Intracellular Processing of Metalloprotease Disintegrin ADAM12
2002; Elsevier BV; Volume: 277; Issue: 29 Linguagem: Inglês
10.1074/jbc.m110814200
ISSN1083-351X
AutoresYi Cao, Qing Kang, Zhefeng Zhao, Anna Żółkiewska,
Tópico(s)HER2/EGFR in Cancer Research
ResumoADAM12 has been implicated in cell-cell interactions in myogenesis and cancer, but the structure of the mature form of ADAM12 is not known, and its localization on the cell surface has been questioned. In this report, we show that full-length ADAM12 isN-glycosylated in the endoplasmic reticulum (ER) and proteolytically processed in the trans-Golgi network to an ∼90-kDa form. The ∼90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. Replacement of Leu73 in the putative α-helical region in the prodomain with proline resulted in retention of ADAM12 in the ER and a complete lack of its processing. However, deletion of the entire pro- and metalloprotease domains did not affect the processing and trafficking of ADAM12. In contrast, replacement of the cytoplasmic domain of ADAM12 with that of ADAM9 or adding a c-Myc tag at the C terminus led to a significant increase in transport of the protein to the cell surface. These results suggest that the cytoplasmic domain of ADAM12 plays an important role in regulating ADAM12 exit from the ER. We conclude that properly folded mouse ADAM12, after passing a rate-limiting step of exit from the ER, is processed in the secretory pathway and reaches the cell surface, where it can mediate adhesion-mediated signaling. ADAM12 has been implicated in cell-cell interactions in myogenesis and cancer, but the structure of the mature form of ADAM12 is not known, and its localization on the cell surface has been questioned. In this report, we show that full-length ADAM12 isN-glycosylated in the endoplasmic reticulum (ER) and proteolytically processed in the trans-Golgi network to an ∼90-kDa form. The ∼90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. Replacement of Leu73 in the putative α-helical region in the prodomain with proline resulted in retention of ADAM12 in the ER and a complete lack of its processing. However, deletion of the entire pro- and metalloprotease domains did not affect the processing and trafficking of ADAM12. In contrast, replacement of the cytoplasmic domain of ADAM12 with that of ADAM9 or adding a c-Myc tag at the C terminus led to a significant increase in transport of the protein to the cell surface. These results suggest that the cytoplasmic domain of ADAM12 plays an important role in regulating ADAM12 exit from the ER. We conclude that properly folded mouse ADAM12, after passing a rate-limiting step of exit from the ER, is processed in the secretory pathway and reaches the cell surface, where it can mediate adhesion-mediated signaling. green fluorescent protein trans-Golgi network endoplasmic reticulum endoglycosidase H brefeldin A Src homology 3 ADAMs, a family of proteins containing adisintegrin and metalloprotease domain, play important roles in many biological processes involving cell-surface proteolysis and cell-cell or cell-matrix interactions (1Wolfsberg T.G. Primakoff P. Myles D.G. White J.M. J. Cell Biol. 1995; 131: 275-278Crossref PubMed Scopus (439) Google Scholar, 2Wolfsberg T.G. White J.M. Dev. Biol. 1996; 180: 389-401Crossref PubMed Scopus (215) Google Scholar, 3Schlöndorff J. Blobel C.P. J. Cell Sci. 1999; 112: 3603-3617Crossref PubMed Google Scholar). ADAMs have been implicated in many vital functions during development (4Fambrough D. Pan D. Rubin G.M. Goodman C.S. Proc. Natl. Acad. 