Furin directly cleaves proMMP-2 in the trans-Golgi network resulting in a nonfunctioning proteinase

Proprotein convertases play an important role in tumorigenesis and invasiveness. Here, we report that a dibasic amino acid convertase, furin, directly cleaves proMMP-2 within the trans-Golgi network leading to an inactive form of matrix metalloproteinase-2 (MMP-2). Co-transfection of COS-1 cells with both proMMP-2 and furin cDNAs resulted in the cleavage of the N-terminal propeptide of proMMP-2. The molecular mass of cleaved MMP-2 (63 kDa), detected in both cell lysates and conditioned medium, is between the intermediate and fully activated forms of MMP-2 induced by membrane type 1-MMP. Furin-cleaved MMP-2 does not possess proteolytic activity as examined in a cell-free assay. Treatment of transfected cells with a furin inhibitor resulted in a dose-dependent inhibition of proMMP-2 cleavage; recombinant tissue inhibitor of metalloproteinase-2, which binds to the active site of membrane type 1-MMP, had no inhibitory effect. Site-directed mutagenesis of amino acids in the furin consensus recognition motif of proMMP-2(R69KPR72) prevented propeptide cleavage, thereby identifying the scissile bond and characterizing the basic amino acids required for cleavage. Other experimental observations were consistent with intracellular furin cleavage of proMMP-2 in the trans-Golgi network. The furin cleavage site in other proMMPs was examined. MMP-3, which contains the RXXR furin consensus sequence, was cleaved in furin co-transfected cells, whereas MMP-1, which lacks an RXXR consensus sequence, was not cleaved. In conclusion, we report the novel observation that furin can directly cleave the RXXR amino acid sequence in the propeptide domain of proMMP-2 leading to inactivation of the enzyme.     

Shedding of collagen XXIII is mediated by furin and depends on the plasma membrane microenvironment

Collagen XXIII belongs to the class of type II orientated transmembrane collagens. A common feature of these proteins is the presence of two forms of the molecule: a membrane-bound form and a shed form. Here we demonstrate that, in mouse lung, collagen XXIII is found predominantly as the full-length form, whereas in brain, it is present mostly as the shed form, suggesting that shedding is tissue-specific and tissue-regulated. To analyze the shedding process of collagen XXIII, a cell culture model was established. Mutations introduced into two putative proprotein convertase cleavage sites showed that altering the second cleavage site inactivated much of the shedding. This supports the idea that furin, a major physiological protease, is predominantly responsible for shedding. Furthermore, our studies indicate that collagen XXIII is localized in lipid rafts in the plasma membrane and that ectodomain shedding is altered by a cholesterol-dependent mechanism. Moreover, newly synthesized collagen XXIII either is cleaved inside the Golgi/trans-Golgi network or reaches the cell surface, where it becomes protected from processing by being localized in lipid rafts. These mechanisms allow the cell to regulate the amounts of cell surface-bound and secreted collagen XXIII.     

MT1-MMP shedding involves an ADAM and is independent of its localization in lipid rafts

The membrane type 1-matrix metalloproteinase (MT1-MMP) is a membrane-anchored protease that its entire ectodomain is shed from the cell surface. Here we show that in HT1080 cells MT1-MMP is shed as two soluble forms of approximately 52 and approximately 50kDa. Analyses in purified HT1080 plasma membranes show that release of these species is a two-step time-dependent process that is mediated by integral membrane metalloprotease(s). Differential sensitivity to TIMP-3 inhibition of the shedding process suggests that the second cleavage step leading to the formation of the 50-kDa soluble species is mediated by an ADAM. We also show that shedding of MT1-MMP is independent of its partition into lipid rafts because both wild type and glycosylphosphatidylinositol (GPI)-anchored MT1-MMP are shed. These studies provide new insights into the process of MT1-MMP ectodomain shedding, which may regulate pericellular proteolysis.     

Complex pattern of membrane type 1 matrix metalloproteinase shedding. Regulation by autocatalytic cells surface inactivation of active enzyme

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.     

Internal Cleavages of the Autoinhibitory Prodomain Are Required for Membrane Type 1 Matrix Metalloproteinase Activation,although Furin Cleavage Alone Generates Inactive Proteinase

The functional activity of invasion-promoting membrane type 1 matrix metalloproteinase (MT1-MMP) is elevated in cancer. This elevated activity promotes cancer cell migration, invasion, and metastasis. MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its prodomain. Excision by furin was considered sufficient for the prodomain release and MT1-MMP activation. We determined, however, that the full-length intact prodomain released by furin alone is a potent autoinhibitor of MT1-MMP. Additional MMP cleavages within the prodomain sequence are required to release the MT1-MMP enzyme activity. Using mutagenesis of the prodomain sequence and mass spectrometry analysis of the prodomain fragments, we demonstrated that the intradomain cleavage of the PGD↓L⁵⁰ site initiates the MT1-MMP activation, whereas the ¹⁰⁸RRKR¹¹¹↓Y¹¹² cleavage by furin completes the removal and the degradation of the autoinhibitory prodomain and the liberation of the functional activity of the emerging enzyme of MT1-MMP.     

