Activation of proMMP‐2 by MT1‐MMP is considered to be a critical event in cancer cell invasion. In the activation step, TIMP‐2 bound to MT1‐MMP on the cell surface acts as a receptor for proMMP‐2. Subsequently, adjacent TIMP‐2‐free MT1‐MMP activates the proMMP‐2 in the ternary complex. In this study, we demonstrate that MT1‐MMP forms a homophilic complex through the hemopexin‐like (PEX) domain that acts as a mechanism to keep MT1‐MMP molecules close together to facilitate proMMP‐2 activation. Deletion of the PEX domain in MT1‐MMP, or swapping the domain with the one derived from MT4‐MMP, abolished the ability to activate proMMP‐2 on the cell surface without affecting the proteolytic activities. In addition, expression of the mutant MT1‐MMP lacking the catalytic domain (MT1PEX‐F) efficiently inhibited complex formation of the full‐length enzymes and activation of pro MMP‐2. Furthermore, expression of MT1PEX‐F inhibited proMMP‐2 activation and Matrigel invasion activity of invasive human fibrosarcoma HT1080 cells. These findings elucidate a new function of the PEX domain: regulating MT1‐MMP activity on the cell surface, which accelerates cellular invasiveness in the tissue.

Pericellular degradation of interstitial collagens is a crucial event for cells to migrate through the dense connective tissue matrices, where collagens exist as insoluble fibers. A key proteinase that participates in this process is considered to be membrane-type 1 matrix metalloproteinase (MT1-MMP or MMP-14), but little is known about the mechanism by which it cleaves the insoluble collagen. Here we report that homodimerization of MT1-MMP through its hemopexin (Hpx) domain is essential for cleaving type I collagen fibers at the cell surface. When dimerization was blocked by coexpressing either a membrane-bound or a soluble form of the Hpx domain, cell surface collagenolytic activity was inhibited in a dose-dependent manner. When MMP-13, a soluble collagenase active as a monomer in solution, was expressed as a membrane-anchored form on the cell surface, homodimerization was also required to cleave collagen. Our results introduce a new concept in that pericellular collagenolysis is regulated by correct molecular assembly of the membrane-anchored collagenase, thereby governing the directionality of the cell to migrate in tissue.

Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.

Background: MT1-MMP promotes cancer cell invasion. Results: Deletion of the MT-LOOP region of MT1-MMP or an antibody to the MT-LOOP inhibits cellular invasion. Conclusion: MT-LOOP-dependent MT1-MMP localization to the cell adhesion complex promotes cellular invasion. Significance: Our work reveals a novel mechanism of MT1-MMP regulation during cellular invasion and identifies the MT-LOOP as a novel target region to develop specific inhibitors.

Activation of proMMP-2 and cell surface collagenolysis are important activities of membrane-type 1 matrix metalloproteinase (MT1-MMP) to promote cell migration in tissue, and these activities are regulated by homodimerization of MT1-MMP on the cell surface. In this study, we have identified the transmembrane domain as a second dimer interface of MT1-MMP in addition to the previously identified hemopexin domain. Our analyses indicate that these two modes of dimerization have different roles; transmembrane-dependent dimerization is critical for proMMP-2 activation, whereas hemopexindependent dimerization is important for degradation of collagen on the cell surface. Our finding provides new insight into the potential molecular arrangement of MT1-MMP contributing to its function on the cell surface.Membrane-type 1 matrix metalloproteinase (MT1-MMP) 2 is a type I transmembrane proteinase that promotes cell migration in tissue (1). MT1-MMP is implicated in many physiological and pathological conditions including wound healing (2), bone development (3, 4), lung development (5, 6), angiogenesis (3,7,8), cancer invasion (9) and growth (10), rheumatoid arthritis (11), and atherosclerosis (12-14). MT1-MMP promotes cellular invasion by degrading barrier extracellular matrix components including collagens I, II, III, fibronectin, laminins, vitronectin,; by activating other MMPs, namely proMMP-2 (9) and proMMP-13 (18); by shedding cell adhesion molecules such as CD44 (19) and syndecan 1 (20); and by activating extracellular signal-regulated kinase (ERK) through as yet undefined mechanisms (21, 22).Having such diverse functions, MT1-MMP is regulated by different mechanisms including gene expression, activation of the zymogen (23, 24), inhibition by endogenous inhibitors, including tissue inhibitor of metalloproteinases (TIMPs) (25), RECK (26), and Testicans (27, 28), localization to the leading edge of migrating cells, including lamellipodia (29 -31) and invadopodia (32), autolytic degradation and processing (33-35), endocytosis through clathrin-and caveolae-dependent mechanisms (36 -38), palmitoylation at its cytoplasmic domain (39), recycling (40), and lysosomal degradation (41). Such regulation is thought to be essential to coordinate MT1-MMP activity with cellular events, enabling it to promote cell invasiveness (42).ProMMP-2 activation is one of the MT1-MMP functions thought to be important in cancer invasion (9, 43) and growth (44), where its significance lays particularly on basement membrane degradation as MT1-MMP itself cannot degrade collagen IV, a major component of the matrix but activated MMP-2 does. In this activation process, MT1-MMP forms a complex with its endogenous inhibitor, TIMP-2 (45-47). TIMP-2 binds to the catalytic site of MT1-MMP through its inhibitory site in the N-terminal domain, leaving the exposed C-terminal domain of TIMP-2 to interact with the hemopexin (Hpx) domain of proMMP-2 (45-47). Thus the MT1-MMP-TIMP-2 complex acts as a receptor for proMMP-2. To activate proMMP-2 in this complex, a sec...