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The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.

Original publication

DOI

10.1074/jbc.m406792200

Type

Journal article

Journal

The Journal of biological chemistry

Publication Date

12/2004

Volume

279

Pages

54944 - 54951

Addresses

CNRS UMR 6198, IFR 53 Biomolecules, Faculty of Medicine, F-51100 Reims, France.

Keywords

Cell Line, Tumor Cells, Cultured, Cell Membrane, Humans, Fibrosarcoma, Collagen, Thrombospondins, Thrombospondin 1, Protein Precursors, Recombinant Proteins, Tissue Inhibitor of Metalloproteinase-2, Culture Media, Conditioned, Transfection, Endocytosis, Gene Expression, Binding Sites, Matrix Metalloproteinase 2, Low Density Lipoprotein Receptor-Related Protein-1