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Co-recycling of MT1-MMP and MT3-MMP through the Trans-Golgi Network

2004; Elsevier BV; Volume: 279; Issue: 10 Linguagem: Inglês

10.1074/jbc.m312369200

1083-351X

Xing Wang, Dawei Ma, Jorma Keski‐Oja, Duanqing Pei,

Peptidase Inhibition and Analysis

Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV582 of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MT-MMPs may coordinate their proteolytic activities through the cellular trafficking machinery. Members of the membrane-type matrix metalloproteinases (MT-MMPs) have been implicated in a wide range of physiological and pathological processes from normal development to tumor growth. Tethered on plasma membrane, these enzymes are potentially regulated by the trafficking machinery of the cells. Here we demonstrate that both MT1-MMP and MT3-MMP are internalized, transported to the trans-Golgi network through early endosomes, and recycled back to cell surface in 60 min in a manner distinct from the one employed by transferrin receptor. Interestingly, co-expressed MT1-MMP and MT3-MMP are localized and routed in the same vesicles throughout the trafficking process. We further demonstrated that the carboxyl-terminal sequence DKV582 of MT1-MMP is required for its recycling, thus defining a novel recycling motif. These results suggest that MT-MMPs may coordinate their proteolytic activities through the cellular trafficking machinery. The cell surface, populated with various protein molecules that mediate communications between the cell and its immediate environment, in many ways defines the cellular phenotype. One class of such molecules is cell surface proteinases that exert functional influence through irreversible cleavage of their substrates (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 2Ellerbroek S.M. Stack M.S. BioEssays. 1999; 21: 940-949Crossref PubMed Scopus (133) Google Scholar, 3Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (226) Google Scholar, 4Koblinski J.E. Ahram M. Sloane B.F. Clin. Chim. Acta. 2000; 291: 113-135Crossref PubMed Scopus (511) Google Scholar). Therefore, these surface proteinases should be regulated properly, and perhaps, differently from other cell surface molecules. The membrane-type matrix metalloproteinases or MT-MMPs 1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; MDCK cells, Madin-Darby canine kidney cells; FITC, fluorescein isothiocyanate.1The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; MDCK cells, Madin-Darby canine kidney cells; FITC, fluorescein isothiocyanate. are good examples of cell surface proteinases employed by various cell types to alter their surrounding environment during angiogenesis, tissue remodeling, tumor invasion, and metastasis (1Werb Z. Cell. 1997; 91: 439-442Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar, 5Seiki M. APMIS. 1999; 107: 137-143Crossref PubMed Scopus (274) Google Scholar, 6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2371) Google Scholar, 7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 8Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 10Velasco G. Cal S. Merlos-Suarez A. Ferrando A.A. Alvarez S. Nakano A. Arribas J. Lopez-Otin C. Cancer Res. 2000; 60: 877-882PubMed Google Scholar). Displayed on the cell surface, these molecules have been shown to mediate a diverse biochemical reactions such as the activation of soluble MMPs, i.e. MMP-2 and MMP-13, or direct degradation of extracellular matrix components (6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2371) Google Scholar, 11Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 12Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (620) Google Scholar, 13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 14Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Abstract Full Text Full Text PDF PubMed Scopus (832) Google Scholar). Ablation of MT1-MMP in mice validated most of these functions in vivo (15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (686) Google Scholar, 16Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1108) Google Scholar). Yet little is known about the cellular mechanisms regulating the trafficking of MT-MMPs to and from the cell surface. The MT-MMP subfamily can be divided into two subgroups, MT1-, MT2-, MT3-, and MT5-MMPs with type I transmembrane domains and MT4- and MT6-MMPs with glycosylphosphatidylinositol anchors (6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2371) Google Scholar, 7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 8Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (447) Google Scholar, 18Okada A. Bellocq J.P. Rouyer N. Chenard M.P. Rio M.C. Chambon P. Basset P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2730-2734Crossref PubMed Scopus (486) Google Scholar, 20Llano E. Pendas A.M. Freije J.P. Nakano A. Knauper V. Murphy G. Lopez-Otin C. Cancer Res. 1999; 59: 2570-2576PubMed Google