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• W2048996380 abstract "It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated. It has been proposed that tissue inhibitor of metalloproteinase-2 (TIMP-2), in stoichiometric concentrations, serves as an intermediate in progelatinase A activation by binding to activated membrane type 1-matrix metalloproteinase 1 (MT1-MMP) on the plasma membrane. An MT1-MMP-independent cell surface receptor for TIMP-2 has also been postulated. To clarify TIMP-2 binding, we have performed 125I-TIMP-2 binding studies on transfected COS-1 cells and endothelial cells. Specific receptors for TIMP-2 were identified on COS-1 cells transfected with MT1-MMP cDNA, but not on vector-transfected cells. Treatment of MT1-MMP transfected COS-1 cells with a hydroxamic acid inhibitor of MMPs, CT-1746, but not an inactive stereoisomer, CT-1915, produced dose-dependent inhibition of specific TIMP-2 binding comparable with that noted with excess unlabeled TIMP-2. This result suggests that TIMP-2 binds to the zinc catalytic site of MT1-MMP. As demonstrated by the limited competition for binding of C-terminal deleted TIMP-2, the C-terminal domain of TIMP-2 participates in binding to MT1-MMP. Cross-linking studies followed by immunoprecipitation using antibodies to MT1-MMP were employed to identify 125I-TIMP-2·MT1-MMP complexes in MT1-MMP-transfected COS-1 cell membrane extracts. TIMP-2 receptors were also identified on concanavalin A-treated human umbilical vein endothelial cells; inhibition of TIMP-2 binding with CT-1746 was demonstrated. Gelatinase A (72-kDa type IV collagenase, matrix metalloproteinase-2 (MMP-2)), 1The abbreviations used are: MMP, matrix metalloproteinase; HUVEC, human umbilical vein endothelial cell(s); MT-MMP, membrane-type MMP; TIMP, tissue inhibitor of metalloproteinases; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; BS3, bis(sulfosuccinimidyl) suberate. 1The abbreviations used are: MMP, matrix metalloproteinase; HUVEC, human umbilical vein endothelial cell(s); MT-MMP, membrane-type MMP; TIMP, tissue inhibitor of metalloproteinases; BSA, bovine serum albumin; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle's medium; BS3, bis(sulfosuccinimidyl) suberate. the dominant MMP released by most epithelial and endothelial cells under basal conditions (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 2Hanemaaijer R. Koolwijk P. Le Clercq L. De Vree W.J.A. Van Hinsbergh V.W.M. Biochem. J. 1993; 296: 803-809Crossref PubMed Scopus (358) Google Scholar), has an important role in turnover of basement membrane type IV collagen and other matrix proteins during angiogenesis, tissue remodeling, and repair (3Unemori E.N. Bouhana K.S. Werb Z. J. Biol. Chem. 1990; 265: 445-451Abstract Full Text PDF PubMed Google Scholar, 4Schnapper H.W. Grant D.S. Stetler-Stevenson W.G. Fridman R. D'Orazi G. Murphy A.N. Bird R.E. Hoythya M. Fuerst T.R. French D.L. Quigley J.P. Kleinman H.K. J. Cell. Physiol. 1993; 156: 235-246Crossref PubMed Scopus (279) Google Scholar). Since progelatinase A secretion is not induced by most cytokines that regulate other MMPs (5Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 42: 197-250Crossref Scopus (2630) Google Scholar), the final activation step appears to exert a more important influence in controlling tissue gelatinase A activity than with other MMPs. Activation of progelatinase A further differs from other MMPs by involving a cell surface activation mechanism (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 6Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (245) Google Scholar, 7Overall C.M. Sodek J. J. Biol. Chem. 1990; 265: 21141-21151Abstract Full Text PDF PubMed Google Scholar, 8Strongin 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 (1434) Google Scholar) that requires the participation of a 63-kDa integral plasma membrane MMP (membrane type-MMP: MT1-MMP) (9Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar). Based on studies in activated tumor cells, Strongin et al. (8Strongin 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 (1434) Google Scholar) proposed that TIMP-2, contributes to the proteolytic activity by binding to activated MT1-MMP in the plasma membrane; this bimolecular complex then binds progelatinase A. Cleavage of the Asn67-Leu68 peptide bond 2The numbering of amino acids of all proteins includes signal peptide sequence. 2The numbering of amino acids of all proteins includes signal peptide sequence. of progelatinase A then occurs to generate an intermediate form that undergoes autoactivation (8Strongin 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 (1434) Google Scholar, 10Atkinson S.J. Crabbe T. Cowell S. Ward R.V. Butler M.J. Sato H. Seiki M. Reynolds J.J. Murphy G. J. Biol. Chem. 1995; 270: 30479-30485Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 11Sato H. Seiki M. J. Biochem. (Tokyo). 1996; 119: 209-215Crossref PubMed Scopus (195) Google Scholar). Despite the presence of TIMP-2 in this system, MT1-MMP has been implicated in the initial cleavage event. Higher concentrations of TIMP-2 inhibit progelatinase A activation. Debate has arisen as to whether TIMP-2 binds initially to MT1-MMP or an unidentified receptor on the cell surface prior to progelatinase A activation (8Strongin 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 (1434) Google Scholar, 12Vassalli J.-D. Pepper M.S. Nature. 1994; 370: 14-15Crossref PubMed Scopus (177) Google Scholar). Independent of its MMP inhibitory effect, TIMP-2 also functions as a regulator of cell proliferation (4Schnapper H.W. Grant D.S. Stetler-Stevenson W.G. Fridman R. D'Orazi G. Murphy A.N. Bird R.E. Hoythya M. Fuerst T.R. French D.L. Quigley J.P. Kleinman H.K. J. Cell. Physiol. 1993; 156: 235-246Crossref PubMed Scopus (279) Google Scholar, 13Corcoran M.L. Stetler-Stevenson W.G. J. Biol. Chem. 1995; 270: 13453-13459Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 14Hayakawa T. Yamashita K. Ohuchi E. Shinagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar).Coexpression of MT1-MMP, gelatinase A, and TIMP-2 cDNA in mesenchymal cells, coinciding with the activation of progelatinase A during embryogenesis, suggests that MT1-MMP functions physiologically to initiate tissue remodeling on the cell surface (15Kinoh H. Sato H. Tsunezuka Y. Takino T. Kawashima A. Okada Y. Seiki M. J. Cell Sci. 1996; 109: 953-959PubMed Google Scholar). Expression of MT1-MMP in tumors also suggests a critical role for this protein in cancer dissemination (9Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 16Okada A. Bellocq J.-B. 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).Transfection of cDNAs for MT1-MMP, TIMP-2, and progelatinase A into COS cells that lack an endogenous system for progelatinase A activation has provided a useful model to explore cell surface protein interactions. MT1-MMP-transfected COS-1 cells or isolated plasma membranes, but not pcDNA3 vector-transfected cells or membranes deficient in MT1-MMP, readily activated exogenous or cell secreted progelatinase A (9Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2365) Google Scholar, 17Cao J. Sato J. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 18Cao J. Rehemtulla A. Bahou W. Zucker S. J. Biol. Chem. 1996; 271: 30174-30180Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Consistent with the hypothesis of Strongin et al. (8Strongin 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 (1434) Google Scholar), low level secretion of TIMP-2 by COS-1 cells was associated with MT1-MMP-induced progelatinase A activation; higher concentrations of TIMP-2, however, interfered in this process (18Cao J. Rehemtulla A. Bahou W. Zucker S. J. Biol. Chem. 1996; 271: 30174-30180Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). In contrast to these findings, using radiolabeled progelatinase A as the ligand, Sato et al. (19Sato H. Takino T. Kinoshita T. Imai K. Okada Y. Stetler Stevenson W.G. Seiki M. FEBS Lett. 1996; 385: 238-240Crossref PubMed Scopus (174) Google Scholar) demonstrated by autoradiography that binding of progelatinase A to COS-1 cells did not require the presence of TIMP-2. However, in another report from the same research group, Imai et al. (20Imai K. Ohuchi E. Aoki T. Nomura H. Fujii Y. Sato H. Seiki M. Okada Y. Cancer Res. 1996; 56: 2707-2710PubMed Google Scholar) reported that COS-1 cells transfected with mutant MT1-MMP cDNA lacking the transmembrane domain (ΔMT1-MMP) secreted ΔMT1-MMP in a complex with TIMP-2, which is then able to bind to progelatinase A. Other investigators reported that high concentrations of soluble forms of MT1-MMP (lacking the transmembrane domain) directly activate progelatinase A (21Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 22Will H. Atkinson S.J. Butler G.S. Smith B. Murphy G. J. Biol. Chem. 1996; 271: 17119-17123Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 23Lichte A. Kolkenbrock M. Tschesche H. FEBS Lett. 1996; 397: 277-282Crossref PubMed Scopus (61) Google Scholar, 24Kinoshita T. Sato H. Takino T. Itoh M. Akizawa T. Seiki M. Cancer Res. 1996; 56: 2535-2538PubMed Google Scholar), thereby bypassing the cell surface activation mechanism. TIMP-2 does not participate in this activation, but can function as an inhibitor of this process. Although stimulated tumor cells have been reported to secrete prodomain deleted MT1-MMP in vitro, the physiologic relevance of this mechanism remains to be determined (20Imai K. Ohuchi E. Aoki T. Nomura H. Fujii Y. Sato H. Seiki M. Okada Y. Cancer Res. 1996; 56: 2707-2710PubMed Google Scholar).The purpose of the current study was to: 1) determine whether MT1-MMP itself serves as a surface receptor for TIMP-2 in transfected COS-1 cells and 2) examine the physiologic receptor binding mechanism in human umbilical vein endothelial cells (HUVEC). In addition to examining the binding of radiolabeled TIMP-2 in HUVEC,125I-labeled TIMP-2 binding was examined in COS-1 cells transfected with MT1-MMP cDNA or pcDNA3 vector in the presence of synthetic inhibitors of MMPs. The results indicate that the functional catalytic site of MT1-MMP serves as the binding site for TIMP-2 in both MT1-MMP transfected COS-1 cells and HUVEC.MATERIALS AND METHODSBovine serum albumin (BSA), polyoxyethylene ethers (W1), and chloramine T were purchased from Sigma. Biotinylated affinity-purified goat anti-rabbit IgG and goat anti-mouse antibodies, alkaline phosphatase-conjugated streptavidin, 5-bromo-4-chloro-3-indolyl phosphate, and nitro blue tetrazolium were obtained from Life Technologies, Inc. Protein A-Sepharose beads were purchased from Pharmacia Biotech Inc. Restriction enzymes were purchased from Stratagene (La Jolla, CA). COS-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.). Bis(sulfosuccinimidyl) suberate (BS3) was purchased from Pierce. NuSerum was purchased from Collaborative Biomedical Products (Bedford, MA). The pcDNA3 expression vector was purchased from Invitrogen (San Diego, CA). Rabbit polyclonal antibodies (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) to a MT1-MMP synthetic peptide CDGNFDTVAMLRGEM were produced as described previously; immunoprecipitation using the rabbit polyclonal antibody to MT1-MMP provides virtually identical results as the mouse monoclonal antibody to the same synthetic peptide (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Recombinant human progelatinase A, recombinant TIMP-1 (6Murphy G. Willenbrock F. Ward R.V. Cockett M.I. Eaton D. Docherty A.J.P. Biochem. J. 1992; 283: 637-641Crossref PubMed Scopus (245) Google Scholar), CT-1746 (C19H28ClN3O4: molecular weight = 398: K i for gelatinase A = 0.04 nm, K i for interstitial collagenase = 122 nm), CT-1915 (molecular weight = 398: K i for gelatinase A = 1000 nm) and CT-1847 (C15H29N3O4S: molecular weight = 347: K i for gelatinase A = 1.55,K i for interstitial collagenase = 2.9 nm) were gifts from CellTech Ltd. (Slough, United Kingdom) (25Anderson I.C. Shipp M.A. Docherty A.J.P. Teicher B.A. Cancer Res. 1996; 56: 715-718PubMed Google Scholar). Recombinant TIMP-2 was purified from the culture medium of Chinese hamster ovary cells transfected with human TIMP-2 cDNA as described previously (26DeClerck Y.A. Yean T.-D. Lu H.S. Ting J. Langley K.E. J. Biol. Chem. 1991; 266: 3893-3899Abstract Full Text PDF PubMed Google Scholar).Construction of Plasmids and COS-1 TransfectionsMT1-MMP cDNA, isolated from endothelial cells, encoding an open reading frame from amino acid residues methionine 1 to valine 582, was cloned in a pcDNA3 expression vector employing a cytomegalovirus promoter as we have previously described (17Cao J. Sato J. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 18Cao J. Rehemtulla A. Bahou W. Zucker S. J. Biol. Chem. 1996; 271: 30174-30180Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). DNA sequence was confirmed by extensive restriction analysis and sequence analysis using an automated DNA sequencer (Applied Biosystems PRISM dye terminator cycle sequencing core kit, Perkin-Elmer).On the day of transfection, cultivated COS-1 cells were washed with phosphate-buffered saline (PBS), pH 7.4, followed by the addition of DMEM containing 10% NuSerum, 300 μg/ml DEAE-dextran, 100 μm chloroquine, and 1.25 μg/ml DNA (17Cao J. Sato J. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar, 18Cao J. Rehemtulla A. Bahou W. Zucker S. J. Biol. Chem. 1996; 271: 30174-30180Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The cells were then incubated for 4 h at 37 °C in an atmosphere of 5% CO2 and 95% air. The cells were washed once with DMEM and incubated for 2 min in 10% Me2SO in Ca2+/Mg2+-free PBS at room temperature and then washed twice with PBS. Finally, the cells were incubated for 1 day in DMEM containing 10% fetal calf serum. For receptor binding studies, COS-1 cells were propagated to confluence in 24-well dishes (Becton Dickinson, Lincoln Park, NJ) and then switched to serum-free conditions. The strategy for generating a truncated form of recombinant TIMP-2 with deletion of the C-terminal region extending from Cys128 to Pro194 (ΔTIMP-2) has been described (27Ko Y.-C. Langley K.E. Mendiaz E.A. Parker V. Taylor S.M. DeClerck Y.A. Biochem. Biophys. Res. Commun. 1997; 236: 100-105Crossref PubMed Scopus (15) Google Scholar).Endothelial Cell CultivationEndothelial cells were routinely cultivated on gelatin-coated plates in M199 medium supplemented with 20% heat-inactivated fetal calf serum, 10 ng/ml endothelial growth factor (Life Technologies, Inc.), penicillin (100 units/ml), and streptomycin (100 μg/ml) in 5% CO2 at 37 °C. For receptor binding studies, HUVEC were propagated in gelatin-coated 24-well dishes and employed in experiments when >90% confluence was achieved. Concanavalin A (40 μg/ml) was added for the final 18 h of HUVEC culture in serum-free M199 or human endothelial serum-free medium.Immunoblotting and Gelatin Substrate ZymographyImmunoblotting was performed using protein A affinity-purified polyclonal antibodies to human MT1-MMP as described previously (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Molecular weights were determined