Paradigmatic identification of MMP-2 and MT1-MMP activation systems in cardiac fibroblasts cultured as a monolayer

Activations of MMP-2 and membrane type 1-matrix metalloproteinase (MT1-MMP) have been correlated with cell migration, a key cellular event in the wound healing and tissue remodeling. We have previously demonstrated furin-dependent MMP-2 and MT1-MMP activations induced by type I collagen in cardiac fibroblasts. To understand mechanistic aspects of the regulation of MMP-2 and MT1-MMP activations by potential non-matrix factor(s) in cardiac fibroblasts, in the present study, we examined the effects of various agents including concanavalin A (ConA), a proteolytic phenotype-producing agent. We showed that treatment of cells with ConA activated pro-MMP-2, and that this activation concurred with elevated levels of cellular MT1-MMP and TIMP-2. The presence of active MT1-MMP and 43 and 36 kDa processed forms of MT1-MMP in a fraction of intracellular proteins prepared from ConA-treated cells suggests the possible internalization of differential forms of MT1-MMP. The appearance of 36 kDa processed form of MT1-MMP in conditioned media prepared from ConA-treated cells indicates the possible extracellular release of the further processed MT1-MMP fragment. Inhibition of furin in ConA-treated cells attenuated pro-MT1-MMP processing and the cellular TIMP-2 level, plus it reduced cell-released active MMP-2 in a time-dependent manner. These results suggest the involvement of furin in the ConA-induced activations of MT1-MMP and MMP-2. Furthermore, the existence of furin inhibitor-insensitive pro- and active MMP-2 species associated with ConA-treated cells implies that a mechanism independent of furin may perhaps account for the binding of the MMP-2 species to the cells. Supplementary material for this article can be found at http://www.mrw.interscience.wiley.com/suppmat/0730-2312/suppmat/94/suppmat_guo.tif.     

Complete restoration of cell surface activity of transmembrane-truncated MT1-MMP by a glycosylphosphatidylinositol anchor. Implications for MT1-MMP-mediated prommp2 activation and collagenolysis in three-dimensions

MT1-MMP is a potent collagenase not only required for skeletal development but also implicated in tumor invasion and metastasis. The mechanism through which cellsdeploy MT1-MMP to mediate collagenolysis remains largely unknown. C-terminally truncated MT1-MMP lacking its transmembrane and cytoplasmic domains, although proteolytic active in purified forms, is known to be deficient in cell-mediated proMMP2 activation and collagenolysis, suggesting that cells regulate its activity through both domains. Indeed, the cytoplasmic domain is recognized by the trafficking machinery that mediates its internalization and recycling. Here we demonstrate that its transmembrane domain can be functionally substituted by the glycosylphosphatidylinositol (GPI)-anchor of MT6-MMP. The GPI-anchored MT1-MMP, or MT1-GPI, activates proMMP2 on the cell surface and promotes cell growth in a three-dimensional type I collagen matrix. On the other hand, a GPI-anchored MMP13 with a functional furin activation signal fails to promote cell growth in a three-dimensional collagen matrix, whereas remaining competent in collagenolysis on a two-dimensional collagen matrix under serum-free conditions. alpha(2) macroglobulin (alpha(2)M) or serum is sufficient to inhibit the collagenase activity of GPI-anchored active MMP13. Our results suggest that both membrane-tethering and proteolytic activity encoded by MT1-MMP are required for its ability to promote cell growth and invasion in a three-dimensional collagen matrix.     

Proteolysis of the membrane type-1 matrix metalloproteinase prodomain: implications for a two-step proteolytic processing and activation

Membrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD downward arrowL50 or P58QS downward arrowL61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111 downward arrowY112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells.     

