Dj. Vines et al., Receptor-mediated regulation of plasminogen activator function: Plasminogen activation by two directly membrane-anchored forms of urokinase, J PEPT SCI, 6(9), 2000, pp. 432-439
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.