The C-terminal Region of Membrane Type Matrix Metalloproteinase Is a Functional Transmembrane Domain Required for Pro-gelatinase A Activation

Abstract

We identified a new matrix metalloproteinase (membrane type matrix metalloproteinase (MT-MMP)) that has a potential transmembrane (TM) domain at the C terminus and reported its expression on the surface of invasive tumor cells. The expression of MT-MMP induced specific activation of 72-kDa pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M.(1994) Nature 370, 61-65). Thus, MT-MMP on the cell surface is thought to play an important role in various physiological and pathological processes accompanying tissue remodeling. In this study, we demonstrated that the potential TM domain deduced from the amino acid sequence functions as a membrane linker when it is fused to a secretory protein, tissue inhibitor of matrix metalloproteinases-1. The pro-gelatinase A activation function of MT-MMP was abolished by truncation of the TM domain and recovered by fusing the MT-MMP mutant with the TM domain of interleukin 2 receptor α-chain. The truncated MT-MMP was released from the cells into the medium and detected as processed or modified forms. In spite of the deletion of the TM domain some portions of the mutant MT-MMP were still retained on the surface of cells. Thus, MT-MMP has an additional device to keep it on the cell surface. The TM domain however, plays an essential role in the pro-gelatinase A activation function of MT-MMP, probably regulating its fine orientation or the localization that is necessary to interact with substrate. We identified a new matrix metalloproteinase (membrane type matrix metalloproteinase (MT-MMP)) that has a potential transmembrane (TM) domain at the C terminus and reported its expression on the surface of invasive tumor cells. The expression of MT-MMP induced specific activation of 72-kDa pro-gelatinase A (Sato, H., Takino, T., Okada, Y., Cao, J., Shinagawa, A., Yamamoto, E., and Seiki, M.(1994) Nature 370, 61-65). Thus, MT-MMP on the cell surface is thought to play an important role in various physiological and pathological processes accompanying tissue remodeling. In this study, we demonstrated that the potential TM domain deduced from the amino acid sequence functions as a membrane linker when it is fused to a secretory protein, tissue inhibitor of matrix metalloproteinases-1. The pro-gelatinase A activation function of MT-MMP was abolished by truncation of the TM domain and recovered by fusing the MT-MMP mutant with the TM domain of interleukin 2 receptor α-chain. The truncated MT-MMP was released from the cells into the medium and detected as processed or modified forms. In spite of the deletion of the TM domain some portions of the mutant MT-MMP were still retained on the surface of cells. Thus, MT-MMP has an additional device to keep it on the cell surface. The TM domain however, plays an essential role in the pro-gelatinase A activation function of MT-MMP, probably regulating its fine orientation or the localization that is necessary to interact with substrate. INTRODUCTIONFor tumor cell invasion, degradation of the extracellular matrix in the vicinity of the cell surface is thought to be essential(1Zucker S. Wieman J.M. Lysik R.M. Wilkie D.P. Ramamurthy N. Lane B. Biochim. Biophys. Acta. 1987; 924: 225-237Crossref PubMed Scopus (49) Google Scholar, 2Nakajima M. Lotan D. Baig M.M. Carralero R.M. Wood W.R. Hendrix M.J. Lotan R. Cancer Res. 1989; 49: 1698-1706PubMed Google Scholar, 3Liotta L.A. Steeg P.S. Stetler-Stevenson W.G. Cell. 1991; 64: 327-336Abstract Full Text PDF PubMed Scopus (2634) Google Scholar, 4Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar, 5Tsuchiya Y. Sato H. Endo Y. Okada Y. Mai M. Sasaki T. Seiki M. Cancer Res. 1993; 53: 1397-1402PubMed Google Scholar, 6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 7Monsky W.L. Kelly T. Lin C.-Y. Yeh Y. Stetler-Stevenson W.G. Mueller S.C. Chen W.-T. Cancer Res. 1993; 53: 3159-3164PubMed Google Scholar). Many investigators have reported that the plasma membrane fractions of invasive tumor cells contain MMP 1The abbreviations used are: MMPmatrix metalloproteinaseMT-MMPmembrane-type matrix metalloproteinaseTIMP-1tissue inhibitor of metalloproteinases-1TMtransmembrane domainIL-2Rinterleukin-2 receptor α-chainmAbmonoclonal antibodyBSAbovine serum albuminPBSphosphate-buffered salineFCSfetal calf serumDMEMDulbecco's modified Eagle's medium. activators(6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4545PubMed Google Scholar, 10Brown P.D. Kleiner D.E. Unsworth E.J. Stetler-Stevenson W.G. Kidney Int. 1993; 43: 163-170Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 11Kleiner D.J. Stetler-Stevenson W.G. Curr. Opin. Cell Biol. 1993; 5: 891-897Crossref PubMed Scopus (327) Google Scholar). Urokinase type plasminogen activator (uPA), associating on the tumor cell surface through a specific receptor(12Roldan A.L. Cubellis M.W. Masucci M.T. Beherendt N. Lund L.D. Dano K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (539) Google Scholar), is an initiator of the MMP activation cascade, activating plasminogen to plasmin, which then activates interstitial collagenase and stromelysin 1(6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 13He C.S. Wilhelm S.M. Pentland A.P. Marmer B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Crossref PubMed Scopus (522) Google Scholar, 14Suzuki K. Enghild J.J. Morodomi T. Salvesen G. Nagase H. Biochemistry. 