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• W2047694858 abstract "The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor α precursor (proTGF-α) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-α, cell adhesion receptor L-selectin, interleukin 6 receptor α subunit, β-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene. The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor α precursor (proTGF-α) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-α, cell adhesion receptor L-selectin, interleukin 6 receptor α subunit, β-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene. INTRODUCTIONThe extracellular domain of a large number of transmembrane proteins can be proteolytically released into the medium. This shedding process regulates the fate and physical location of membrane-anchored growth factors(1.Massagué J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (598) Google Scholar), growth factor receptors(2.Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (682) Google Scholar), cell adhesion molecules, ectoenzymes(3.Ehlers M.R.W. Riordan J.F. Biochemistry. 1991; 30: 10065-10074Crossref PubMed Scopus (255) Google Scholar), and proteins of unknown function such as the β-amyloid precursor protein (βAPP) 1The abbreviations used are: βAPPβ-amyloid precursor proteinTNF-αtumor necrosis factor αIL-6interleukin 6IL-6 RIL-6 receptorCHOChinese hamster ovaryPMAphorbol 12-myristate 13-acetateDFPdiisopropylfluorophosphateTPCKtosylphenylalanyl chloromethyl ketoneMEMminimum essential mediumPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisFACSfluorescence-activated cell sorterDCIdichloroisocoumarineTAPIN-{D,L-[2-9hydroxyamino-carbonyl)methyl]-4-methypentanoyl}L-3(2′naphthyl)-alanyl-L-alanine, 2-aminoethyl amideTAPI-2analog of TAPI with the naphthyl-alanine side chain replaced by a terbutyl groupproTGF-αtransforming growth factor α precursor. (4.Haass C. Selkoe D.J. Cell. 1993; 75: 1039-1042Abstract Full Text PDF PubMed Scopus (736) Google Scholar). Many of these proteins are of practical importance. For example, βAPP is implicated in the pathogenesis of Alzheimer's disease(5.Sisodia S. Price D.L. FASEB J. 1995; 9: 366-370Crossref PubMed Scopus (224) Google Scholar), angiotensin converting enzyme plays an important role in the regulation of blood pressure(6.Skeggs Jr., L.T.D. Kahn J.R. Shumway N.P. J. Exp. Med. 1956; 103: 295-299Crossref PubMed Scopus (708) Google Scholar), and tumor necrosis factor α (TNF-α) and the homing receptor L-selectin are implicated in inflammatory responses(7.Vassalli P. Annu. Rev. Immunol. 1992; 10: 411-452Crossref PubMed Scopus (1797) Google Scholar, 8.Gearing A.J.H. Newman W. Immunol. Today. 1993; 14: 506-512Abstract Full Text PDF PubMed Scopus (1273) Google Scholar). Ectodomain shedding can convert membrane-anchored growth factors into diffusible factors, membrane receptors into soluble competitors of their own ligand (9.Fernández-Botran R. FASEB J. 1991; 5: 2567-2574Crossref PubMed Scopus (256) Google Scholar) or accessories to ligand binding(2.Rose-John S. Heinrich P.C. Biochem. J. 1994; 300: 281-290Crossref PubMed Scopus (682) Google Scholar), and cell adhesion receptors into products no longer capable of mediating physical interactions with other cells or the extracellular matrix(8.Gearing A.J.H. Newman W. Immunol. Today. 1993; 14: 506-512Abstract Full Text PDF PubMed Scopus (1273) Google Scholar). Membrane protein ectodomain shedding is now recognized as an important aspect of cell regulation and cell-cell interaction.Despite its broad interest, this shedding mechanism involves molecular components of unknown identity. Shedding appears to occur at or near the cell surface and does not require cytosolic factors that are essential for many forms of membrane traffic(10.Bosenberg M.W. Pandiella A. Massagué J. J. Cell Biol. 1993; 122: 95-101Crossref PubMed Scopus (54) Google Scholar). Shedding is often stimulated by protein kinase C activators and other agents(11.Pandiella A. Massagué J. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1726-1730Crossref PubMed Scopus (181) Google Scholar, 12.Huang E.J. Nocka K.H. Buck J. Besmer P. Mol. Biol. Cell. 1992; 3: 349-362Crossref PubMed Scopus (273) Google Scholar, 13.Stein J. Rettenmier C.W. Oncogene. 1991; 6: 601-605PubMed Google Scholar, 14.Downing J.R. Roussel M.F. Sherr C.J. Mol. Cell. Biol. 1989; 9: 2890-2896Crossref PubMed Scopus (164) Google Scholar, 15.Porteu F. Brockhaus M. Wallach D. Engelmann H. Nathan C.F. J. Biol. Chem. 1991; 266: 18846-18853Abstract Full Text PDF PubMed Google Scholar, 16.Müllberg J. Schooltink H. Stoyan T. Heinrich P.C. Rose-John S. Biochem. Biophys. Res. Commun. 1992; 189: 794-800Crossref PubMed Scopus (98) Google Scholar, 17.Serra-Pages C. Saito H. Streuli M. J. Biol. Chem. 1994; 269: 23632-23641Abstract Full Text PDF PubMed Google Scholar, 18.Buxbaum J.D. Gandy S.E. Cicchetti P. Ehrlich M.E. Czernik A.J. Fracasso R.P. Ramabhadran T.V. Unterbeck A.J. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6003-6006Crossref PubMed Scopus (426) Google Scholar, 19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar, 20.Ramchandran R. Sen G.C. Misono K. Sen I. J. Biol. Chem. 1994; 269: 2125-2130Abstract Full Text PDF PubMed Google Scholar). However, the proteins that are shed are not the targets of phosphorylation in this process. The nature of the proteases involved is of great interest because they might constitute ideal targets for therapy in various disease conditions. Given the diversity of amino acid sequences that are cleaved, many different proteases could be involved in this process, each endowed with a specific substrate recognition capacity. This notion has been reinforced by recent reports that the shedding of different ectodomains appears to be inhibited by different proteinase inhibitors(19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar, 20.Ramchandran R. Sen G.C. Misono K. Sen I. J. Biol. Chem. 1994; 269: 2125-2130Abstract Full Text PDF PubMed Google Scholar, 21.Bazil V. Strominger J.L. J. Immunol. 1994; 152: 1314-1322PubMed Google Scholar, 22.Leca G. Mansur S.E. Bensussan A. J. Immunol. 1995; 154: 1069-1077PubMed Google Scholar, 23.Gearing A.J.H. Beckett P. Christodoulou M. Churchill M. Clements J. Davidson A.H. Drummond A.H. Galloway W.A. Gilbert R. Gordon J.L. Leber T.M. Mangan M. Miller K. Nayee P. Owen K. Patel S. Thomas W. Wells G. Wood L.M. Wolley K. Nature. 1994; 370: 555-557Crossref PubMed Scopus (1104) Google Scholar, 24.McGeehan G.M. Becherer J.D. Bast Jr., R.C. Boyer C.M. Champion B. Connolly K.M. Conway J.G. Furdon P. Karp S. Kidao S. McElroy A.B. Nichols J. Pryzwansky K.M. Schoenen F. Sekut L. Truesdale A. Verghese M. Warner J. Ways J.P. Nature. 1994; 370: 558-561Crossref PubMed Scopus (544) Google Scholar, 25.Mohler K. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarma K. Kronhein S.R. Petersen M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Crossref PubMed Scopus (571) Google Scholar, 26.Crowe P. Walter B.N. Mohler K.M. Otten-Evans C. Black R.A. Ware C.F. J. Exp. Med. 1995; 181: 1205-1210Crossref PubMed Scopus (245) Google Scholar, 27.Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massagué J. J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar, 28.Müllberg J. Oberthür W. Lottspeich F. Mehl E. Dittrich E. Graeve L. Heinrich P.C. Rose-John S. J. Immunol. 1994; 152: 4958-4968PubMed Google Scholar).Despite expectations that many different proteolytic activities may be involved in membrane protein ectodomain shedding, recent genetic evidence suggests that these processes may share certain components. We recently isolated a mutant cell line that is defective in the shedding of at least two unrelated molecules, βAPP and proTGF-α, thus providing evidence that the shedding mechanisms of these two molecules share a common component(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar). In the present report, we show that independent selection of cell mutants defective in proTGF-α shedding yields cell lines that have identical phenotypes and belong to the same genetic complementation group, indicating a repeated isolation of mutations in the same gene. The defect in these cells prevents shedding of all membrane proteins tested. Furthermore, recently described compounds that inhibit certain metalloproteases and prevent shedding of TNF-α (25.Mohler K. Sleath P.R. Fitzner J.N. Cerretti D.P. Alderson M. Kerwar S.S. Torrance D.S. Otten-Evans C. Greenstreet T. Weerawarma K. Kronhein S.R. Petersen M. Gerhart M. Kozlosky C.J. March C.J. Black R.A. Nature. 1994; 370: 218-220Crossref PubMed Scopus (571) Google Scholar) and the 80-kDa TNF-α receptor (26.Crowe P. Walter B.N. Mohler K.M. Otten-Evans C. Black R.A. Ware C.F. J. Exp. Med. 1995; 181: 1205-1210Crossref PubMed Scopus (245) Google Scholar) prevent also the shedding of TGF-α, βAPP, L-selectin, IL-6 Rα, and a large group of endogenous membrane proteins in parental CHO cells. Previously observed differences in protease inhibitor sensitivity of these various molecules are shown here to result from effects of the inhibitors on membrane protein maturation and transport. These results suggest the existence of a common shedding mechanism involving one or several components sensitive to metalloprotease inhibitors.EXPERIMENTAL PROCEDURESMaterialsPhorbol 12-myristate 13-acetate (PMA), ethyl methane sulfonate, diisopropylfluorophosphate (DFP), L-1-chloro-3-[4-tosylamido]-4-phenyl-2-butanone, tosylphenylalanyl chloromethyl ketone (TPCK), and 1,10-phenanthroline were from Sigma. 3,4-Dichloroisocoumarine and phosphoramidon were from Boehringer Mannheim. TAPI-2 was kindly provided by Immunex.Cell Transfection, Mutagenesis, Selection, and FusionsCHO cells were cultured in monolayers in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum (Life Technologies, Inc.). pCDM8 vectors containing L-selectin or IL-6 R cDNAs were cotransfected with the selectable plasmid pCEP-4 at a DNA ratio of 1:10 into CHO cells by electroporation. Stable transfectants were selected in 700 μg/ml of hygromicin and subcloned. Clones expressing L-selectin or IL-6 Rα at high levels were used for subsequent experiments. CHO cell mutants defective in TGF-α cleavage were isolated as described previously(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar).For cell fusions, 2 × 106 cells of a hygromicin-resistant clone and 2 × 106 cells of a histidinol-resistant clone were plated in 60-mm dishes. 16 h later, the cultures were briefly covered with 3 ml of 45% polyethylene glycol (Mr 1300-1600, American Type Culture Collection) in MEM, 10 mM Hepes with a final pH of 7.3. The polyethylene glycol/MEM solution was immediately aspirated, leaving only the minimum amount needed to cover the cells, and the cultures were incubated for 10 min at 37°C. Cells were washed three times with MEM and twice with MEM containing nonessential amino acids and 10% fetal bovine serum using warm medium. After 10 h of incubation in the latter medium, the cultures were trypsinized and plated into 150-mm dishes. Hybrid cell clones were selected in histidine-free MEM containing nonessential amino acids, 10% dialyzed fetal bovine serum, 0.5 mM histidinol, and 800 μg/ml of hygromicin for 2 weeks.Flow Cytometry AnalysisCells were washed with Dulbecco's modified Eagle medium for 1 h at 37°C and then treated with or without protease inhibitors for 5 min and treated with or without PMA and/or protease inhibitors cells for an additional 20 min. Cells were then incubated for 45 min at 4°C with 10 μg/ml of anti-HA monoclonal antibody (12CA5, Babco) or 25 μg/ml anti-L-selectin DREG-200 monoclonal antibodies(19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar), in phosphate-buffered saline (PBS) containing 5% bovine serum albumin and stained for 30 min at 4°C with fluorescein isothiocyanate-conjugated anti-mouse IgG (Becton Dickinson) in PBS containing 5% bovine serum albumin. Flow cytometry was done on a FACscan instrument and software (Becton Dickinson).Metabolic Labeling, Biotinylation, and Immunoprecipitation of Cell Surface ProteinsApproximately 106 exponentially growing CHO cells expressing TGF-α, L-selectin, or IL-6 Rα were labeled for 30 min with 250 μCi/ml [35S]cysteine and 250 μCi/ml translabel (DuPont NEN) in methionine- and cysteine-free medium at 37°C. The label was chased in complete medium for the indicated times in the presence or the absence of protease inhibitors and/or 1 μM PMA as indicated. Cells were then washed with cold PBS and lysed in PBS containing 1% Nonidet P-40 and 5 mM EDTA (lysis buffer). Aliquots from the medium and from cell lysates were immunoprecipitated with anti-HA monoclonal antibody or polyclonal antibodies against the extracellular domain or the cytoplasmic domain of TGF-α(35.Ignotz R.A. Kelly B. Davis R.J. Massagué J. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 6307-6311Crossref PubMed Scopus (87) Google Scholar), L-selectin(19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar), or IL-6 Rα (36.Stoyan T. Michaelis U. Schooltink H. Vam Dam M. Rudolph R. Heinrich P.C. Rose-John S. Eur. J. Biochem. 1993; 236: 239-245Crossref Scopus (67) Google Scholar) as indicated. For immunoprecipitation of cell surface of HA-tagged proTGF-α or L-selectin, metabolically labeled cells were chased for the indicate times in complete medium, shifted to 4°C, and incubated for 30 min in PBS containing 5% bovine serum albumin and 10 μg/ml of anti-HA antibody or a 1:100 dilution of antiserum against the L-selectin ectodomain. Cells were washed with PBS and directly lysed in lysis buffer or further incubated for 5 min with or without the addition of the indicated protease inhibitors and for an additional 15 min with or without addition of 1 μM PMA. Cells were then washed three times with PBS and lysed in lysis buffer containing 5 μg/ml of HA peptide in order to block any unbound anti-HA antibody. Insoluble material was removed by centrifugation. Immune complexes were collected by incubation of cell lysates and medium samples with protein A-Sepharose for 45 min at 4°C, washed three times with PBS containing 0.1% Triton X-100 and 0.1% SDS, and analyzed by SDS-PAGE.For biotinylation of cell surface proteins, cells were labeled with 250 μCi/ml of [35S]methionine and 250 μCi/ml of [35S]cysteine for 2 h in methionine- and cysteine-free medium, chased for 30 min in complete medium, shifted to 4°C, and incubated with 1 mg/ml sulfo-NHS-LC-biotin (Pierce) for 1 h at 4°C. Unreacted biotinylating agent was quenched by washing the cells with PBS containing 50 mM Tris. Cells were then incubated for 30 min at 37°C in complete medium, 5 min with or without 200 μM TAPI-2, and an additional 20 min with or without 1 μM PMA and/or 200 μM TAPI-2. Cells were washed three times with cold PBS and lysed in lysis buffer. Medium samples and cell lysates were incubated with streptavidin-agarose beads, and the beads were washed with 0.1% Triton X-100 and 0.1% SDS and analyzed on 12-18% gradient polyacrylamide gels.RESULTSRecessive Loss of Ectodomain Shedding Activity in Independently Isolated Cell MutantsRegulated shedding of membrane protein ectodomains was studied using two independently isolated mutant CHO cell clones. One of these clones, M1, was previously isolated from CHO cells expressing a transfected proTGF-α tagged with the HA epitope (HA-proTGF-α)(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar). M1 was isolated by treating these transfectants with the single base mutagen ethyl methane sulfonate and then sorting by FACS those cells that retain cell surface anti-HA staining after treatment with the phorbol ester PMA, a known inducer of proTGF-α cleavage. After four consecutive rounds of sorting, the mutagenized population yielded cells that are defective in TGF-α shedding. A control nonmutagenized cell population run in parallel did not yield shedding defective cells. Interestingly, the M1 line and other cell lines clonally propagated from the mutant pool are also defective in βAPP shedding activity. Activation of shedding by protein kinase C-independent mechanisms is also defective in these cells. However, these cells are normal with respect to membrane protein biosynthesis and transport(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar).Although M1 and other cell lines established from the original mutant pool have the same phenotype, the entire mutant pool could be derived from a single mutant clone enriched during the consecutive rounds of sorting. In order to determine the frequency of isolation of a shedding defective phenotype by mutation of the same gene, we isolated an independent cell line, M2, by repeating the mutagenesis and sorting protocol with a fresh batch of CHO cells. Like M1 cells, M2 cells were unable to shed cell surface proTGF-α (Fig. 1A) or βAPP (data not shown) in response to PMA. Furthermore, hybrids generated by fusion of M1 or M2 with parental CHO cells had wild type shedding activity (Fig. 1A). M1 × M2 cell hybrids lacked shedding activity, showing that they belong to the same genetic complementation group (Fig. 1A). Thus, the M1 and M2 cell lines have the same recessive phenotype and belong to the same complementation group. These results argue that the defect in membrane protein ectodomain cleavage in these independent cell lines is caused by recessive mutations in the same gene.General Loss of Ectodomain Shedding ActivityPrevious results showed that the defect in the M1 mutant cell line inhibited PMA-dependent shedding of various CHO cell surface proteins of unknown identity(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar). To better establish the shedding defect, the M1 and M2 cell lines and parental CHO cells were transfected with expression vectors encoding L-selectin or IL-6 Rα, two proteins that undergo PMA-induced ectodomain cleavage and are structurally and functionally unrelated to each other and to proTGF-α or βAPP(19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar, 28.Müllberg J. Oberthür W. Lottspeich F. Mehl E. Dittrich E. Graeve L. Heinrich P.C. Rose-John S. J. Immunol. 