Scholar). Because the first group MT-MMPs have similar structures at their carboxyl termini with transmembrane domains and cytoplasmic tails, it is expected that their activities should be regulated by the trafficking machinery that presumably interacts with their cytoplasmic tails (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 24Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). Given their apparent divergence in their cytoplasmic domains, the trafficking pattern of each MT-MMP may differ significantly (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar). Indeed, although MT1-MMP and MT3-MMP are regulated by endocytosis, MT5-MMP does not appear to internalize well in the cells analyzed (24Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). Functionally, we have recently shown that MT1-MMP and MT3-MMP have distinct activities toward two well established substrates, proMMP-2 and type I collagen (25Jiang A. Pei D. J. Biol. Chem. 2003; 278: 38765-38771Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Furthermore, these distinctions are encoded in their individual domains, i.e. catalytic and hemopexin domains, as analyzed by domain-swapping experiments (25Jiang A. Pei D. J. Biol. Chem. 2003; 278: 38765-38771Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Based on these findings, we hypothesize that the type I MT-MMPs may be regulated coordinately by the cellular trafficking machinery to deliver their distinct proteolytic activities to the cell surface. In this report we demonstrate that MT1-MMP and MT3-MMP are co-internalized and co-routed through the recycling pathway back to the cell surface, providing the cells a unique way to regulate their activity dynamically under physiological as well as pathological conditions. Cell Culture—Madin-Darby canine kidney cells (MDCK, ATCC, Manassas, VA) were maintained as described (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 26Kang T. Nagase H. Pei D. Cancer Res. 2002; 62: 675-681PubMed Google Scholar). Cell culture media and supplements were purchased from Invitrogen. Antibodies and Reagents—Rabbit anti-MT1-MMP antibody (Ab3) has been described (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 27Lehti K. Lohi J. Juntunen M.M. Pei D. Keski-Oja J. J. Biol. Chem. 2002; 277: 8440-8448Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). FITC-labeled mouse anti-MT1-MMP monoclonal antibody was purchased from R&D Systems (Minneapolis, MN). Rabbit anti-MT3-MMP antibody was from Chemicon (Temecula, CA). Mouse anti-p230 antibody was purchased from Transduction Laboratories (Lexington, KY). Rabbit anti-furin antibody was from Affinity Bioreagents (Golden, CO). Texas Red-conjugated human transferrin and Alexa-488- and -595-conjugated secondary antibodies were from Molecular Probes (Eugene, OR). Proteinase inhibitors and secondary antibody conjugates were from Sigma. BB-94 was a gift from British Biotechnology (Oxford, UK). MP070 is a novel MMP inhibitor that will be described (17Ma' D. Wu W. Yang G. Li J. Li J. Ye Q. Bioorg. Med. Chem. Lett. 2004; (in press)Google Scholar). Expression Constructs and Transfection—The expression constructs for MT1-MMP and MT3-MMP have been described (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 26Kang T. Nagase H. Pei D. Cancer Res. 2002; 62: 675-681PubMed Google Scholar). MT1-MMPΔ3 was constructed by a two-step process using high fidelity PCR and inserted in the EcoRV site of pCR3.1 expression vector as described (7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). RUFY expression construct is a gift from Dr. Qiu (7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). The DNA constructs were transfected into various cells by Effectene (Qiagen, Valencia, CA) according to manufacturer's protocol. Select experiments were performed in the presence of MMP inhibitor, BB-94 and MP-070, to prevent the autocatalytic decay of MT1-MMP. Western Blotting, Immunoprecipitation, and Gelatin Zymography— These procedures have been described previously (7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). In brief, serum-free media supplemented with purified pro-MMP-2 or proMMP-2 in fetal bovine serum (5% v/v) were added to cells. After the indicated time period, conditioned media were collected, cleared of cell debris by centrifugation and analyzed by SDS/PAGE impregnated with gelatin (1 mg/ml) as described (7Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). For immunoprecipitation and Western blotting, cells were lysed in radioimmune precipitation assay buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, protease inhibitors mixture tablet Complete (Roche Applied Science). The lysates were centrifuged (14,000 × g, 20 min) to remove debris, and the resulting supernatants were immunoprecipitated and blotted as described (28Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). Immunostaining and Confocal Microscopy—For internalization