The TIMP2 Membrane Type 1 Metalloproteinase “Receptor” Regulates the Concentration and Efficient Activation of Progelatinase A

doi:10.1074/jbc.273.2.871

The TIMP2 Membrane Type 1 Metalloproteinase “Receptor” Regulates the Concentration and Efficient Activation of Progelatinase A

A KINETIC STUDY*

From the ^‡Strangeways Research Laboratory, Worts’ Causeway, Cambridge CB1 4RN and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom,^§InVitek GmbH, Robert-Roessle-Strasse 10, D-13125 Berlin-Buch, Germany, ^¶Chugai Pharmaceuticals Co. Ltd., 135 Komakado, 1 Chome, Gotemba-Shi, Shizuoka 412, Japan, ^‖Celltech Therapeutics Ltd., 216 Bath Road, Slough SL1 4EN, United Kingdom,^**British Biotech Pharmaceuticals Ltd., Watlington Road, Oxford OX4 5LY, United Kingdom, and ^‡INSERM U296, Faculté de Médecine, Avenue du Général Sarrail, 94010 Créteil, France

Abstract

We have used C-terminal domain mutants to further define the role of interactions of progelatinase A and membrane type 1 matrix metalloproteinase (MT1 MMP) in the binding of TIMP2 and in the cell-associated activation of progelatinase A. Soluble constructs of MT1 MMP were used to demonstrate that binding with TIMP2 occurs primarily through N-terminal domain interactions, leaving the C-terminal domain free for interactions with progelatinase A. The rate of autolytic activation of progelatinase A initiated by MT1 MMP cleavage could be potentiated by concentration of the proenzyme by binding to heparin. Residues 568–631 of the progelatinase A C-terminal domain are important in formation of the heparin binding site, since replacement of this region with the corresponding stromelysin-1 sequence abolished binding to heparin and the potentiation of activation. The same region of gelatinase A was required for binding of latent and active enzyme to TIMP2, but residues 418–474 were not important. A similar pattern was seen using cell membrane-associated MT1 MMP; residues 568–631 were required for binding and activation of progelatinase A, whereas residues 418–474 were not. Neither region was required for activation in solution. The addition of TIMP2 to HT1080 membrane preparations expressing MT1 MMP, but depleted of endogenous TIMP2, resulted in potentiation of progelatinase A activation. This effect was dependent upon TIMP2 binding to MT1 MMP rather than at an independent membrane site. Together, the data suggest that TIMP2 forms a receptor with MT1 MMP that regulates the concentration and efficient generation of functionally active gelatinase A.

Footnotes

↵* This work was supported by the Wellcome Trust, the Arthritis and Rheumatism Council, the Medical Research Council (United Kingdom), the Deutsche Forschungsgemeinschaft (Germany), and INSERM (France).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵§§ To whom correspondence should be addressed: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom. Tel.: 44-1603-456161; Fax: 44-1603-592250; E-mail:g.murphy{at}uea.ac.uk.
↵1 The abbreviations used are: MMP, matrix metalloproteinase; MT1 MMP, membrane type 1 matrix metalloproteinase; MT1 MMP_cat, catalytic domain of MT1 MMP (Δ269–559); ΔTM MT1 MMP, MT1 MMP lacking the transmembrane and cytoplasmic domains (Δ501–559); TIMP, tissue inhibitor of metalloproteinases; N-GLA, N-terminal domain of gelatinase A (Δ418–631); C-GLA, C-terminal domain of gelatinase A (Δ1–414); N-GL.C-SL, gelatinase A (residues 1–417) fused to stromelysin-1 (residues 248–460); N-G.C-SGG, gelatinase A (residues 1–417) fused to stromelysin-1 (residues 248–305) then gelatinase A (residues 475–631); N-G.C-SGS, gelatinase A (residues 1–417) fused to stromelysin-1 (residues 248–305) followed by gelatinase A (residues 475–567) and stromelysin-1 (residues 400–460); E375A-gelatinase A, inactive mutant with active site Glu mutated to Ala; (Δ128–194)TIMP2, N-terminal three loops of TIMP2; (Δ186–194)TIMP2, TIMP2 lacking the C-terminal charged tail QEFLDIEDP; APMA, 4-aminophenylmercuric acetate; PAGE, polyacrylamide gel electrophoresis; HBSS, Hanks’ balanced saline solution; BSA, bovine serum albumin; CHO, Chinese hamster ovary.
↵2 d’Ortho, M.-P., Will, H., Atkinson, S., Butler, G., Messent, A., Gavriloić, J., Smith, B., Timpl, R., Zardi, L., and Murphy, G. (1997) Eur. J. Biochem., in press.
↵3 M.-P. d’Ortho and R. M. Hembry, manuscript in preparation.
↵4 M. Cockett and G. Murphy, unpublished observation.
↵5 G. Butler and G. Murphy, unpublished results.
↵6 G. Butler and G. Murphy, unpublished observation.
↵7 A. J. Messent, G. Murphy, and J. Gavrilović, unpublished observations.
- Received July 14, 1997.
- Revision received September 30, 1997.
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