1994; 152: 4958-4968PubMed Google Scholar). The cell-bound and released forms of these proteins and of TGF-α as a control were immunoprecipitated from metabolically labeled cells treated with or without PMA. As described previously(29.Arribas J. Massagué J. J. Cell Biol. 1995; 128: 433-441Crossref PubMed Scopus (130) Google Scholar), PMA induced a loss of the two cell surface proTGF-α forms (17 and 20-22 kDa) with a concomitant increase in soluble TGF-α in parental CHO cells but had no effect in the mutants (Fig. 1B, bottom).Immunoprecipitation of metabolically labeled L-selectin transfectants with antibodies against the L-selectin intracellular domain yielded products of 50 and 74 kDa (Fig. 1B). Based on previous characterization, these products correspond to the biosynthetic precursor of L-selectin and the fully glycosilated cell surface form, respectively (19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar) (see also Fig. 3B). Upon cell treatment with PMA, the cell surface form is converted into a soluble form (Fig. 1B), leaving a cell-associated 6-kDa transmembrane/cytoplasmic fragment(19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar). In contrast to parental CHO cells, M1 and M2 cells showed a lack of basal or activated L-selectin shedding activity (Fig. 1B and data not shown). Likewise, IL-6 Rα, which is expressed in transfected CHO cells as an 80-kDa cell surface protein and shed as a 57-kDa soluble ectodomain in response to PMA(28.Müllberg J. Oberthür W. Lottspeich F. Mehl E. Dittrich E. Graeve L. Heinrich P.C. Rose-John S. J. Immunol. 1994; 152: 4958-4968PubMed Google Scholar), was not shed in the mutants (Fig. 1B, middle panel). The results obtained with M1 and M2 transfectants were identical, and only one of the mutants (M2) is shown here for simplicity. These results indicate that the gene affected in these mutants is essential not only for proTGF-α and βAPP shedding but also for the shedding of L-selectin and IL-6 Rα.Figure 33,4-DCI does not prevent shedding of L-selectin or proTGF-α. A, flow cytometry analysis of CHO cells expressing L-selectin and HA/proTGF-α treated with or without PMA and/or 100 μM 3,4-DCI for 20 min. Cells were immunostained with antibodies against the L-selectin ectodomain or the HA epitope and analyzed by flow cytometry. B, metabolically labeled CHO cells expressing HA/proTGF-α and L-selectin were chased for 25 min in complete medium and then incubated with anti HA monoclonal antibodies or polyclonal antibodies against the ectodomain of L-selectin at 4°C. Cells were then shifted to 37°C, briefly washed with medium with or without 3,4-DCI, and incubated in the presence or the absence of PMA and/or 100 μM 3,4-DCI for 15 min as indicated. Immune complexes present in cell lysates and medium were precipitated with protein A-agarose and analyzed by SDS-PAGE.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Effect of Serine Protease Inhibitors on Ectodomain Shedding ActivityPrevious studies showed that TGF-α release can be blocked by the addition of certain serine protease inhibitors(27.Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massagué J. J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar), whereas shedding of IL-6 Rα (28.Müllberg J. Oberthür W. Lottspeich F. Mehl E. Dittrich E. Graeve L. Heinrich P.C. Rose-John S. J. Immunol. 1994; 152: 4958-4968PubMed Google Scholar) or L-selectin ((19.Kahn J. Ingraham R.H. Shirley F. Migaki G.I. Kishimoto T.K. J. Cell Biol. 1994; 125: 461-470Crossref PubMed Scopus (182) Google Scholar) and references therein) is not sensitive to such inhibitors. These results could be interpreted as evidence for the involvement of different proteolytic activities. However, these results were obtained using different methods and cell lines. For example, 3,4-DCI inhibited the release of TGF-α from CHO cells as determined by immunoprecipitation of metabolically labeled proteins (27.Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massagué J. J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar) but did not inhibit the shedding of L-selectin as determined by FACS analysis of immunostained human granulocytes(21.Bazil V. Strominger J.L. J. Immunol. 