title: 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.
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 Gly(285), 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 (Glu(153)-Pro(221)) and the gelatinase A hemopexin C domain (Gly(446)-Cys(660)) 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 (Gly(285)-Cys(508)), 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.
authors.affiliation: 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. chris.overall@ubc.ca
authors.forename: C M
authors.initials: CM
authors.lastname: Overall
authors.forename: E
authors.initials: E
authors.lastname: Tam
authors.forename: G A
authors.initials: GA
authors.lastname: McQuibban
authors.forename: C
authors.initials: C
authors.lastname: Morrison
authors.forename: U M
authors.initials: UM
authors.lastname: Wallon
authors.forename: H F
authors.initials: HF
authors.lastname: Bigg
authors.forename: A E
authors.initials: AE
authors.lastname: King
authors.forename: C R
authors.initials: CR
authors.lastname: Roberts
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002845:Chromatography; D003208:Concanavalin A / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D000939:Epitopes; D005998:Glycine / Q000737:chemistry; D007150:Immunohistochemistry; D007700:Kinetics; D020778:Matrix Metalloproteinase 2 / Q000737:chemistry* / Q000378:metabolism*; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases / Q000737:chemistry* / Q000378:metabolism* / Q000502:physiology; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D018000:Receptors, Peptide / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000737:chemistry*; D014407:Tumor Cells, Cultured; D020798:Two-Hybrid System Techniques
chemical_list: D000939:Epitopes; D020033:Protein Isoforms; D018000:Receptors, Peptide; D011994:Recombinant Proteins; C047453:hemopexin receptor; D003208:Concanavalin A; D019716:Tissue Inhibitor of Metalloproteinase-2; D053504:Matrix Metalloproteinases, Membrane-Associated; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D005998:Glycine