using prestained protein standards.Zymography was performed in 10% polyacrylamide gels that had been cast in the presence of 0.1% gelatin as described previously (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). After electrophoresis, SDS was replaced by Triton X-100, thus renaturing gelatinases, followed by incubation in a Tris-based buffer for 24 h. Gels were stained with Coomassie Brilliant Blue, and gelatinolytic activity was detected as a clear band in the background of uniform staining.Preparation of Plasma Membrane-enriched Cell FractionsCell membranes were prepared from endothelial cells following nitrogen cavitation as described previously (28Zucker S. Wieman J.M. Lysik R.M. Wilkie D. Ramamurthy N.S. Golub L.M. Lane B. Cancer Res. 1987; 47: 1608-1614PubMed Google Scholar). The post-nuclear supernatant (770 × g × 10 min) was collected, and heavy organelles were removed by centrifugation at 6,000 × gfor 15 min. This supernatant was centrifuged at 100,000 ×g for 1 h at 4 °C to recover the plasma membrane enriched lighter cell organelles in the pellet. Cell organelles were characterized by electron microscopy as described previously (28Zucker S. Wieman J.M. Lysik R.M. Wilkie D. Ramamurthy N.S. Golub L.M. Lane B. Cancer Res. 1987; 47: 1608-1614PubMed Google Scholar). The experimental design included incubating cell organelles with either buffer (to determine spontaneous release of membrane-associated gelatinase) or with 72-kDa recombinant progelatinase A for 18 h at 37 °C to determine membrane-induced activation of progelatinase A. After incubating membranes with progelatinase A, conditioned medium was recovered and subjected to gelatin zymography. Protein determinations were made using the bicinchoninic acid reagent as per the manufacturer's instructions (Pierce BCA protein assay reagent).Cell Surface Binding of TIMP-2Recombinant TIMP-2 (rTIMP-2) was iodinated to a specific activity of 5.5 × 1010dpm/mg by adding 0.25 mCi of Na125I to a tube containing 10 μg of rTIMP-2 and 100 μg of chloramine T. After 5-min incubation at 23 °C, 200 μg of sodium metabisulfite was added, and125I-labeled TIMP-2 in PBS containing 0.1% BSA was separated from free 125I by chromatography over a G-25 Sephadex column (Pharmacia Biotech. Inc.). In an independent study using the Nanorange protein quantitation kit (Molecular Probes, Eugene, OR), this radiolabeling procedure resulted in >95% recovery of125I-TIMP-2. Biological activity of radiolabeled TIMP-2 was confirmed by measuring the ability of 125I-labeled TIMP-2 to inhibit the activation of progelatinase A as compared with unlabeled TIMP-2 (demonstrated by zymography). Binding of125I-labeled TIMP-2 to cells propagated in 24-well dishes (Corning Costar, Wilmington, MA) to >90% confluence was performed in duplicate (∼10% variation between duplicates) as follows. Cells were washed thoroughly and treated with 3 m glycine buffer in 0.9% saline, pH 3, for 3 min to dissociate preformed receptor-ligand complexes. Cells were then washed with cold PBS with 0.1% BSA. For equilibrium binding experiments, dilutions of 125I-labeled TIMP-2 (0.1–16 nm) in PBS-BSA buffer were added to cells in 200 μl of serum-fee medium (total volume) in the presence or absence of excess of unlabeled rTIMP-2 or rTIMP-1 at 4 and 22 °C. After 30–240 min of incubation, supernatant fluid was collected and dishes were washed three times with PBS; washes were collected and added to the unbound 125I-TIMP-2 fraction. Cell monolayers were then lysed in 0.1% SDS in 0.5 m NaOH and collected as the bound fraction. Bound and unbound 125I were measured by γ counting. The residual radioactivity associated with cells in the nonspecific binding experiment (50-fold excess TIMP-2) was subtracted from the total bound fraction (no