ALPHA-(2)-MACROGLOBULIN RECEPTOR LDL-RECEPTOR-RELATED-PROTEIN (LRP)-DEPENDENT INTERNALIZATION OF THE UROKINASE RECEPTOR

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound comp lexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator i nhibitor type-1) or PN-1 (protease nexin-l) are readily internalized i n several cell types. Here we address the question whether uPAR is int ernalized as well upon binding of uPA-serpin complexes. Both LB6 clone 19 cells, a mouse cell line transfected with the human uPAR cDNA, and the human U937 monocytic cell line, express in addition to uPAR also the endocytic alpha(2)-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/alpha(2)-MR) which is required to intern alize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of c ell surface uPAR molecules in U937 cells was detected by cytofluorimet ric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37 degrees C; this effect was blocked by preincubation with the ligand of LRP/alpha(2)-MR, RAP (LRP alpha(2)-MR-associated protein), known to b lock the binding of the uPA complexes to LRP/alpha(2)-MR. Downregulati on correlated in time with the intracellular appearance of uPAR as ass essed by confocal microscopy and immune-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), con focal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinucle ar position. This effect was inhibited by the LRP/alpha(2)-MR RAP. Per inuclear uPAR did not represent newly synthesized nor a preexisting in tracellular pool of uPAR, since this fluorescence pattern was not modi fied by treatment with the protein synthesis inhibitor cycloheximide, and since in LB6 clone 19 cells all of uPAR was expressed on the cell surface. Immuno-electron microscopy confirmed the plasma membrane to i ntracellular translocation of uPAR, and its dependence on LRP/alpha(2) -MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37 degrees C with uPA-PAI-1, 93% of the sp ecific immunogold particles were present in cytoplasmic vacuoles vs 17 .6% in the case of DFP-uPA. We conclude therefore that in the process of uPA-serpin internalization, uPAR itself is internalized, and that i nternalization requires the LRP/alpha(2)-MR.