Mutation analysis of membrane type-1 matrix metalloproteinase (MT1-MMP). The role of the cytoplasmic tail Cys(574), the active site Glu(240), and furin cleavage motifs in oligomerization, processing, and self-proteolysis of MT1-MMP expressed in breast carcinoma cells

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a key enzyme in the activation pathway of matrix prometalloproteinase-2 (pro-MMP-2). Both activation and autocatalytic maturation of pro-MMP-2 in trans suggest that MT1-MMP should exist as oligomers on the cell surface. To better understand the functions of MT1-MMP, we designed mutants with substitutions in the active site (E240A), the cytoplasmic tail (C574A), and the RRXR furin cleavage motifs (R89A, ARAA, and R89A/ARAA) of the enzyme. The mutants were expressed in MCF7 breast carcinoma cells that are deficient in both MMP-2 and MT1-MMP. Our results supported the existence of MT1-MMP oligomers and demonstrated that a disulfide bridge involving the Cys(574) of the enzyme's cytoplasmic tail covalently links MT1-MMP monomers on the MCF7 cell surface. The presence of MT1-MMP oligomers also was shown for the enzyme naturally expressed in HT1080 fibrosarcoma cells. The single (R89A and ARAA) and double (R89A/ARAA) furin cleavage site mutants of MT1-MMP were processed in MCF7 cells into the mature proteinase capable of activating pro-MMP-2 and stimulating cell locomotion. This suggested that furin cleavage is not a prerequisite for the conversion of pro-MT1-MMP into the functionally active enzyme. A hydroxamate class inhibitor (GM6001, or Ilomastat) blocked activation of MT1-MMP in MCF7 cells but not in HT1080 cells. This implied that a matrixin-like proteinase sensitive to hydroxamates could be involved in a furin-independent, alternative pathway of MT1-MMP activation in breast carcinoma cells. The expression of the wild type MT1-MMP enhanced cell invasion and migration, indicating a direct involvement of this enzyme in cell locomotion. In contrast, both the C574A and E240A mutations render MT1-MMP inefficient in stimulating cell migration and invasion. In addition, the C574A mutation negatively affected cell adhesion, thereby indicating critical interactions involving the cytosolic part of MT1-MMP and the intracellular milieu.     

Cleavage of amyloid-beta precursor protein (APP) by membrane-type matrix metalloproteinases

Amyloid-beta precursor protein (APP) was identified on expression cloning from a human placenta cDNA library as a gene product that modulates the activity of membrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with APP in HEK293T cells induced cleavage and shedding of the APP ectodomain when co-expressed with APP adaptor protein Fe65. Among the MT-MMPs tested, MT3-MMP and MT5-MMP also caused efficient APP shedding. The recombinant APP protein was cleaved by MT3-MMP in vitro at the A463-M464, N579-M580, H622-S623, and H685-Q686 peptide bonds, which included a cleavage site within the amyloid beta peptide region known to produce a C-terminal fragment. The Swedish-type mutant of APP, which produces a high level of amyloid beta peptide, was more effectively cleaved by MT3-MMP than wild-type APP in both the presence and absence of Fe65; however, amyloid beta peptide production was not affected by MT3-MMP expression. Expression of MT3-MMP enhanced Fe65-dependent transactivation by APP fused to the Gal4 DNA-binding and transactivation domains. These results suggest that MT1-MMP, MT3-MMP and MT5-MMP should play an important role in the regulation of APP functions in tissues including the central nervous system.     

Role for furin in tumor necrosis factor alpha-induced activation of the matrix metalloproteinase/sphingolipid mitogenic pathway

Neutral sphingomyelinase (nSMase), the initial enzyme of the sphingolipid signaling pathway, is thought to play a key role in cellular responses to tumor necrosis factor alpha (TNF-alpha), such as inflammation, proliferation, and apoptosis. The mechanism of TNF-alpha-induced nSMase activation is only partly understood. Using biochemical, molecular, and pharmacological approaches, we found that nSMase activation triggered by TNF-alpha is required for TNF-alpha-induced proliferation and in turn requires a proteolytic cascade involving furin, membrane type 1 matrix metalloproteinase (MT1-MMP), and MMP2, and leading finally to extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and DNA synthesis, in smooth muscle cells (SMC) and fibroblasts. Pharmacological and molecular inhibitors of MMPs (batimastat), furin (alpha1-PDX inhibitor-transfected SMC), MT1-MMP (SMC overexpressing a catalytically inactive MT1-MMP), MMP2 (fibroblasts from MMP2(-/-) mice), and small interfering RNA (siRNA) strategies (siRNAs targeting furin, MT1-MMP, MMP2, and nSMase) resulted in near-complete inhibition of the activation of nSMase, sphingosine kinase-1, and ERK1/2 and of subsequent DNA synthesis. Exogenous MT1-MMP activated nSMase and SMC proliferation in normal but not in MMP2(-/-) fibroblasts, whereas exogenous MMP2 was active on both normal and MMP2(-/-) fibroblasts. Altogether these findings highlight a pivotal role for furin, MT1-MMP, and MMP2 in TNF-alpha-induced sphingolipid signaling, and they identify this system as a possible target to inhibit SMC proliferation in vascular diseases.     