unlabeled TIMP-2) to give specific binding. Scatchard plot analysis of binding data employed best-fit curves using the Sigma Plot program (Jandel Scientific, San Rafael, CA). In competition experiments, cells were preincubated with hydroxamic acid inhibitors or TIMPs for 30 min prior to the addition of125I-TIMP-2.Cross-linking ExperimentsThe 100,000 × gplasma membrane-enriched fraction from COS-1 was isolated as described above for HUVEC. Membrane proteins were extracted using 0.25% (final concentration) polyoxyethylene ether (W-1) in 10 mm HEPES buffer, pH 7.5, containing 150 mm KCl and 1 mmCaCl2 for 1 h at 4 °C. Following centrifugation at 13,000 × g for 15 min, extracted proteins were incubated for 3 h at 4 °C with 125I-labeled TIMP-2. Cross-linking was performed in 10 μl of total volume containing indicated amounts of radiolabeled ligand, competing ligand, and detergent-extracted membrane proteins in HEPES buffer. The reaction was incubated with 2 mm BS3 for 1 h at 4 °C to allow cross-linking (covalent amide bond formed when theN-hydroxysuccinimide ester conjugation reagent reacts with primary amines); the cross-linking was quenched by the addition of 1m Tris for 10 min. Affinity-purified rabbit polyclonal antibodies to MT1-MMP (1Zucker S. Conner C. DiMassmo B.I. Ende H. Drews M. Seiki M. Bahou W.F. J. Biol. Chem. 1995; 270: 23730-23738Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar) were added to the reaction mixture and incubated at 4 °C for 16 h to immunoprecipitate MT1-MMP and MT1-MMP-containing complexes. The mixture was then incubated with protein A-coated Sepharose beads (Pharmacia Biotech Inc.) for 2 h with constant rocking at 4 °C. The beads were then washed in HEPES buffer five times by centrifugation until release of radiolabeled125I was minimal. Beads were then added to SDS-PAGE sample buffer containing β-mercaptoethanol. Samples were heated for 2 min in a boiling water bath prior to loading on a 8–12% gradient gel. The gels were subjected to SDS-PAGE followed by autoradiography.DISCUSSIONIn this study we have employed radiolabeled recombinant TIMP-2 to demonstrate specific TIMP-2 receptors on MT1-MMP transfected COS-1 cells and human endothelial cells. The absence of specific TIMP-2 binding to pcDNA3 vector transfected COS-1 cells identifies MT1-MMP as the TIMP-2 receptor. The K d of 1.39 and 0.77 nm (derived from Scatchard plot analysis) for the TIMP-2 receptor on COS-1 cells and concanavalin A treated-endothelial cells, respectively, is similar to previous data derived from phorbol 12-myristate 13-acetate-treated wild-type HT-1080 fibrosarcoma cell and MCF-7 breast cancer cell lines, but the number of receptor sites identified per MT1-MMP transfected COS-1 cell (450,000) and per endothelial cell (183,000) are considerably higher than previously reported with cancer cells (25,000–40,000/cell) (8Strongin 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 (1434) Google Scholar, 30Emmert-Buck M.R. Emonard H.P. Corcoran M.L. Foidart J.-M. Stetler-Stevenson W.G. FEBS Lett. 1995; 364: 28-32Crossref PubMed Scopus (98) Google Scholar). These differences may be due to our use of a low pH buffer to strip surface receptors of bound ligand prior to adding 125I-TIMP-2, which would have the effect of increasing the number of available receptor sites per cell. Of note, based on the ∼20–30% efficiency of MT1-MMP transfection in COS-1 cells (data not shown), the actual number of TIMP-2 receptor sites per transfected cell is many fold higher than noted with nontransfected cells (endothelial cells, tumor cells), which reflects the high level of expression of MT1-MMP resulting from transient transfection with MT1-MMP cDNA. In contrast to the above data, Hayakawa et al. (14Hayakawa T. Yamashita K. Ohuchi E. Shinagawa