alpha-2 Macroglobulin receptor/Ldl receptor-related protein(Lrp)-dependent internalization of the urokinase receptor.

Abstract

Abstract. The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA). On the contrary, uPAR-bound complexes of uPA with its serpin inhibitors PAI-1 (plasminogen activator inhibitor type-l) or PN-1 (protease nexin-1) are readily internalized in several cell types. Here we address the question whether uPAR is internalized 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 et2-macroglobulin receptor/low density lipoprotein receptor-related protein (LRP/a2-MR) which is required to internalize uPAR-bound uPA-PAI-1 and uPA-PN-1 complexes. Downregulation of cell surface uPAR molecules in U937 cells was detected by cytofluorimetric analysis after uPA-PAI-1 and uPA-PN-1 incubation for 30 min at 37°C; this effect was blocked by preincubation with the ligand of LRP/ aE-MR, RAP (LRP/a2-MR-associated protein), known to block the binding of the uPA complexes to LRP/et E-MR. Downregulation correlated in time with the intracellular appearance of uPAR as assessed by confocal microscopy and immuno-electron microscopy. After 30 min incubation with uPA-PAI-1 or uPA-PN-1 (but not with free uPA), confocal microscopy showed that uPAR staining in permeabilized LB6 clone 19 cells moved from a mostly surface associated to a largely perinuclear position. This effect was inhibited by the LRP/a2-MR RAP. Perinuclear uPAR did not represent newly synthesized nor a preexisting intracellular pool of uPAR, since this fluorescence pattern was not modified 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 intracellular translocation of uPAR, and its dependence on LRP/ot2-MR in LB6 clone 19 cells only after binding to the uPA-PAI-1 complex. After 30 min incubation at 37°C with uPA-PAI-1, 93 % of the specific 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 internalization requires the LRP/a2-MR.

Embryonic Fibroblasts That Are Genetically Deficient in Low Density Lipoprotein Receptor-related Protein Demonstrate Increased Activity of the Urokinase Receptor System and Accelerated Migration on Vitronectin

“…The MEF-2 cultures accumulated increased amounts of uPA in the medium, and the cells expressed increased amounts of cell-surface uPAR. These findings are consistent with previous studies demonstrating binding of free uPA (not uPAR-associated) to LRP (30,32) and endocytosis of uPAR in complex with LRP under certain conditions (35).…”

“…Expression of increased amounts of cell-surface uPAR by MEF-2 cells is consistent with the studies of Conese et al (35), which demonstrated a role for LRP in mediating the internalization of cell-surface uPAR. We hypothesized that LRP deficiency, in the MEF-2 cells, allowed these cells to establish a higher equilibrium level of cell-surface uPAR.…”

“…When uPAR-associated uPA⅐PAI-1 complex undergoes LRP-mediated endocytosis, the uPAR may be internalized as well (35). Thus, LRP may shuttle large macromolecular complexes from the cell surface into intracytoplasmic pools.…”

Arf6-independent GPI-anchored Protein-enriched Early Endosomal Compartments Fuse with Sorting Endosomes via a Rab5/Phosphatidylinositol-3′-Kinase–dependent Machinery

“…However, the study demonstrated that PrP C followed a different endocytic route than other GPIanchored proteins, such as CD59, which showed no enrichment in caveolae (Peters et al, 2003). Other instances of such protein-specific modulation are binding of a complex of the urokinase-type plasminogen activator (uPA) and its GPIanchored uPA receptor to the ␣-2 macroglobulin receptor/ low-density lipoprotein receptor-related protein (Nykjaer et al, 1992;Conese et al, 1995); and GPI-anchored CD14, which interacts with bacterial lipopolysaccharide (LPS) bound to the plasma-LPS-binding protein (Poussin et al, 1998;Triantafilou et al, 2001). In the absence of lateral associations of the type described above, or cross-linking and internalization through the caveolar/raft-dependent pathway (Nabi and Le, 2003), GPI-anchored proteins seem to be selectively internalized through a dynamin-independent pathway (Fivaz et al, 2002;Sabharanjak et al, 2002;Guha et al, 2003).…”

Anthocyanidins inhibit migration of glioblastoma cells: Structure‐activity relationship and involvement of the plasminolytic system

“…The process of plasminogen activation in a healthy organism is controlled through the availability of PAs, localized activation, and interaction with specific inhibitors (PAIs). At the cell surface, urokinase-type plasminogen activator (uPA) binds to its specific receptor (urokinasetype plasminogen activator receptor (uPAR)), then binds its inhibitor plasminogen activator inhibitor-1 (PAI-1), localized in the matrix, and the complex is internalized by endocytic receptors such as the low-density lipoprotein receptor-related protein (LRP) [Orth et al, 1994;Conese et al, 1995]. Recent evidence suggests that the uPA/uPAR system plays a role in the regulation of cell-matrix interactions such as cell adhesion and migration [Irigoyen et al, 1999].…”