Dissecting Mannose 6-Phosphate-Insulin-like Growth Factor 2 Receptor Complexes That Control Activation and Uptake of Plasminogen in Cells

Vladimir Leksa,Karin Pfisterer,Gabriela Ondrovičová,Brigitte Binder,Silvia Lakatošová,Clemens Donner,Herbert B Schiller,Alexander Zwirzitz,Katarína Mrvová,Vladimir Pevala,Eva Kutejová,Hannes Stockinger

doi:10.1074/jbc.m112.339663

Abstract

The plasminogen (Plg) activation cascade on the cell surface plays a central role in cell migration and is involved in a plethora of physiological and pathological processes. Its regulation is coordinated by many receptors, in particular the urokinase-type plasminogen activator receptor (uPAR, CD87), receptors that physically interact and functionally cooperate with uPAR, and Plg binding molecules. Here we studied the impact of one of the Plg binding molecules, the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P-IGF2R, CD222), on cellular Plg activation. By developing both in vitro and in vivo Plg activation assays on size-fractionated lysates of M6P-IGF2R-silenced cells, we identified Plg-associated complexes with M6P-IGF2R as the regulatory factor. Using lipid raft preserving versus dissolving detergents, we found lipid dependence of the Plg regulatory function of these complexes. Furthermore, M6P-IGF2R-silencing in uPAR-positive human cell lines reduced internalization of Plg, resulting in elevated Plg activation. In contrast, the expression of human M6P-IGF2R in mouse embryonic fibroblasts derived from M6P-IGF2R knock-out mice enhanced Plg internalization. Finally, peptide 18-36 derived from the Plg-binding site within M6P-IGF2R enhanced Plg uptake. Thus, by targeting Plg to endocytic pathways, M6P-IGF2R appears to control Plg activation within cells that might be important to restrict plasmin activity to specific sites and substrates.

Highlights

The plasminogen system is central in cell migration and is involved in many patho/physiological processes
This was true for the D2D3 form of uPAR that was in line with the previous observation that the uPAR cleavage was accelerated in lipid rafts [6]
Our results obtained from lipid raft flotation analysis indicate that M6P-IGF2R is not implicated in the uPAR partitioning into lipid rafts (Fig. 1)

Summary

Background

The plasminogen system is central in cell migration and is involved in many patho/physiological processes. We investigated the impact of M6P-IGF2R on the reorganization of the cell membrane protein complexes associated with Plg conversion For this purpose, we silenced M6P-IGF2R in human cell lines by RNA interference, and we expressed human M6P-IGF2R, both the wild type form and a variant containing the lysine 25 point mutation within the Plg binding region, in mouse embryonic fibroblasts derived from M6PIGF2R knock-out mice. We silenced M6P-IGF2R in human cell lines by RNA interference, and we expressed human M6P-IGF2R, both the wild type form and a variant containing the lysine 25 point mutation within the Plg binding region, in mouse embryonic fibroblasts derived from M6PIGF2R knock-out mice Employing these cells for lipid raft flotation analysis, two-dimensional blue native polyacrylamide gel electrophoresis (BN/SDS-PAGE) followed by zymography, gel filtration analysis combined with cellular proteolysis assays, and internalization assays, we show here that M6P-IGF2R contributes to regulation of pericellular proteolysis by Plg internalization

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION