Membrane-type matrix metalloproteinases (MT-MMPsTIMP-4 associates with MMP-2 and MT1-MMP in a manner similar to TIMP-3, but its deletion had no effect on pro-MMP-2 processing. Thus, TIMP-3 provides an inherent regulation over the kinetics of pro-MMP-2 processing, serving at a level distinct from that of TIMP-2 and TIMP-4.Matrix metalloproteinases (MMPs) 2 are fundamental to biological processes because of their ability to cleave and remodel the extracellular matrix (ECM). MT1-MMP is one of six cell surface membrane-type MMPs (MT-MMP). Its activity and regulation have been widely studied in the context of cell surface MMP activation, cell migration, and invasion (1-3), independently and in conjunction with other cell adhesion molecules (4 -10). A key function of MT1-MMP is to process pro-MMP-2, whose activity also correlates with an invasive propensity in several cancers and is predictive of poor survival (reviewed in . Understanding the regulation of pro-MMP-2 processing by MT1-MMP and other members of the MT-MMP family is central to defining their role in cancer biology.The classical model for the cell surface activation of MMP-2 is through the formation of a trimolecular complex comprising MT1-MMP, TIMP-2, and pro-MMP-2 (15). The transmembrane MT1-MMP interacts via its N-terminal domain to the N terminus of TIMP-2, forming a "receptor" onto which pro-MMP-2 (72 kDa) binds. Pro-MMP-2 is initially cleaved to its intermediate form (64 kDa) by an adjacent active MT1-MMP. The second stage of MMP-2 processing, resulting in its fully active form (62 kDa), involves an autocatalytic event that requires an MMP-2 molecule in trans (16). It is known that of the six MT-MMPs, MT2-, 3-, 5-, and 6-MMP (17-21) also have the capacity to activate pro-MMP-2. Alternative mechanisms of MMP-2 processing at the plasma membrane, such as the urokinase plasminogen system (22, 23) or TIMP-independent processing involving MT2-MMP, have also been suggested (24). Although the dynamics of the trimolecular complex have been well studied, new insights into its regulation and control are continually being discovered. More recently, cell surface processing of pro-MMP-2 is reported to occur through formation of a trimolecular complex comprised of MT3-MMP, TIMP-3, and pro-MMP-2 (25), although TIMP-3 is known not to form similar complexes with MT1-MMP (26).TIMPs are the naturally occurring inhibitors of metalloproteinase activity. There are four members of the TIMP family, and each has a specific niche with respect to function. Studies have focused on the dual role of TIMP-2 in regulating the processing of pro-MMP-2. A threshold level of TIMP-2 is required in relation to MT1-MMP to construct the trimolecular complex, which still leaves sufficient MT1-MMP uninhibited to cleave pro-MMP-2. At higher concentrations, TIMP-2 prevents MMP-2 processing by inhibiting all free MT1-MMP (27-29). The presence of TIMP-2 was initially considered necessary to achieve any form of MMP-2 processing (30, 31), but recently Timp-2 Ϫ/Ϫ cells were shown capable of some pro...