experiments cells seeded on coverslips in 6-well plates were washed 3 times with phosphate-buffered saline and shifted to 4 °C. Anti-MT-MMP antibodies were added to the cells at 0.2 μg/ml for 2 h. Antibody was subsequently removed, and cells were washed before being shifted to 37 °C with prewarmed media for the indicated times. Cells were then fixed with 3.7% paraformaldehyde in phosphate-buffered saline, pH 7.4, for 30 min at room temperature and blocked with phosphate-buffered saline diluent (0.3% Triton X-100, 1% normal donkey serum, 1% bovine serum albumin, and 0.01% sodium azide, pH 7.2) for 1 h followed by staining with secondary antibodies (Molecular Probes). The coverslips were mounted by NO-FADE (10% glycerol in phosphate-buffered saline, 0.1% p-phenylenediamine, pH 8.0). For colocalization experiments, cells were labeled with M2 anti-FLAG or anti-p230 antibodies as the primary antibodies after fixation followed by Alex-595-conjugated secondary antibody (Molecular Probes). Confocal microscopy was carried out in the Biomedical Image Processing laboratories at the University of Minnesota using a Bio-Rad MRC 1024 system attached to an Olympus microscope (Melville, NY) with a 60× oil objective. The images were processed in Photoshop 7.0 (Adobe, San Jose, CA). Quantification was carried out in Openlab (Improvision, Coventry, UK). The statistic analysis was done with GraphPad Prism software program (San Diego, CA). Recycling Patterns for MT1-MMP and MT3-MMP—We have recently demonstrated that MT1-MMP and MT3-MMP differ greatly in their activities against type I collagen and proMMP-2 (25Jiang A. Pei D. J. Biol. Chem. 2003; 278: 38765-38771Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). It is not clear if they are regulated similarly by the cellular trafficking machinery, given the apparent divergence between their cytoplasmic domains (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar). To determine the difference between these two enzymes, we extended our internalization experiments up to 90 min as described (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar). Surprisingly, both MT1-MMP and MT3-MMP virtually followed the same pattern of recycling as shown in Fig. 1A. Both molecules were labeled on the cell surface at 4 °C (Fig. 1A, panels a and b). Upon shifting to 37 °C to commence internalization, a lag period of 10 min was observed (Fig. 1A, panels c and d). In the next 20 min most of the cell surface MT1-MMP and MT3-MMP were internalized (Fig. 1A, panels e and f). Between 50–70 min the internalized molecules appeared to have recycled back to the cell surface (Fig. 1A, panels g, h, i, and j). Further incubations suggest that the recycled MT1-MMP and MT3-MMP are re-internalized again, resembling the steady state pattern observed for MT1-MMP and MT3-MMP (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar, 26Kang T. Nagase H. Pei D. Cancer Res. 2002; 62: 675-681PubMed Google Scholar) (Fig. 1A, panels k and l), suggesting that an equilibrium is achieved through the recycling of MT-MMPs between intracellular pools and the cell surface. Because internalized MT-MMPs may enter intracellular compartments where MT1-MMP or MT3-MMP dissociates from their respective antibodies, the observed findings in Fig. 1A may reflect the route of the dissociated antibody rather than that of the two enzymes. To address this concern, we re-stained the cells that underwent internalization with rabbit anti-MT1-MMP antibody and found that the internalized MT1-MMPs labeled with mouse anti-MT1-MMP antibody remained reactive with the rabbit antibody (Fig. 1B, panel b), strongly suggesting that the internalized MT1-MMP-antibody complex remains intact. Similar results were obtained for the MT3-MMP-antibody complex (data not shown). Further quantification supports the conclusion that both MT1-MMP and MT3-MMP undergo internalization and recycling in a similar fashion (Fig. 1C). To confirm whether the recycled MT1-MMPs are indeed re-exposed on cell surface, we labeled MT1-MMP on the cell surface with FITC-conjugated mouse monoclonal antibody (Fig. 1D, panels a–c), allowed them to internalize for 30 min, stripped off any residual antibodies on the cell surface (Fig. 1D, panels g–l), and then incubated for a further 30 min to allow the internalized MT1-MMP to resurface (Fig. 1D, panel j–l). Should the resurfaced MT1-MMP molecules remain associated with the mouse antibodies, anti-mouse IgG should be able to detect these resurfaced molecules. Indeed, we detected the resurfaced mouse antibodies as shown in Fig. 1D, panel l. Similar results were obtained when MT3-MMP recycling was analyzed in a similar fashion (data not shown). Together, we conclude that both MT1-MMP and MT3-MMP recycles through the intracellular compartment. Recycling of MT-MMPs through the Trans-Golgi Network—To understand the route of MT1-MMP recyling we traced the internalized MT1-MMP with