Membrane-type Matrix Metalloproteinase-3 Regulates Neuronal Responsiveness to Myelin through Nogo-66 Receptor 1 Cleavage

Nogo-66 receptor 1 (NgR1) is a glycosylphosphatidylinositol-anchored receptor for myelin-associated inhibitors that restricts plasticity and axonal regrowth in the CNS. NgR1 is cleaved from the cell surface of SH-SY5Y neuroblastoma cells in a metalloproteinase-dependent manner; however, the mechanism and physiological consequence of NgR1 shedding have not been explored. We now demonstrate that NgR1 is shed from multiple populations of primary neurons. Through a loss-of-function approach, we found that membrane-type matrix metalloproteinase-3 (MT3-MMP) regulates endogenous NgR1 shedding in primary neurons. Neuronal knockdown of MT3-MMP resulted in the accumulation of NgR1 at the cell surface and reduced the accumulation of the NgR1 cleavage fragment in medium conditioned by cortical neurons. Recombinant MT1-, MT2-, MT3-, and MT5-MMPs promoted NgR1 shedding from the surface of primary neurons, and this treatment rendered neurons resistant to myelin-associated inhibitors. Introduction of a cleavage-resistant form of NgR1 reconstitutes the neuronal response to these inhibitors, demonstrating that specific metalloproteinases attenuate neuronal responses to myelin in an NgR1-dependent manner.     

Regulation of membrane type-1 matrix metalloproteinase activation by proprotein convertases

Membrane type-1 matrix metalloproteinase (MT1-MMP) is the prototypical member of a subgroup of membrane-anchored proteinases that belong to the matrix metalloproteinase family. Although synthesized as a zymogen, MT1-MMP plays an essential role in extracellular matrix remodeling after an undefined process that unmasks its catalytic domain. We now report the existence of a proprotein convertase-MT1-MMP axis that regulates the processing and functional activity of the metalloproteinase. Two sets of basic motifs in the propeptide region of MT1-MMP are identified that potentially can be recognized by the proprotein convertase family of subtilisin-like proteases. Processing of proMT1-MMP as well as the expression of its proteolytic activity were blocked by mutating these recognition motifs or by inhibiting the proprotein convertases furin and PC6 with the serpin-based inhibitor alpha(1) antitrypsin Portland. Furthermore, both furin-dependent and furin-independent MT1-MMP processing pathways are identified that require tethering of the metalloproteinase to the cell surface. These findings demonstrate the existence of a proprotein convertase-MT1-MMP axis that can regulate extracellular matrix remodeling.     

Activation-coupled membrane-type 1 matrix metalloproteinase membrane trafficking

The transmembrane collagenase MT1-MMP (membrane-type 1 matrix metalloproteinase), also known as MMP-14, has a critical function both in normal development and in cancer progression, and is subject to extensive controls at the post-translational level which affect proteinase activity. As zymogen activation is crucial for MT1-MMP activity, an alpha1-PI (alpha1-proteinase inhibitor)-based inhibitor was designed by incorporating the MT1-MMP propeptide cleavage sequence into the alpha1-PI reactive-site loop (designated alpha1-PI(MT1)) and this was compared with wild-type alpha1-PI (alpha1-PI(WT)) and the furin inhibitory mutant alpha1-PI(PDX). Alpha1-PI(MT1) formed an SDS-stable complex with furin and inhibited proMT1-MMP activation. A consequence of the loss of MT1-MMP activity was the activation of proMMP-2 and the inhibition of MT1-MMP-mediated collagen invasion. alpha1-PI(MT1) expression also resulted in the intracellular accumulation of a glycosylated species of proMT1-MMP that was retained in the perinuclear region, leading to significantly decreased cell-surface accumulation of proMT1-MMP. These observations suggest that both the subcellular localization and the activity of MT1-MMP are regulated in a coordinated fashion, such that proMT1-MMP is retained intracellularly until activation of its zymogen, then proMT1-MMP traffics to the cell surface in order to cleave extracellular substrates.     