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These ADAMs are predicted to be active proteases involved in shedding of the ectodomains of membrane proteins, which is critical for cell-surface remodeling, regulating growth factor availability, and modulating the capacity of cells to respond to extracellular stimuli (14Black R.A. White J.M. Curr. Opin. Cell Biol. 1998; 10: 654-659Crossref PubMed Scopus (428) Google Scholar, 15Blobel C.P. Curr. Opin. Cell Biol. 2000; 12: 606-612Crossref PubMed Scopus (224) Google Scholar). At least six members of the ADAM family have been demonstrated to have proteolytic activity. ADAM17 (tumor necrosis factor-α-converting enzyme) releases soluble tumor necrosis factor-α from its membrane precursor (16Black R.A. Rauch C.T. Kozlosky C.J. Peschon J.J. Slack J.L. Wolfson M.F. Castner B.J. Stocking K.L. Reddy P. Srinivasan S. Nelson N. Boiani N. Schooley K.A. Gerhart M. Davis R. Fitzner J.N. Johnson R.S. Paxton R.J. March C.J. Cerretti D.P. 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Science. 1999; 283: 91-94Crossref PubMed Scopus (374) Google Scholar); ephrin A2 (21Hattori M. Osterfield M. Flanagan J.G. Science. 2000; 289: 1360-1365Crossref PubMed Scopus (460) Google Scholar); and amyloid precursor protein (22Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (977) Google Scholar). ADAM9 has been implicated in shedding of the membrane-anchored heparin-binding epidermal growth factor-like growth factor (23Izumi Y. Hirata M. Hasuwa H. Iwamoto R. Umata T. Miyado K. Tamai Y. Kurisaki T. Sehara-Fujisawa A. Ohno S. Mekada E. EMBO J. 1998; 17: 7260-7272Crossref PubMed Scopus (474) Google Scholar). Members of the ADAMTS subfamily of ADAM proteins degrade the extracellular matrix proteins aggrecan (ADAMTS-4 and ADAMTS-5) and procollagen (ADAMTS-2) (11Tortorella M.D. Burn T.C. Pratta M.A. Abbaszade I. Hollis J.M. Liu R. Rosenfeld S.A. Copeland R.A. Decicco C.P. Wynn R. Rockwell A. Yang F. Duke J.L. Solomon K. George H. Bruckner R. Nagase H. Itoh Y. Ellis D.M. Ross H. Wiswall B.H. Murphy K. Hillman M.C. Hollis G.F. Newton R.C. Magolda R.L. Trzaskos J.M. Arner E.C. Science. 1999; 284: 1664-1666Crossref PubMed Scopus (617) Google Scholar, 24Tang B.L. Int. J. Biochem. Cell Biol. 2001; 33: 33-44Crossref PubMed Scopus (268) Google Scholar). ADAM19 participates in the cleavage of membrane-anchored neuregulin (25Shirakabe K. Wakatsuki S. Kurisaki T. Fujisawa-Sehara A. J. Biol. Chem. 2001; 276: 9352-9358Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). Finally, ADAM12 cleaves insulin-like growth factor-binding protein-3 (26Shi Z., Xu, W. Loechel F. Wewer U.M. Murphy L.J. J. Biol. Chem. 2000; 275: 18574-18580Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar) and -5 (27Loechel F. Fox J.W. Murphy G. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 278: 511-515Crossref PubMed Scopus (276) Google Scholar) and the heparin-binding epidermal growth factor-like growth factor (28Asakura M. Kitakaze M. Takashima S. Liao Y. Ishikura F. Yoshinaka T. Ohmoto H. Node K. Yoshino K. Ishiguro H. Asanuma H. Sanada S. Matsumura Y. Takeda H. Beppu S. Tada M. Hori M. Higashiyama S. Nat. Med. 2002; 8: 35-40Crossref PubMed Scopus (639) Google Scholar).ADAM12 has been previously implicated in differentiation of mesenchymal cells such as skeletal myoblasts (29Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (438) Google Scholar, 30Kurisaki T. Masuda A. Osumi N. Nabeshima Y. Fujisawa-Sehara A. Mech. Dev. 1998; 73: 211-215Crossref PubMed Scopus (94) Google Scholar, 31Gilpin B.J. Loechel F. Mattei M.-G. Engvall E. Albrechtsen R. Wewer U. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 32Bornemann A. Kuschel R. Fujisawa-Sehara A. J. Muscle Res. Cell Motil. 2000; 21: 475-480Crossref PubMed Scopus (36) Google Scholar) and osteoblasts (33Inoue D. Reid M. Lum L. Krätzschmar J. Weskamp G. Myung Y.M. Baron R. Blobel C.P. J. Biol. Chem. 1998; 273: 4180-4187Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). In mouse embryo, ADAM12 expression is temporally and spatially restricted, and it is the most prominent in the regions of skeletal muscle and bone formation (30Kurisaki T. Masuda A. Osumi N. Nabeshima Y. Fujisawa-Sehara A. Mech. Dev. 1998; 73: 211-215Crossref PubMed Scopus (94) Google Scholar). In adult skeletal muscle, the expression level of ADAM12 is very low in both differentiated muscle fibers and quiescent satellite cells (29Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (438) Google Scholar, 31Gilpin B.J. Loechel F. Mattei M.