markers for various vesicles. Consistent with our previously conclusion that the internalized MT1-MMP enters the endosomes (23Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (224) Google Scholar), we observed that the newly internalized MT1-MMPs co-localize with RUFY, a rab4-interacting protein associated with early endosomes (29Yang J. Kim O. Wu J. Qiu Y. J. Biol. Chem. 2002; 277: 30219-30226Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar) (Fig. 2A). When most of the cell surface labels were internalized, a significant portion of the internalized MT1-MMP became co-localized with p230, a marker for trans-Golgi network (Fig. 2B, panel b). Interestingly, we also found that a majority portion of the internalized MT1-MMPs are co-localized with furin, a resident protein in the trans-Golgi network (Fig. 2C). Similar results were obtained with internalized MT3-MMP (data not shown). Given these results, we conclude that MT1-MMP and MT3-MMP are internalized through the early endosomes, routed through the trans-Golgi network, where they are recycled back to the cell surface. Co-internalization of MT1-MMP and MT3-MMP—The recycling patterns of MT1-MMP and MT3-MMP are very similar as shown in Figs. 1 and 2, suggesting that they may recycle through the same vesicles. To test this possibility, we co-transfected cells with both MT1-MMP and MT3-MMP and performed internalization experiments with mouse anti-MT1-MMP antibody and rabbit anti-MT3-MMP antibodies as described in Fig. 1. A time course study revealed that MT1-MMP and MT3-MMP are virtually co-localized throughout the recycling process. As shown in Fig. 3A, signals of internalized MT1-MMP and MT3-MMP overlapped in almost 100% of the vesicles observed. This observation is consistent with the recent report that MT1-MMP and MT3-MMP share similar motifs in their cytoplasmic domains for internalization (24Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar). To rule out the possibility that the observed co-internalization is due to passive segregation of cell surface molecules in the same vesicles, we compared the internalization of MT1-MMP with that of transferrin. As shown in Fig. 3B, transferrin is internalized much faster than MT1-MMP, with the internalization process virtually completed in 5–10 min, compared with a time frame of 30–40 min for MT1-MMP. These data strongly suggest that MT1-MMP and MT3-MMP share the same internalization pathway, distinct from that employed by transferrin. The Carboxyl Motif DKV582 in MT1-MMP Is Required for Its Recycling—To begin to probe the cellular machinery regulating MT1-MMP recycling, we analyzed a panel of MT1-MMP mutants with carboxyl-terminal truncations. One of the mutants, MT1-MMPΔ3 (Fig. 4A), revealed an interesting pattern of trafficking. Distinct from the pattern exhibited by wild type MT1-MMP shown in Fig. 1A, panels g and i, this mutant fails to recycle to the cell surface after an apparently normal internalization process (Fig. 4B). Further quantitative analysis confirmed that MT1-MMPΔ3 follows the same kinetics as wild type MT1-MMP during internalization (Fig. 4C, 10 and 30 min) but remains intracellular when the wild type molecules recycle to the cell surface (Fig. 4C, 50 and 70 min). Similar results were obtained with MT3-MMP (data not shown). Together these data suggest that the well conserved DKV582 motif in MT1-MMP encodes a signal for recycling. In this report we describe the recycling of MT1-MMP and MT3-MMP in MDCK cells in a coordinated fashion. Both molecules recycle every 60 min between plasma membrane and intracellular vesicles, mostly trans-Golgi and endosomal vesicles, in a pattern distinct from the transferrin receptor. These findings outline a basic trafficking map for the MT-MMPs, which may be exploited for the development of drugs against MT-MMP-dependent pathological conditions such as cancer metastasis. Furthermore, our data raise the possibility that MT-MMPs can be mobilized in a coordinated fashion to specialized areas of the cell membrane where focalized proteolysis is a prerequisite for many pathological and physiological conditions. One concern about the experimental system described here is the bivalent nature of the antibodies used, which may bind to both the antigens (MT1-MMP or MT3-MMP) and the antibody receptors on cell surface, thus altering the trafficking pattern. Fortunately, MDCK is a well characterized cell line utilized routinely for trafficking-related studies (30Jou T.S. Leung S.M. Fung L.M. Ruiz W.G. Nelson W.J. Apodaca G. Mol. Biol. Cell. 2000; 11: 287-304Crossref PubMed Scopus (57) Google Scholar, 31Hunziker W. Male P. Mellman I. EMBO J. 1990; 9: 3515-3525Crossref PubMed Scopus (128) Google Scholar). So far, no Fc receptors have been reported for MDCK cells, and Fc receptor had to be transfected into MDCK cells when IgG transcytosis was investigated (30Jou T.S. Leung S.M. Fung L.M. Ruiz W.G. Nelson