two mouse u‐PAR variant proteins by diisopropylfluorophosphate‐inactivated mouse u‐PA ‐ Sepharose affinity chromatography yielded two silver‐stained bands when analysed by SDS/PAGE, corresponding in electrophoretic mobility to those seen by ligand‐blotting analysis. The present identification and characterization of mouse u‐PAR will facilitate experimental studies in vivo on the role of u‐PAR and u‐PA/u‐PAR interaction in normal and pathological conditions, including cancer invasion.