1990; 29: 10261-10270Crossref PubMed Scopus (384) Google Scholar, 15Vassalli J.D. Sappino A.P. Belin D. J. Clin. Invest. 1991; 88: 1067-1072Crossref PubMed Scopus (1091) Google Scholar). The 72-kDa gelatinase A is another MMP that is reportedly activated on the invasive tumor cell surface(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 16Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar) and retained there through interaction with a receptor-like molecule (7Monsky W.L. Kelly T. Lin C.-Y. Yeh Y. Stetler-Stevenson W.G. Mueller S.C. Chen W.-T. Cancer Res. 1993; 53: 3159-3164PubMed Google Scholar, 18Emonard H.P. Remacle A.G. Noel A.C. Grimaud J.A. StetlerStevenson W.G. Foidart J.M. Cancer Res. 1992; 52: 5845-5848PubMed Google Scholar). However, neither uPA nor plasmin can activate pro-gelatinase A. Instead, it is activated by the cells cultivated with 12-O-tetradecanoylphorbol-13-acetate(16Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar), concanavalin A(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 19Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar), or in collagen gel (9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4545PubMed Google Scholar) and by invasive tumor cells(10Brown P.D. Kleiner D.E. Unsworth E.J. Stetler-Stevenson W.G. Kidney Int. 1993; 43: 163-170Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Azzam H.S. Arand G. Lippman M.E. Thompson E.W. J. Natl. Cancer Inst. 1993; 85: 1758-1764Crossref PubMed Scopus (207) Google Scholar). The plasma membrane fraction prepared from the cells can activate pro-gelatinase A, and the reaction was sensitive to chelating reagents and tissue inhibitor of metalloproteinases-2 (TIMP-2)(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Thus, the putative pro-gelatinase A activator may be a member of the MMP family.We isolated a cDNA clone that encodes a hitherto unknown MMP named MT-MMP(21Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1994; (in press)Google Scholar, 22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). MT-MMP is expressed as a 63-kDa protein on the surface of transfected cells with MT-MMP cDNA. At the same time, the cells caused specific activation of 72-kDa pro-gelatinase A(22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). Since MT-MMP has a potential TM domain and was not released into the culture medium, the TM domain is thought to play a direct role in MT-MMP integration into the plasma membrane. In this study, we examined whether the potential TM domain is indeed required for anchoring MT-MMP onto the cell surface by constructing deletions and chimeric molecules. The pro-gelatinase A activation function was also examined in to the localization of MT-MMP of of the was used to MT-MMP as H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). A. Murphy G. Reynolds J.J. Nature. PubMed Scopus Google Scholar) and the TM of MT-MMP or interleukin 2 receptor J.M. S. J. M. Nature. PubMed Scopus Google Scholar, M. S. T. G. T. Nature. PubMed Scopus Google Scholar) were as The cDNA the C terminus of MT-MMP to the deduced TM domain to or the TM domain of to were by were then to the of the cDNA to as and into the expression A deletion mutant the deduced TM domain of MT-MMP was by a linker and a at amino from the linker were to the truncated C A and the TM domain of was the in the and the TM The were for for for and for and cells were in modified Eagle's calf serum and 2 a were transfected into cells as H. M. Seiki M. J. Biol. Chem. 1993; 268: Full Text PDF PubMed Google as H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). for the cells were with in and for at the medium was and by were then in the and cell were with or MT-MMP were by were with protein A to by with were and in and then in an gel with a The was detected by a of Cell cell membrane was as A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). cells transfected with mutant and type MT-MMP expression were for in were and a The was at for and the fraction was The cell fraction was by at for 2 The was in and onto a and and at for The plasma fraction at the was and The were in at a protein of fractions of the cells transfected with various were with an