Low Density Lipoprotein Receptor-related Protein Mediates Endocytic Clearance of Pro-MMP-2·TIMP-2 Complex through a Thrombospondin-independent Mechanism

doi:10.1074/jbc.M406792200

Low Density Lipoprotein Receptor-related Protein Mediates Endocytic Clearance of Pro-MMP-2·TIMP-2 Complex through a Thrombospondin-independent Mechanism^*

^‡CNRS UMR 6198, IFR 53 Biomolecules, Faculty of Medicine, F-51100 Reims, France, the ^§Cell Biology Unit, Christian de Duve Institute of Cellular Pathology and Université catholique de Louvain, B-1200 Brussels, Belgium, the ^∥Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College London, London W6 8LH, United Kingdom, the ^‡‡Institute of Medical Biochemistry, University of Aarhus, Aarhus, Denmark, and the ^§§Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, H-1518 Budapest, Hungary

¶¶ To whom correspondence should be addressed: CELL Unit, UCL-ICP 7541, Ave. Hippocrate, 75, B-1200 Brussels, Belgium. Tel.: 32-2-764-75-41; Fax: 32-2-764-75-43; E-mail: courtoy{at}cell.ucl.ac.be.

Abstract

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 ¹²⁵I-pro-MMP-2·TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of ¹²⁵I-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 ¹²⁵I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on ¹²⁵I-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 ¹²⁵I-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.

Footnotes

↵1 The abbreviations used are: MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinases; TSP, thrombospondin; LRP, lipoprotein receptor-related protein; RAP, receptor-associated protein; CBD, collagen-binding domain; SPR, surface plasmon resonance; EGF, epidermal growth factor; DMEM, Dulbecco's modified Eagle medium; TBS, Tris-buffered saline; MT, membrane-type.
↵* This work was supported in part by the CNRS, France (to H. E., G. B., A. R. and W. H.); by the Wellcome Trust Grant 057508, UK (to H. N.); by the International Center for Genetic Engineering and Biotechnology Grant CRP/HUN98-03 and Hungarian National Research Fund Grant (to L. P.); and by the Fonds National de la Recherche Scientifique, Concerted Research Actions, and Interuniversity Attraction Poles, Belgium (to P. H., E. M., Y. E., and P. J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¶ Both authors contributed equally to this work.
↵** Recipient of a Marie Curie Individual Fellowship from the European Community (HPMF-CT 2002-01790).
- Received June 17, 2004.
- Revision received October 13, 2004.
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