A. J. Cell Sci. 1994; 107: 2373-2379Crossref PubMed Google Scholar) described two classes of TIMP-2 receptors on Raji cells with the majority of receptors having low affinity with a K d of 35 nm and 140,000 sites/cell; the high affinity receptor had aK d of 0.15 nm and 20,000 sites/cell.To further characterize MT1-MMP as the TIMP-2 receptor,125I-TIMP 2 binding to MT1-MMP transfected COS-1 cells was performed in the presence of CT-1746, a hydroxamic acid inhibitor with greater activity against gelatinases. CT-1746, but not the inactive stereoismomer CT-1915, inhibited 125I-TIMP 2 binding to cells in a dose-dependent fashion that was comparable with that achieved with unlabeled TIMP-2. CT-1847, an MMP inhibitor with broad spectrum activity, likewise readily inhibited125I-TIMP 2 binding to cells. These data are consistent with the concept that TIMP-2 binds directly to the catalytic site of MT1-MMP on the cell surface. The fact that both a general and a more specific hydroxamic acid-based inhibitor displayed approximately equivalent activity in blocking TIMP 2 binding to MT1-MMP suggests that the catalytic site of MT1-MMP differs considerably from other well known MMPs. The N-terminal domain of TIMP-2 that consists of the first three disulfide loops and has an OB barrel-like structure (homologous to that seen in proteins of the oligosaccharide/oligonucleotide binding (OB) fold family) (32Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar) has been shown to behave as a fully active inhibitor of several MMPs, including intestitial collagenase (27Ko Y.-C. Langley K.E. Mendiaz E.A. Parker V. Taylor S.M. DeClerck Y.A. Biochem. Biophys. Res. Commun. 1997; 236: 100-105Crossref PubMed Scopus (15) Google Scholar), MMP-7, stromelysin-1, and gelatinase A (33Murphy G. Houbrechts A. Cockett M.I. Williamson R.A. O'Shea M. Docherty A.J.P. Biochemistry. 1991; 30: 8097-8102Crossref PubMed Scopus (283) Google Scholar). These data therefore suggest that the N-terminal domain of TIMP-2 contains the domain interacting with the active catalytic domain of these MMPs. However, in the case of MT-MMP, our data suggest that the C-terminal domain of TIMP-2 positively influences the association between the N-terminal domain of TIMP-2 and the catalytic domain of MT-MMP. Of relevance for this possibility is the observation of Nguyen et al. (34Nguyen Q. Willenbrook F. Cockett M.I. O'Shea M. Docherty A.J.P. Murphy G. Biochemistry. 1994; 33: 2089-2095Crossref PubMed Scopus (82) Google Scholar) who demonstrated that the C-terminal domain of TIMP-1 and TIMP-2 act to increase the association constant by binding to the C-terminal domains of gelatinase A or the N-terminal domain of stromelysin-2. Additional studies of the C-terminal domain structures of TIMP-2 and TIMP-1 (32Williamson R.A. Martorell G. Carr M.D. Murphy G. Docherty A.J.P. Freedman R.B. Feeney J. Biochemistry. 1994; 33: 11745-11759Crossref PubMed Scopus (95) Google Scholar) will be helpful in explaining their differences in inhibiting MT1-MMP. Of note, competition for binding to active collagenase (soluble) has likewise been observed between TIMP-1 and low molecular weight synthetic inhibitors that are directed at the catalytic zinc of collagenase (35Lelievre Y. Bouboutou R. Boiziau J. Faucher D. Achard D. Cartwright T. Matrix. 1990; 10: 292-299Crossref PubMed Scopus (24) Google Scholar).To characterize the physical interaction between125I-labeled TIMP-2 and MT1-MMP-transfected COS-1 c" @default.
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• W2048996380 title "Tissue Inhibitor of Metalloproteinase-2 (TIMP-2) Binds to the Catalytic Domain of the Cell Surface Receptor, Membrane Type 1-Matrix Metalloproteinase 1 (MT1-MMP)" @default.
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