of 2 The were by gel The were onto a membrane a The membrane was with serum in for at and then with and MT-MMP for at The of was in with the membrane was with with in for at The was in a of of The reaction was by the in was as A. E. J. J. 1992; PubMed Google Scholar). cells were with and or MT-MMP expression and then with for at The cells were with and then with and at for with and in on The cell were by and with protein as of membrane fractions were with of as a of pro-gelatinase A and for 2 at The reaction was by the of and the were by was as J.M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). were with in the of a and for to and on and at a of were in for to the this activated were then for at in a reaction and with The of is as a in the of TM of MT-MMP as a the role of the putative TM domain of MT-MMP as a membrane we prepared a protein the TM domain and a secretory protein, by the of a was that expressed chimeric The TM domain of the receptor was used as a and a protein was and were expressed in cells by of the with cell were prepared and examined by can be detected as a of the P. R. J. Biol. Chem. 1990; 265: Full Text PDF PubMed Google Scholar). and were detected in cell as and that were to the and was detected in the culture medium, the chimeric were Thus, of the TM fused to to the of the into the medium. However, it is that chimeric the secretory in the cells. this the of chimeric protein expression was by the chimeric with that was were detected in the cell of and its in cells. and its and were transfected into cells. were then with for and in the cell and in the medium were with a were on and the was detected by of in the A, the transfected were the expression for and of the were and as in the were was with or and were the same as in the localization of the on the cell transfected cells were on and examined by by the cells with H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). cells were not with and cells were on the cell surface not that the TM domain of can a secretory on the cell surface and that the of the TM domain of MT-MMP is also with that of Thus, the TM domain of MT-MMP is a membrane of the TM on A the role of the TM domain of MT-MMP in pro-gelatinase A activation the C terminus the TM domain was truncated as and cells that pro-gelatinase A into the culture were used as the for A activation was by with the MT-MMP induced of pro-gelatinase A into the activated an A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, 22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar), the truncated mutant to activate The was not of the of expression as demonstrated by and pro-gelatinase A activation was not induced when the of the transfected were not Thus, the TM domain of MT-MMP is for pro-gelatinase A of the TM domain from MT-MMP and its on pro-gelatinase A cells were transfected with the expression and A, transfected cells were with for and cell were prepared and with cells transfected with MT-MMP, or were in for medium was with and in a gel as and the gel was with of the TM of is still to that the TM domain of MT-MMP is required for pro-gelatinase A activation in a as a membrane this the TM domain of receptor was fused to and the of pro-gelatinase A activation function was expression of in cells induced pro-gelatinase A activation as as plasma membrane fractions of the transfected cells were and the pro-gelatinase A that is in calf serum were examined membrane fractions from the transfected cells not contain by with from the cells that MT-MMP and retained the to of gelatinase A, that from the cells not Thus, we that the TM domain was to the function of MT-MMP and that in the TM domain were not essential for this A activation by the plasma membrane cells transfected with were and plasma membrane fractions were prepared by fractions of were with or calf serum for at and were by of the MT-MMP and were transfected into cells with the and the in the culture were examined by with MT-MMP and were detected at in the cell MT-MMP was not released into the culture medium as and the secretory protein was released and in the medium H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). However, was detected in the culture medium, at as with the and detected in the medium were with and and were not the same as in the cell is not at this of or is with the released into the culture medium. However, it is that deletion of the TM domain from MT-MMP the of the into the of and its into culture medium. cells were with MT-MMP, or with were with and in the or cell were with and detected by in the from by were detected by another not the of cell surface expression of the cell surface A. E. J. J. 1992; PubMed Google Scholar) were transfected cells were with and were The cells were then and that on the surface were with protein The was a protein, and it was detected in the cell the MT-MMP and were by this as in The secretory protein was not not Thus, a fraction of on the cell it is thought to be into the medium. that MT-MMP has an additional device to keep it on the surface of cells its TM of the plasma membrane which were used for the as in the demonstrated that and MT-MMP with the membrane fractions is also another that is still associating with the cell of and MT-MMP on the plasma membrane. A, the expression was into cells with and MT-MMP cells were then with MT-MMP and and then were and the cells were were with protein were prepared from the cells that were not with and as in plasma membrane fractions of cells transfected with and MT-MMP were by and is an MMP by cDNA T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1994; (in press)Google Scholar, 22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). We deduced the cell surface expression of MT-MMP from its at the C which of a amino acid it indeed on the cell surface and specific activation of pro-gelatinase A, we not have to the protein is to the plasma membrane and its in the function of the The function of the putative TM domain of MT-MMP as a membrane linker was demonstrated by constructing a protein with the secretory protein The TM domain the localization from culture medium onto the cell as the TM domain of Thus, we that the TM domain is to MT-MMP on the cell membrane integration of MT-MMP through the TM domain was essential for its pro-gelatinase A activation the was abolished by truncation of the domain and recovered by a of the mutant with a TM domain of receptor of the TM domain of the into the culture medium. However, a of the was still retained on the cell surface as by the Thus, MT-MMP to have additional the TM domain to it on the cell surface. However, not activate pro-gelatinase A its localization on the cell surface. we not this the cell surface MT-MMP and is however, that membrane integration is essential to the orientation of the molecule on the cell the molecule to interact with or at that released into the medium have a and from that in and on the cells Since of the released were specific or of the to with the at or from the cells. The medium the not pro-gelatinase A activation not expressed in the transfected cells is thought be in the a the is to that of the a deletion in the which be it is in a as from the protein sequence not The activation of MT-MMP from that of the the sequence can be by A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar, H. H. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar, H. Enghild J.J. K. Salvesen G. Biochemistry. 1990; 29: PubMed Scopus Google Scholar, Y. Morodomi T. Enghild J.J. K. A. Salvesen G. Nagase H. J. 1990; PubMed Scopus Google Scholar, D.E. A. K. Stetler-Stevenson W.G. Biochemistry. 1993; PubMed Scopus Google Scholar). MT-MMP and stromelysin P. P. J.M. P. Nature. 1990; PubMed Scopus Google Scholar) have an sequence the and T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1994; (in press)Google Scholar, 22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). that reported to be the for M. M. T. K. J. K. K. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, D. G. 1993; PubMed Scopus Google Scholar) were the Many secretory and have this and processed J.M. M.J. R.M. Gene (Amst.). 1991; PubMed Scopus Google Scholar, D. C.M. 1992; PubMed Scopus Google Scholar, Annu. Rev. 1992; PubMed Scopus Google Scholar). However, this to be at for some of the of can be processed into in the Proc. Natl. Acad. Sci. U. S. A. 85: PubMed Scopus Google Scholar). Thus, MT-MMP may be at in the transfected and a of the can be that to be is whether MT-MMP the domain of pro-gelatinase A. activation may be by a molecule that is the of We to MT-MMP from the membrane fraction and direct pro-gelatinase A. A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar) reported that an in the domain and Thus, it be of to whether MT-MMP can this this study, we demonstrated the function of the TM domain of MT-MMP as a membrane linker and as an essential for pro-gelatinase A A chimeric molecule as be in the specific of MT-MMP INTRODUCTIONFor tumor cell invasion, degradation of the extracellular matrix in the vicinity of the cell surface is thought to be essential(1Zucker S. Wieman J.M. Lysik R.M. Wilkie D.P. Ramamurthy N. Lane B. Biochim. Biophys. Acta. 1987; 924: 225-237Crossref PubMed Scopus (49) Google Scholar, 2Nakajima M. Lotan D. Baig M.M. Carralero R.M. Wood W.R. Hendrix M.J. Lotan R. Cancer Res. 1989; 49: 1698-1706PubMed Google Scholar, 3Liotta L.A. Steeg P.S. Stetler-Stevenson W.G. Cell. 1991; 64: 327-336Abstract Full Text PDF PubMed Scopus (2634) Google Scholar, 4Stetler-Stevenson W.G. Aznavoorian S. Liotta L.A. Annu. Rev. Cell Biol. 1993; 9: 541-573Crossref PubMed Scopus (1515) Google Scholar, 5Tsuchiya Y. Sato H. Endo Y. Okada Y. Mai M. Sasaki T. Seiki M. Cancer Res. 1993; 53: 1397-1402PubMed Google Scholar, 6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 7Monsky W.L. Kelly T. Lin C.