CELL-SURFACE ACCELERATION OF UROKINASE-CATALYZED RECEPTOR CLEAVAGE

The urokinase-type plasminogen activator (uPA) binds to a specific cel l-surface receptor, uPAR. On several cell types uPAR is present both i n the full-length form and a cleaved form, uPAR(2+3), which is devoid of binding activity. The formation of uPAR(2+3) on cultured U937 cells is either directly or indirectly mediated by uPA itself. In a soluble system, uPA can cleave purified uPAR, but the low efficiency of this reaction has raised doubts as to whether uPA is directly responsible f or uPAR cleavage on the cells. We now report that uPA-catalyzed cleava ge of uPAR on the cell surface is strongly favored relative to, the re action in solution. The time course of uPA-catalyzed cleavage of cell- bound uPAR was studied using U937 cells stimulated with phorbol 12-myr istate 13-acetate. Only 30 min was required for 10 nM uPA to cleave 50 % of the cell-bound uPAR. This uPA-catalyzed cleavage reaction was inh ibited by a prior. incubation of the cells with uPA inactivated by dii sopropyl fluorophosphate, demonstrating a a requirement for specific r eceptor binding of the active uPA to obtain the high-efficiency cleava ge of cell-bound uPAR. Furthermore, amino-terminal sequence analysis r evealed that uPAR(2+3), purified from U937 cell lysates, had the same amino termini as uPAR(2+3), generated by uPA in a purified system. In both cases cleavage had occurred at two positions in the hinge region connecting domain 1 and 2, between Arg83-Ala84 and Arg89-Ser90, respec tively. The uPA-catalyzed cleavage of uPAR is a new negative-feedback regulation mechanism for cell-surface plasminogen activation. We propo se that this mechanism plays a physiological role at specific sites wi th high local concentrations of uPA, thus adding another step to the c omplex regulation of this cascade reaction.