-G. Engvall E. Albrechtsen R. Wewer U. J. Biol. Chem. 1998; 273: 157-166Abstract Full Text Full Text PDF PubMed Scopus (279) Google Scholar, 32Bornemann A. Kuschel R. Fujisawa-Sehara A. J. Muscle Res. Cell Motil. 2000; 21: 475-480Crossref PubMed Scopus (36) Google Scholar). ADAM12 expression is, however, dramatically up-regulated in regenerating muscle, where ADAM12 is readily detected in activated satellite cells undergoing differentiation and fusion (32Bornemann A. Kuschel R. Fujisawa-Sehara A. J. Muscle Res. Cell Motil. 2000; 21: 475-480Crossref PubMed Scopus (36) Google Scholar) and in newly formed muscle fibers (34Galliano M.F. Huet C. Frygelius J. Polgren A. Wewer U.M. Engvall E. J. Biol. Chem. 2000; 275: 13933-13939Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). In addition, a strong up-regulation of ADAM12 expression has been observed in human carcinomas, raising a possibility that ADAM12 may play a role in cell-cell and/or cell-matrix interactions in cancer (35Iba K. Albrechtsen R. Gilpin B.J. Loechel F. Wewer U.M. Am. J. Pathol. 1999; 154: 1489-1501Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar).Proteolytic processing is an inherent element of maturation of ADAM proteins in mammalian cells. Most notably, many ADAMs are cleaved between the pro- and metalloprotease domains as they progress through the secretory pathway. Because the presence of the prodomain in an unprocessed ADAM protein inhibits the catalytic activity of the metalloprotease domain via a cysteine-switch mechanism (36Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1190) Google Scholar), the cleavage of ADAMs at the boundary between the pro- and metalloprotease domains, followed by the dissociation of the free prodomain, constitutes an important step in the activation of the metalloprotease. Proteolytic removal of the prodomain has been best studied for ADAM15 (37Lum L. Reid M.S. Blobel C.P. J. Biol. Chem. 1998; 273: 26236-26247Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar), ADAM9 (38Roghani M. Becherer J.D. Moss M.L. Atherton R.E. Erdjument-Bromage H. Arribas J. Blackburn R.K. Weskamp G. Tempst P. Blobel C.P. J. Biol. Chem. 1999; 274: 3531-3540Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar), ADAM17 (39Schlöndorff J. Becherer J.D. Blobel C.P. Biochem. J. 2000; 347: 131-138Crossref PubMed Scopus (307) Google Scholar), and ADAM28 (40Howard L. Maciewicz R.A. Blobel C.P. Biochem. J. 2000; 348: 21-27Crossref PubMed Scopus (108) Google Scholar), but it has been also observed for ADAM10 (22Lammich S. Kojro E. Postina R. Gilbert S. Pfeiffer R. Jasionowski M. Haass C. Fahrenholz F. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3922-3927Crossref PubMed Scopus (977) Google Scholar) and ADAM19 (25Shirakabe K. Wakatsuki S. Kurisaki T. Fujisawa-Sehara A. J. Biol. Chem. 2001; 276: 9352-9358Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). In addition, ADAM15 (37Lum L. Reid M.S. Blobel C.P. J. Biol. Chem. 1998; 273: 26236-26247Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar) and ADAM2 (41Lum L. Blobel C.P. Dev. Biol. 1997; 191: 131-145Crossref PubMed Scopus (89) Google Scholar) have been reported to undergo further processing, resulting in mature forms in which the entire metalloprotease or a large part of it was missing.Proteolytic processing seems to play a particularly important role in the regulation of ADAM12 function. The extent of the processing and the intracellular localization of the mature form of ADAM12 are, however, unclear. According to the original report, both the pro- and metalloprotease domains of ADAM12 need to be removed in order for ADAM12 to stimulate myoblast fusion (29Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (438) Google Scholar). Overexpression of ADAM12 lacking its pro- and metalloprotease domains in the mouse myoblastic cell line C2C12 leads to accelerated cell differentiation and fusion, whereas transfection of C2C12 cells with full-length ADAM12 results in inhibition of fusion (29Yagami-Hiromasa T. Sato T. Kurisaki T. Kamijo K. Nabeshima Y. Fujisawa-Sehara A. Nature. 