-Y. Yeh Y. Stetler-Stevenson W.G. Mueller S.C. Chen W.-T. Cancer Res. 1993; 53: 3159-3164PubMed Google Scholar). Many investigators have reported that the plasma membrane fractions of invasive tumor cells contain MMP 1The abbreviations used are: MMPmatrix metalloproteinaseMT-MMPmembrane-type matrix metalloproteinaseTIMP-1tissue inhibitor of metalloproteinases-1TMtransmembrane domainIL-2Rinterleukin-2 receptor α-chainmAbmonoclonal antibodyBSAbovine serum albuminPBSphosphate-buffered salineFCSfetal calf serumDMEMDulbecco's modified Eagle's medium. activators(6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4545PubMed Google Scholar, 10Brown P.D. Kleiner D.E. Unsworth E.J. Stetler-Stevenson W.G. Kidney Int. 1993; 43: 163-170Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 11Kleiner D.J. Stetler-Stevenson W.G. Curr. Opin. Cell Biol. 1993; 5: 891-897Crossref PubMed Scopus (327) Google Scholar). Urokinase type plasminogen activator (uPA), associating on the tumor cell surface through a specific receptor(12Roldan A.L. Cubellis M.W. Masucci M.T. Beherendt N. Lund L.D. Dano K. Appella E. Blasi F. EMBO J. 1990; 9: 467-474Crossref PubMed Scopus (539) Google Scholar), is an initiator of the MMP activation cascade, activating plasminogen to plasmin, which then activates interstitial collagenase and stromelysin 1(6Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar, 13He C.S. Wilhelm S.M. Pentland A.P. Marmer B.L. Grant G.A. Eisen A.Z. Goldberg G.I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2632-2636Crossref PubMed Scopus (522) Google Scholar, 14Suzuki K. Enghild J.J. Morodomi T. Salvesen G. Nagase H. Biochemistry. 1990; 29: 10261-10270Crossref PubMed Scopus (384) Google Scholar, 15Vassalli J.D. Sappino A.P. Belin D. J. Clin. Invest. 1991; 88: 1067-1072Crossref PubMed Scopus (1091) Google Scholar). The 72-kDa gelatinase A is another MMP that is reportedly activated on the invasive tumor cell surface(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 16Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar) and retained there through interaction with a receptor-like molecule (7Monsky W.L. Kelly T. Lin C.-Y. Yeh Y. Stetler-Stevenson W.G. Mueller S.C. Chen W.-T. Cancer Res. 1993; 53: 3159-3164PubMed Google Scholar, 18Emonard H.P. Remacle A.G. Noel A.C. Grimaud J.A. StetlerStevenson W.G. Foidart J.M. Cancer Res. 1992; 52: 5845-5848PubMed Google Scholar). However, neither uPA nor plasmin can activate pro-gelatinase A. Instead, it is activated by the cells cultivated with 12-O-tetradecanoylphorbol-13-acetate(16Brown P.D. Levy A.T. Margulies I.M. Liotta L.A. Stetler-Stevenson W.G. Cancer Res. 1990; 50: 6184-6191PubMed Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar), concanavalin A(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 19Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar), or in collagen gel (9Azzam H.S. Thompson E.W. Cancer Res. 1992; 52: 4540-4545PubMed Google Scholar) and by invasive tumor cells(10Brown P.D. Kleiner D.E. Unsworth E.J. Stetler-Stevenson W.G. Kidney Int. 1993; 43: 163-170Abstract Full Text PDF PubMed Scopus (126) Google Scholar, 20Azzam H.S. Arand G. Lippman M.E. Thompson E.W. J. Natl. Cancer Inst. 1993; 85: 1758-1764Crossref PubMed Scopus (207) Google Scholar). The plasma membrane fraction prepared from the cells can activate pro-gelatinase A, and the reaction was sensitive to chelating reagents and tissue inhibitor of metalloproteinases-2 (TIMP-2)(8Ward R.V. Atkinson S.J. Slocombe P.M. Docherty A.J. Reynolds J.J. Murphy G. Biochim. Biophys. Acta. 1991; 1079: 242-246Crossref PubMed Scopus (193) Google Scholar, 17Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Thus, the putative pro-gelatinase A activator may be a member of the MMP family.We isolated a cDNA clone that encodes a hitherto unknown MMP named MT-MMP(21Takino T. Sato H. Yamamoto E. Seiki M. Gene (Amst.). 1994; (in press)Google Scholar, 22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). MT-MMP is expressed as a 63-kDa protein on the surface of transfected cells with MT-MMP cDNA. At the same time, the cells caused specific activation of 72-kDa pro-gelatinase A(22Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). Since MT-MMP has a potential TM domain and was not released into the culture medium, the TM domain is thought to play a direct role in MT-MMP integration into the plasma membrane. In this study, we examined whether the potential TM domain is indeed required for anchoring MT-MMP onto the cell surface by constructing deletions and chimeric molecules. The pro-gelatinase A activation function was also examined in to the localization of MT-MMP