Receptor-mediated regulation of plasminogen activator function: Plasminogen activation by two directly membrane-anchored forms of urokinase

The generation of the broad specificity serine protease plasmin in the peri cellular environment is regulated by binding of the urokinase-type plasmino gen activator (uPA) to its specific glycosylphosphatidylinositol (GPI)-anch ored cell-surface receptor, uPAR. This interaction potentiates the reciproc al activation of the cell-associated zymogens pro-uPA and plasminogen. To f urther study the role of uPAR in this mechanism, we have expressed two dire ctly membrane-anchored chimeric forms of uPA, one anchored by a C-terminal GPI-moiety (GPI-uPA), the other with a C-terminal transmembrane peptide (TM -uPA), These were expressed in the monocyte-like cell lines U937 and THP-1, which are excellent models for kinetic and mechanistic studies of cell-sur face plasminogen activation. In both cell-lines, GPI-uPA activated cell-ass ociated plasminogen with characteristics both qualitatively and quantitativ ely indistinguishable from those of uPAR-bound uPA. By contrast, TM-uPA act ivated cell-associated plasminogen less efficiently. This was due to effect s on the K-m for plasminogen activation (which was increased up to five-fol d] and the efficiency of pro-uPA activation (which was decreased approximat ely four-fold). These observations suggest that uPAR serves two essential r oles in mediating efficient cell-surface plasminogen activation. in additio n to confining uPA to the cell-surface, the GPI-anchor plays an important r ole by increasing accessibility to substrate plasminogen and, thus, enhanci ng catalysis. However, the data also demonstrate that, in the presence of a n alternative mechanism for uPA localization, uPAR is dispensable and, ther efore, unlikely to participate in any additional interactions that may be n ecessary for the efficiency of this proteolytic system. In these experiment s zymogen pro-uPA was unexpectedly found to be constitutively activated whe n expressed in THP-1 cells, suggesting the presence of an alternative plasm in-independent proteolytic activation mechanism in these cells. Copyright ( C) 2000 European Peptide Society and John Wiley & Sons, Ltd.