1995; 377: 652-656Crossref PubMed Scopus (438) Google Scholar). However, when expressed as a green fluorescent protein (GFP)1fusion protein in COS-7 cells, full-length human ADAM12 is cleaved predominantly between the pro- and metalloprotease domains (42Hougaard S. Loechel F., Xu, X. Tajima R. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 275: 261-267Crossref PubMed Scopus (52) Google Scholar), suggesting that mature ADAM12 contains an intact metalloprotease domain. Surprisingly, the processed form of ADAM12-GFP is retained in the trans-Golgi network (TGN), with very little protein present at the cell surface (42Hougaard S. Loechel F., Xu, X. Tajima R. Albrechtsen R. Wewer U.M. Biochem. Biophys. Res. Commun. 2000; 275: 261-267Crossref PubMed Scopus (52) Google Scholar). When overexpressed in C2C12 myoblasts or 10T1/2 fibroblasts, mouse ADAM12 does not undergo any proteolytic processing and localizes exclusively in the endoplasmic reticulum (ER) (43Kadota N. Suzuki A. Nakagami Y. Izumi T. Endo T. J. Biochem. (Tokyo). 2000; 128: 941-949Crossref PubMed Scopus (15) Google Scholar). In clear contrast, a mature form of ADAM12 is expressed at the cell surface of Chinese hamster ovary cells transfected with full-length mouse ADAM12, where it supports the interactions with integrin α9-expressing cells (44Eto K. Puzon-McLaughlin W. Sheppard D. Sehara-Fujisawa A. Zhang X.P. Takada Y. J. Biol. Chem. 2000; 275: 34922-34930Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar).In this report, we have investigated the intracellular processing and localization of ADAM12 in mouse C2C12 myoblasts and COS-7 cells. We have found that full-length ADAM12 was processed in both cell lines to an ∼90-kDa form. The 90-kDa form, which lacks the prodomain, was the predominant form present at the cell surface. We also found that the processing of endogenous ADAM12 in C2C12 cells was similar to the processing of overexpressed ADAM12. Immunofluorescence analysis of transfected cells using anti-ADAM12 antibody further demonstrated that ADAM12 was present both at the cell surface and in the intracellular compartments of the secretory pathway and that the cytoplasmic domain of ADAM12 was a limiting factor for the export of ADAM12 from the ER. In contrast, replacement of a leucine residue (Leu73) in the putative α-helical region in the prodomain with a helix-breaking proline resulted in retention of the ADAM12 mutant in the ER and a complete lack of its proteolytic processing. We conclude that properly folded mouse ADAM12 is targeted to the plasma membrane, where it can mediate cell-cell adhesion or adhesion-mediated signaling.RESULTSADAM12 contains the sequence RHKR at the boundary between the prodomain and the metalloprotease, which corresponds to the consensus cleavage site (RX(R/K)R) of furin, a member of the subtilisin-like family of endoproteases (49Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar). The subtilisin-like proteases are ubiquitously expressed, are localized predominantly in the TGN or secretory granules, and function as mammalian proprotein convertases (49Nakayama K. Biochem. J. 1997; 327: 625-635Crossref PubMed Scopus (701) Google Scholar, 50Zhou A. Webb G. Zhu X. Steiner D.F. J. Biol. Chem. 1999; 274: 20745-20748Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). To examine whether ADAM12, similarly to several other ADAM proteins, undergoes a proteolytic cleavage between the pro- and metalloprotease domains, C2C12 myoblasts or COS-7 cells were transfected with an expression vector encoding full-length ADAM12. Analysis of total cell lysates (Fig.2 A) or cellular glycoproteins (Fig. 2 B) by Western blotting with an antibody raised against a peptide from the cytoplasmic domain of ADAM12 revealed the presence of two major forms of ADAM12 of ∼120 and ∼90 kDa, respectively. (Several protein bands observed on immunoblots of total cell lysates were absent in the