Domain interactions in the gelatinase A center dot TIMP-2 center dot MT1-MMP activation complex - The ectodomain of the 44-kDa form of membrane type-I matrix metalloproteinase does not modulate gelatinase A activation

On the cell surface, the 59-kDa membrane type I-matrix metalloproteinase (M T1-MMP) activates the 72-kDa progelatinase A (MMP-2) after binding the tiss ue inhibitor of metalloproteinases (TIMP)-2, A 44-kDa remnant of MT1-MMP, w ith 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, bu t its role in gelatinase A activation is unknown. We investigated intermole cular interactions in the gelatinase A activation complex using recombinant proteins, domains, and peptides, yeast two-hybrid analysis, solid- and sol ution-phase assays, cell culture, and immunocytochemistry, A strong interac tion between the TIMP-2 C domain (Glu(153)-Pro(221)) and the gelatinase A h emopexin C domain (Gly(446)-Cys(660)) was demonstrated by the yeast two-hyb rid system, Epitope masking studies showed that the anionic TIMP-2 C tail l ost 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 activatio n 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 ina ctive, stable degradation product.