W.J. Apodaca G. Mol. Biol. Cell. 2000; 11: 287-304Crossref PubMed Scopus (57) Google Scholar, 31Hunziker W. Male P. Mellman I. EMBO J. 1990; 9: 3515-3525Crossref PubMed Scopus (128) Google Scholar). Furthermore, Seiki and co-workers (24Uekita T. Itoh Y. Yana I. Ohno H. Seiki M. J. Cell Biol. 2001; 155: 1345-1356Crossref PubMed Scopus (215) Google Scholar) observe no difference between intact IgG and its Fab fragment in internalization and trafficking of MT1-MMP in several cell lines. Finally, the fact that both the wild type MT1-MMP and MT1-MMPΔ3 underwent distinct routes of trafficking, as detected by the same antibodies in the same cells, also suggests that the Fc moiety has no effect on MT1-MMP trafficking. In agreement, a report was published using antibody labeling to demonstrate that MT1-MMP does recycle back to cell surface (32Remacle A. Murphy G. Roghi C. J. Cell Sci. 2003; 116: 3905-3916Crossref PubMed Scopus (214) Google Scholar). Together, the data presented in this report reflect the trafficking pattern of MT-MMPs without the influence from any potential Fc-Fc receptor interaction. Recycling of MT-MMPs may be one of the strategies to mobilize their proteolytic activity rapidly in response to physiological and pathological signals. This mechanism may allow the cells to maintain a dynamic pool of MT-MMPs readily deployable to the cell surface. Recycling may also enable the cells to redistribute cell surface molecules as described for endothelin-converting enzyme (33Muller L. Barret A. Etienne E. Meidan R. Valdenaire O. Corvol P. Tougard C. J. Biol. Chem. 2003; 278: 545-555Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Redistribution of the MT-MMPs may mobilize pre-existent molecules to cell surface locations or compartments undergoing active proteolysis, thus affording the cells the capability to regulate proteolytic activity without altering the profile of gene expression. The observed co-recycling of MT1-MMP and MT3-MMP suggests that both enzymes could be coordinated at the cellular level through trafficking events to focus their distinct proteolytic activities on the extracellular matrix barriers encountered by cells such as metastasizing tumor cells. Indeed, both MT1-MMP and MT3-MMP have been localized together in malignant tumor specimens (19Sato H. Okada Y. Seiki M. Thromb. Haemostasis. 1997; 78: 497-500Crossref PubMed Scopus (103) Google Scholar, 34Ueno H. Nakamura H. Inoue M. Imai K. Noguchi M. Sato H. Seiki M. Okada Y. Cancer Res. 1997; 57: 2055-2060PubMed Google Scholar). Given the irreversible nature of proteolysis, a selective advantage may be achieved by re-directing MT-MMPs to focal area where the underling extracellular matrix is hydrolyzed abnormally. The hydrolysis of extracellular matrix would certainly lead to alteration of the cell microenvironment, which could confer growth advantages for the surrounding cells such that subsequent mutations and genetic alterations can be accumulated. Thus, therapeutic or preventive intervention may be designed against the trafficking pathway of MT-MMPs. Finally, we defined a recycling-deficient mutant in MT1-MMP, i.e. MT1-MMPΔ3. Urena et al. (22Urena J.M. Merlos-Suarez A. Baselga J. Arribas J. J. Cell Sci. 1999; 112: 773-784Crossref PubMed Google Scholar) note that the carboxyl-terminal residue of MT1-MMP and transforming growth factor α determines their subcellular localization. However, the carboxyl-terminal mutant of MT1-MMP analyzed by these investigators failed to mature properly (22Urena J.M. Merlos-Suarez A. Baselga J. Arribas J. J. Cell Sci. 1999; 112: 773-784Crossref PubMed Google Scholar), in contrast to our finding that MT1-MMPΔ3 behaves just like the wild type molecule except for its recycling to cell surface. More work is needed to reconcile our observation with that of Urena et al. (22Urena J.M. Merlos-Suarez A. Baselga J. Arribas J. J. Cell Sci. 1999; 112: 773-784Crossref PubMed Google Scholar). The cellular proteins interacting with this motif remain unknown. However, we have identified an adaptor protein with two PDZ motifs that can interact with the analogous EWV motif of MT5-MMP. 2X. Wang, P. Wang, and D. Pei, unpublished results. Interestingly, through a phage display screen, the DKV582 motif of MT1-MMP was identified as a binding site for the PDZ domain of the nNOS protein (21Stricker N.L. Christopherson K.S. Yi B.A. Schatz P.J. Raab R.W. Dawes G. Bassett Jr., D.E. Bredt D.S. Li M. Nat. Biotechnol. 1997; 15: 336-342Crossref PubMed Scopus (218) Google Scholar). However, we have not been able to implicate nNOS in the trafficking of MT1-MMP. 3A. Jiang and D. Pei, unpublished results. Given the specific defect identified for MT1-MMPΔ3 in this report, we are currently searching for the cellular factor that can interact with the DKV582 motif of MT1-MMP and regulate its trafficking in cells. We thank members of the Pei laboratory for encouragement and discussions.