1994; 152: 1314-1322PubMed Google Scholar). Therefore, we decided to test the effect of 3,4-DCI on the shedding of L-selectin, IL-6 Rα, TGF-α, and βAPP under the same experimental conditions. As in previous studies on proTGF-α cleavage(27.Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massagué J. J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar), metabolically labeled cells were treated with 3,4-DCI for 30 min prior to PMA addition. Under these conditions, 3,4-DCI inhibited the release of L-selectin and IL-6 Rα as well as TGF-α (Fig. 2). Two other serine protease inhibitors, TPCK and DFP, previously shown to prevent TGF-α release under these conditions (27.Pandiella A. Bosenberg M.W. Huang E.J. Besmer P. Massagué J. J. Biol. Chem. 1992; 267: 24028-24033Abstract Full Text PDF PubMed Google Scholar) prevented the PMA-induced release of L-selectin and IL-6 Rα as well as endogenous βAPP (data not shown).Figure 2Effect of 3,4-DCI on the release of L-selectin, IL-6 Rα, and proTGF-α ectodomains. CHO cells transfected with L-selectin, IL-6 Rα, or HA/proTGF-α were metabolically labeled and chased for 45 min in the presence or the absence of 100 μM 3,4-DCI. Where indicated, PMA was added during the last 30 min of the chase period. Aliquots from medium samples were immunoprecipitated with antiserum against the corresponding protein ectodomains.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The current availability of a HA-tagged proTGF-α construct that can be recognized on the cell surface by anti-HA antibody allowed us to assess the effect of these protease inhibitors on the shedding of cell surface proTGF-α using FACS analysis. In marked contrast with the results obtained by immunoprecipitation of metabolically labeled products, FACS analysis of CHO transfectants surface-stained with anti-HA antibody showed that 3,4-DCI did not inhibit the PMA-induced shedding of cell surface proTGF-α (Fig. 3A). As described previously(21.Bazil V. Strominger J.L. J. Immunol. 1994; 152: 1314-1322PubMed Google Scholar), 3,4-DCI did not prevent the PMA-induced loss of cell surface L-selectin as determined by FACS analysis. The same results were obtained when TPCK or DFP were analyzed in this type of assay (data not shown).To confirm the inability of 3,4-DCI to prevent ectodomain cleavage at the cell surface, a protocol was designed to specifically follow the fate of proteins that are present on the cell surface at the time of PMA addition. Cells were metabolically labeled and then chased long enough to allow labeled membrane proteins to reach the cell surface. Cells were then incubated with antibodies against proteins of interest and treated with PMA and/or 3,4-DCI. The immune complexes formed on the cell surface were recovered by precipitation from cell lysates and medium samples and analyzed by SDS-PAGE. When tested in this manner, 3,4-DCI did not inhibit PMA-induced loss of either proTGF-α or L-selectin from the cell surface (Fig. 3B, left panels) and did not inhibit the release of soluble L-selectin and TGF-α into the medium (Fig. 3B, right panels).These results suggested that 3,4-DCI and related protease inhibitors prevented the release of newly synthesized membrane proteins but not the shedding of these proteins once they have reached the cell surface. To determine whether these protease inhibitors interfered with transport of membrane proteins to the cell surface, a proTGF-α pulse-chase metabolic labeling experiment was done in the presence or the absence of the inhibitors. ProTGF-α is synthesized as an 18-kDa precursor that matures into forms of 20-22 kDa that reach the cell surface and are converted to a 17-kDa product by remov" @default.
• W2047694858 created "2016-06-24" @default.
• W2047694858 creator A5027957849 @default.
• W2047694858 creator A5055628562 @default.
• W2047694858 creator A5064557246 @default.
• W2047694858 creator A5077169627 @default.
• W2047694858 creator A5086215911 @default.
• W2047694858 creator A5089057275 @default.
• W2047694858 date "1996-05-01" @default.
• W2047694858 modified "2023-10-11" @default.
• W2047694858 title "Diverse Cell Surface Protein Ectodomains Are Shed by a System Sensitive to Metalloprotease Inhibitors" @default.
• W2047694858 cites W1484493440 @default.
• W2047694858 cites W1534418440 @default.
• W2047694858 cites W1577001387 @default.
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• W2047694858 cites W1578939248 @default.
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