Cleavage of syndecan-1 by membrane type matrix metalloproteinase-1 stimulates cell migration

The transmembrane heparan sulfate proteoglycan syndecan-1 was identified from a human placenta cDNA library by the expression cloning method as a gene product that interacts with membrane type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with syndecan-1 in HEK293T cells promoted syndecan-1 shedding, and concentration of cell-associated syndecan-1 was reduced. Treatment of cells with MMP inhibitor BB-94 or tissue inhibitor of MMP (TIMP)-2 but not TIMP-1 interfered with the syndecan-1 shedding promoted by MT1-MMP expression. In contrast, syndecan-1 shedding induced by 12-O-tetradecanoylphorbol-13-acetate treatment was inhibited by BB-94 but not by either TIMP-1 or TIMP-2. Shedding of syndecan-1 was also induced by MT3-MMP but not by other MT-MMPs. Recombinant syndecan-1 core protein was shown to be cleaved by recombinant MT1-MMP or MT3-MMP preferentially at the Gly245-Leu246 peptide bond. HT1080 fibrosarcoma cells stably transfected with the syndecan-1 cDNA (HT1080/SDC), which express endogenous MT1-MMP, spontaneously shed syndecan-1. Migration of HT1080/SDC cells on collagen-coated dishes was significantly slower than that of control HT1080 cells. Treatment of HT1080/SDC cells with BB-94 or TIMP-2 induced accumulation of syndecan-1 on the cell surface, concomitant with further retardation of cell migration. Substitution of Gly245 of syndecan-1 with Leu significantly reduced shedding from HT1080/SDC cells and cell migration. These results suggest that the shedding of syndecan-1 promoted by MT1-MMP through the preferential cleavage of Gly245-Leu246 peptide bond stimulates cell migration.     

The shedding of betaglycan is regulated by pervanadate and mediated by membrane type matrix metalloprotease-1

Betaglycan is a membrane-anchored proteoglycan that binds transforming growth factor-beta (TGF-beta) via its core protein. A soluble form of betaglycan can be released by proteolytic cleavage (also known as shedding) of the membrane-bound form, yielding soluble betaglycan. The mechanism leading to the generation of soluble betaglycan is poorly understood. Because the membrane and soluble forms of betaglycan have opposite effects regulating the availability of TGF-beta, it is important to characterize the shedding of betaglycan further. Here we present evidence showing that in certain cell types, pervanadate, a general tyrosine phosphatase inhibitor, induces the release of the previously described fragment that encompasses almost the entire extracellular domain of betaglycan (sBG-120). In addition, treatment with pervanadate unveils the existence of a novel 90-kDa fragment (sBG-90). Noticeably, the cleavage that generates sBG-90 is mediated by a tissue inhibitor of metalloprotease-2-sensitive protease. Overexpression of all membrane type matrix metalloproteases (MT-MMPs) described to date indicates that MT1-MMP and MT3-MMP are endowed with ability to generate sBG-90. Furthermore, the patterns of expression of different MT-MMPs in the cell lines used in this study suggest that MT1-MMP is the protease involved in the shedding of sBG-90. Overexpression of MT1-MMP in COS-1 cells, which do not express detectable levels of this metalloprotease, confirms the feasibility of this hypothesis. Unexpectedly, during the course of these experiments, we observed that MT2-MMP decreases the levels of MT1-MMP and betaglycan. Finally, binding competition experiments indicate that, similar to the wild type membrane betaglycan, sBG-90 binds the TGF-beta2 isoform with greater affinity than TGF-beta1, suggesting that once released, it could affect the cellular availability of TGF-beta.     

Prointegrin maturation follows rapid trafficking and processing of MT1-MMP in Furin-Negative Colon Carcinoma LoVo Cells

Understanding the function of invasion-promoting membrane type-1 matrix metalloproteinase (MT1-MMP) is of paramount importance for understanding cancer biology. MT1-MMP is synthesized in cells as a latent zymogen that requires the cleavage of its prodomain to exert the proteolytic activity. The mature alphav integrin subunit is also generated by endoproteolytic cleavage of the alphav subunit precursor (pro-alphav). Cleavage by furin is considered to be a principal event in the activation of both MT1-MMP and pro-alphav. To elucidate the alternative activation pathway of MT1-MMP and pro-alphav, we employed furin-negative LoVo cells, which co-express MT1-MMP with integrin alphavbeta3. In these cells the MT1-MMP proenzyme was rapidly trafficked to the plasma membrane via an unconventional Brefeldin A-resistant pathway and, then, autocatalytically processed on the cell surface. Next, the MT1-MMP activity converted the cell surface-associated pro-alphav into the mature alphav integrin, represented by the disulfide-bonded heavy and light chains, and promoted the formation of the functional integrin alphavbeta3 heterodimer. These events stimulated cell motility in vitro, and malignant invasion and tumor growth in vivo. Our data suggest that in furin-negative colon carcinoma cells MT1-MMP is autocatalytically processed and the active protease then operates as a prointegrin convertase. Our findings argue strongly that the processing by furin is not a prerequisite for the activation of MT1-MMP.