eluates from the concanavalin A column; and therefore, they represented nonspecific antibody binding rather than endogenous ADAM12.) The ∼120-kDa form corresponded to full-length ADAM12. The ∼90-kDa form was larger than ADAM12(Δ1–424), i.e. ADAM12 lacking the pro- and metalloprotease domains and containing an exogenous secretion signal (Fig. 2, lanes 3; see also Fig. 1 A), indicating that the ∼90-kDa form most likely represented ADAM12 without the prodomain. (The predicted molecular mass of the prodomain is ∼20 kDa, and two potential N-linked glycosylation sites are present.)Remarkably, substitution of Leu73 in the prodomain with proline resulted in complete inhibition of ADAM12 processing, as only the 120-kDa form was detected both in C2C12 and COS-7 cells transfected with the ADAM12(L73P) construct (Fig. 2, lanes 2). Leu73 resides in a region in the prodomain that is predicted to form an α-helix (amino acids 68–89). Analysis of the ADAM12 amino acid sequence with a secondary structure prediction software (GOR IV) 2Available at abs.cit.nih.gov. indicated that the probability of Leu73 to participate in an α-helix was ∼70%. Substitution of Leu73 with a helix-breaking proline decreased this probability to ∼30%; and therefore, it might have affected the proper folding of ADAM12. Because misfolded proteins are routinely retained in the ER and later degraded (51Ellgaard L. Molinari M. Helenius A. Science. 1999; 286: 1882-1888Crossref PubMed Scopus (1061) Google Scholar, 52Nehls S. Snapp E.L. Cole N.B. Zaal K.J. Kenworthy A.K. Roberts T.H. Ellenberg J. Presley J.F. Siggia E. Lippincott-Schwartz J. Nat. Cell Biol. 2000; 2: 288-295Crossref PubMed Scopus (215) Google Scholar, 53Ellgaard L. Helenius A. Curr. Opin. Cell Biol. 2001; 13: 431-437Crossref PubMed Scopus (333) Google Scholar), the L73P mutation of ADAM12 most likely prevented the export of ADAM12 from the ER and its further processing in post-ER compartments (see also Figs. 4, 7, and 8).Figure 4Deglycosylation of the recombinant forms of ADAM12. COS-7 cells were transfected with an expression vector encoding ADAM12 (lanes 1–3), ADAM12(L73P) (lanes 4–6), or ADAM12(Δ1–424) (lanes 7–9). Forty-eight hours after transfection, recombinant forms of ADAM12 were partially purified by affinity chromatography on glutathione-Sepharose columns containing immobilized glutathione S-transferase that was fused to the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase (47Kang Q. Cao Y. Zolkiewska A. J. Biol. Chem. 2001; 276: 24466-24472Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Column eluates without any further treatment (lanes 1, 4, and 7) or treated with Endo H (lanes 2, 5, and 8) or peptideN-glycanase F (PNGase F; lanes 3,6, and 9) were analyzed by SDS-PAGE and Western blotting with anti-ADAM12 antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Immunolocalization of ADAM12 in COS-7 cells. Cells were transfected with an expression vector encoding full-length ADAM12 (A–F), ADAM12(L73P) (G–I), or ADAM12(Δ1–424) (J–O). Two days after transfection, cells were fixed with 3.7% paraformaldehyde; permeabilized with 0.1% Triton X-100; and immunostained with anti-ADAM12 antibody (A, D, G,J, and M) and either anti-KDEL antibody (B, H, and K), a marker for the ER, or anti-TGN38 antibody (E and N), a marker for thetrans-Golgi network. To detect ADAM12, cells were incubated with rhodamine-conjugated anti-rabbit IgG antibody. To detect KDEL-containing proteins or TGN38, cells were incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG antibody.C, F, I, L, andO represent merged images of A and B,D and E, G and H,J and K, and M and N, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8Immunolocalization of ADAM12 in C2C12 cells. C2C12 cells were transfected with an expression vector encoding full-length ADAM12 (A–C), the L73P mutant of ADAM12 (D–F), or ADAM12(Δ1–424) (G–I). Cells were stained with anti-ADAM12 antibody (A, D, andG) and anti-KDEL antibody (B, E, andH) as described in the legend to Fig. 7. C,F, and I represent