Domain Interactions in the Gelatinase A·TIMP-2·MT1-MMP Activation Complex

doi:10.1074/jbc.M005932200

Domain Interactions in the Gelatinase A·TIMP-2·MT1-MMP Activation Complex

THE ECTODOMAIN OF THE 44-kDa FORM OF MEMBRANE TYPE-1 MATRIX METALLOPROTEINASE DOES NOT MODULATE GELATINASE A ACTIVATION*

From the^‡Department of Oral Biological and Medical Sciences and the ^§Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

Abstract

On the cell surface, the 59-kDa membrane type 1-matrix metalloproteinase (MT1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tissue inhibitor of metalloproteinases (TIMP)-2. A 44-kDa remnant of MT1-MMP, with an N terminus at Gly²⁸⁵, is also present on the cell after autolytic shedding of the catalytic domain from the hemopexin carboxyl (C) domain, but its role in gelatinase A activation is unknown. We investigated intermolecular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and solution-phase assays, cell culture, and immunocytochemistry. A strong interaction between the TIMP-2 C domain (Glu¹⁵³-Pro²²¹) and the gelatinase A hemopexin C domain (Gly⁴⁴⁶-Cys⁶⁶⁰) was demonstrated by the yeast two-hybrid system. Epitope masking studies showed that the anionic TIMP-2 C tail lost immunoreactivity after binding, indicating that the tail was buried in the complex. Using recombinant MT1-MMP hemopexin C domain (Gly²⁸⁵-Cys⁵⁰⁸), no direct role for the 44-kDa form of MT1-MMP in cell surface activation of progelatinase A was found. Exogenous hemopexin C domain of gelatinase A, but not that of MT1-MMP, blocked the cleavage of the 68-kDa gelatinase A activation intermediate to the fully active 66-kDa enzyme by concanavalin A-stimulated cells. The MT1-MMP hemopexin C domain did not form homodimers nor did it bind the gelatinase A hemopexin C domain, the C tail of TIMP-2, or full-length TIMP-2. Hence, the ectodomain of the remnant 44-kDa form of MT1-MMP appears to play little if any role in the activation of gelatinase A favoring the hypothesis that it accumulates on the cell surface as an inactive, stable degradation product.

Footnotes

↵* This work was supported in part by grants from the National Cancer Institute of Canada with funds raised by the Canadian Cancer Society.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.
↵¶ Recipient of a Medical Research Council of Canada Scientist Award. To whom correspondence should be addressed: University of British Columbia, 2199 Wesbrook Mall, Vancouver BC, Canada V6T 1Z3. Tel.: 604-822-2958; Fax: 604-822-3562; E-mail: chris.overall@ubc.ca.
↵‖ Recipient of a Medical Research Council of Canada/B.C. Lung Association Scientist Award.
Published, JBC Papers in Press, September 15, 2000, DOI 10.1074/jbc.M005932200
↵2 E. Tam, C. R. Roberts, and C. M. Overall, manuscript in preparation.
↵3 C. M. Morrison, H. F. Bigg, G. S. Butler, and C. M. Overall, unpublished data.
↵4 H. F. Bigg, C. J. Morrison, G. S. Butler, M. A. Bogoyevitch, Z. Wang, P. D. Saloway, and C. M. Overall, submitted for publication.
Abbreviations:

MT

membrane-type

MMP

matrix metalloproteinase

BSA

bovine serum albumin

C domain

carboxyl-terminal domain

PAGE

polyacrylamide gel electrophoresis

PBS

phosphate-buffered saline

TIMP

tissue inhibitor of metalloproteinases

N-T2

peptide present in the N domain of TIMP-2

C-T2

peptide representing the anionic carboxyl-terminal tail of TIMP-2

ConA

concanavalin A
- Received July 6, 2000.
The American Society for Biochemistry and Molecular Biology, Inc.