merged images of Aand B, D and E, G andH, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate the proteolytic processing of mouse ADAM12 in the secretory pathway, ADAM12-transfected COS-7 cells were pulse-labeled with [35S]methionine/cysteine, followed by incubation for various periods of time in 35S-free medium. Cell lysates from 35S-labeled cells were then subjected to immunoprecipitation with anti-ADAM12 antibody, SDS-PAGE, and autoradiography. As shown in Fig.3 A (lanes 2 and3), within 1 h after metabolic labeling, only the ∼120-kDa form of ADAM12 was immunoprecipitated. The ∼90-kDa form was detected after a 3-h chase period (lane 4). With the increasing chase time, the amount of 35S-labeled ADAM12 protein in the immunoprecipitate gradually decreased (lanes 5 and 6). These results suggested that the ∼90-kDa form of ADAM12 was generated from the ∼120-kDa ADAM12 and that the proteolytic event occurred ∼3 h after the initiation of ADAM12 synthesis.Figure 3Pulse-chase labeling of ADAM12. A, COS-7 cells were transfected with an expression vector (Vect.) without an insert (lane 1) or with a vector encoding ADAM12 (lanes 2–6). Forty-eight hours after transfection, cells were pulse-labeled for 20 min with [35S]methionine/cysteine, followed by a chase for the indicated times in medium without radioactive precursors, immunoprecipitation with anti-ADAM12 antibody, SDS-PAGE, and autoradiography. B, COS-7 cells transfected with an empty vector (lane 1) or with a vector with an ADAM12 insert (lanes 2 and 3) were pulse-labeled with [35S]methionine/cysteine as described for Aand chased for 3 h without (lanes 1 and 2) or with (lane 3) BFA, followed by immunoprecipitation with anti-ADAM12 antibody, SDS-PAGE, and autoradiography. Molecular mass markers (in kilodaltons) are shown on the left.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To identify the intracellular compartment where ADAM12 was processed, COS-7 cells were transfected with a vector encoding ADAM12, pulse-labeled with [35S]methionine/cysteine, and incubated in the presence of BFA. BFA inhibits transport through the secretory pathway by redistributing proteins from the cis-,medial-, and trans-Golgi cisternae, but not from the trans-Golgi network, into the ER (54Lippincott-Schwartz J. Yuan L.C. Bonifacino J.S. Klausner R.D. Cell. 1989; 56: 801-813Abstract Full Text PDF PubMed Scopus (1306) Google Scholar). Fig.3 B demonstrates that BFA treatment resulted in the immunoprecipitation of only unprocessed ADAM12 after a 3-h chase (lane 3), whereas without BFA, the processed form could be easily detected (lane 2). These results suggested that the proteolytic processing of ADAM12 occurred in the trans-Golgi network. Because furin is a ubiquitous proprotein convertase that resides in the trans-Golgi network, it has been postulated that it might be directly involved in the proteolytic processing of ADAM12. However, we have observed that expression of ADAM12 in LoVo cells, a furin-deficient cell line, resulted in a similar processing of ADAM12 as in C2C12 and COS-7 cells (data not shown), which strongly suggested that furin was not responsible for the cleavage of ADAM12.To further define the subcellular localization of individual proteolytic forms of ADAM12 and ADAM12 mutants, we assessed their sensitivity to two glycosidases: peptide N-glycosidase F and Endo H. The sensitivity to peptide N-glycosidase F, an enzyme that removes all types of N-linked oligosaccharides from glycoproteins, is an indication of N-linked protein glycosylation. Endo H selectively removes high mannose-type oligosaccharides from glycoproteins, but it does not cleave complex glycans. Because the conversion of high mannose oligosaccharides into complex glycans occurs in the medial-Golgi compartment, resistance to Endo H is an indication that a glycoprotein has reached this compartment (55Maley F. Trimble R.B. Tarentino A.L. Plummer T.H. Anal. Biochem. 1989; 180: 195-204Crossref PubMed Scopus (6
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