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• W1606112682 abstract "neu differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF.In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium. neu differentiation factor (NDF), also known as heregulin, is structurally related to the epidermal growth factor family of growth factors; it stimulates tyrosine phosphorylation of the neu/HER-2 oncogene and causes differentiation of certain human breast cancer cell lines. Alternative splicing of a single gene gives rise to multiple isoforms of NDF/heregulin, as well as the neuronal homologues, designated ARIA (acetylcholine receptor inducing activity) and GGF (glial growth factor); at least 15 structural variants are known. All but two of the NDF/heregulin cDNAs are predicted to encode transmembrane, glycosylated precursors of soluble NDF. In this report we characterized the biosynthetic processing of different NDF isoforms in stably transfected Chinese hamster ovary cells expressing individual NDF isoforms, and in the native cell line Rat 1-EJ, which expresses at least six different NDF isoforms. We found that the precursors for NDF undergo typical glycosylation and trafficking. A portion of the molecules are proteolytically cleaved intracellularly leading to the constitutive secretion of soluble, mature NDF into the culture media. However, a significant portion of the newly synthesized NDF precursor molecules escape intracellular cleavage and are transported to the cell surface of both transfected and native cells, where they reside as full-length, transmembrane proteins. Finally we show that these full-length, transmembrane NDF molecules can undergo phorbol ester regulated cleavage from the membrane, releasing the soluble growth factor into the medium. neu differentiation factor (NDF) 1The abbreviations used are: NDFneu differentiation factorARIAacetylcholine receptor inducing activityGGFglial growth factorSCFstem cell factorTGFtransforming growth factorCHAPS3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonic acidCHOChinese hamster ovaryEGFepidermal growth factorPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisPMAphorbol 12-myristate 13-acetate. 1The abbreviations used are: NDFneu differentiation factorARIAacetylcholine receptor inducing activityGGFglial growth factorSCFstem cell factorTGFtransforming growth factorCHAPS3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonic acidCHOChinese hamster ovaryEGFepidermal growth factorPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisPMAphorbol 12-myristate 13-acetate. was identified and purified from the conditioned medium of Rat 1-EJ cells, based on its ability to stimulate tyrosine phosphorylation of the neu oncogene (also known as HER-2 and c-erbB-2), and was shown to cause differentiation of certain human breast cancer cell lines(1Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Ben Levy R. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 2Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar). A similar strategy was employed to identify the human homologue of NDF, heregulin(3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar). In addition to growth and differentiation activities, one recent study found that NDF/heregulin may function as a paracrine mediator in wound healing and tissue repair in vivo(4Danilenko D.M. Ring B.D. Lu J.Z. Tarpley J.E. Chang D. Liu N. Wen D. Pierce G.F. J. Clin. Invest. 1995; 95: 842-851Crossref PubMed Google Scholar). Subsequent cloning and sequencing of NDF and heregulin predicted that the secreted factor was derived from a membrane-bound precursor protein belonging to the EGF family of growth factors(2Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar). neu differentiation factor acetylcholine receptor inducing activity glial growth factor stem cell factor transforming growth factor 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonic acid Chinese hamster ovary epidermal growth factor phosphate-buffered saline polyacrylamide gel electrophoresis phorbol 12-myristate 13-acetate. neu differentiation factor acetylcholine receptor inducing activity glial growth factor stem cell factor transforming growth factor 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonic acid Chinese hamster ovary epidermal growth factor phosphate-buffered saline polyacrylamide gel electrophoresis phorbol 12-myristate 13-acetate. Until recently, NDF/heregulin was thought to be the direct ligand for HER-2/neu(1Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Ben Levy R. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (478) Google Scholar, 2Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar, 3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepard H.M. Kuang W.-J. Wood W.I. Goeddel D.V. Vandlen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar); however, several lines of investigation suggested that NDF did not directly stimulate the phosphorylation of HER-2/neu(5Peles E. Ben-Levy R. Tzahar E. Liu N. Wen D. Yarden Y. EMBO J. 1993; 12: 961-971Crossref PubMed Scopus (213) Google Scholar, 6Plowman G.D. Green J.M. Culouscou J.-M. Carlton G.W. Rothwell V.M. Buckley S. Nature. 1993; 366: 473-475Crossref PubMed Scopus (438) Google Scholar). It now appears that NDF binds directly to two other members of the EGF receptor family, HER-3 and HER-4, and stimulates phosphorylation of HER-2, HER-3, and HER-4 through the formation of homo- and heterodimeric receptor molecules at the cell surface ((6Plowman G.D. Green J.M. Culouscou J.-M. Carlton G.W. Rothwell V.M. Buckley S. Nature. 1993; 366: 473-475Crossref PubMed Scopus (438) Google Scholar, 7Carraway III, K.L. Sliwkowski M.X. Akita R. Platko J.V. Guy P.M. Nuijens A. Diamonti A.J. Vandlen R.L. Cantley L.C. Cerione R.A. J. Biol. Chem. 1994; 269: 14303-14306Abstract Full Text PDF PubMed Google Scholar, 8Sliwkowski M.X. Schaefer G. Akita R.W. Lofgren J.A. Fitzpatrick V.D. Nuijens A. Fendly B.M. Cerione R.A. Vandlen R.L. Carraway III, K.L. J. Biol. Chem. 1994; 269: 14661-14665Abstract Full Text PDF PubMed Google Scholar, 9Tzahar E. Levkowitz G. Karunagaran D. Yi L. Peles E. Lavi S. Chang D. Liu N. Yayon A. Wen D. Yarden Y. J. Biol. Chem. 1994; 269: 25226-25233Abstract Full Text PDF PubMed Google Scholar, 10Kita Y.A. Barff J. Luo Y. Wen D. Brankow D. Hu S. Liu N. Prigent S.A. Gullick W.J. Nicolson M. FEBS Lett. 1994; 349: 139-143Crossref PubMed Scopus (68) Google Scholar); for review, see (11Carraway III, K.L. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (585) Google Scholar)). The HER-2/neu oncogene has been of keen interest to tumor biologists because specific mutations in this receptor tyrosine kinase lead to the development of neuroblastomas and glioblastomas in rats. In humans, it has been shown that amplification and/or overexpression of neu/HER-2 occurs in approximately 25% of breast, ovarian, stomach, pancreatic, and bladder carcinomas. Furthermore, HER-2 overexpression in breast and ovarian cancer is associated with poor prognosis for survival ((12Slamon D.J. Clark G.M. Wong S.G. Levin W.J. Ullrich A. McGuire W.L Science. 1987; 235: 177-182Crossref PubMed Scopus (9936) Google Scholar, 13Slamon D.J. Godolphin W. Jones L.A. Holt J.A. Wong S.G. Keith D.E. Levin W.J. Stuart S.G. Udove J. Ullrich A. Press M.F. Science. 1989; 244: 707-712Crossref PubMed Scopus (6256) Google Scholar, 14Berger M.S. Locher G.W. Saurer S. Gullick W.J. Waterfield M.D. Groner B. Hynes N.E. Cancer Res. 1988; 48: 1238-1243PubMed Google Scholar); see (15Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar) for review). Less is known about the expression of the newer members of this family in human cancers (HER-3 and HER-4), but studies suggest that these too may be amplified in mammary tumors(16Kraus M.H. Issing W. Miki T. Popescu N.C. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9193-9197Crossref PubMed Scopus (674) Google Scholar). The potential role played by the ligand(s) for these receptors, including NDF/heregulin, in normal and malignant cell growth is of great interest and is an area of intense research (for reviews, see (11Carraway III, K.L. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (585) Google Scholar) and (15Peles E. Yarden Y. Bioessays. 1993; 15: 815-824Crossref PubMed Scopus (260) Google Scholar)). Two other homologues of NDF have been identified from neuronal sources, one from chicken brain called ARIA (for acetylcholine receptor inducing activity; (17Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (552) Google Scholar)), and one from bovine brain named GGF (for glial growth factor; (18Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonough O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (682) Google Scholar)). ARIA was discovered by its ability to induce the synthesis of acetylcholine receptors in muscle. It has subsequently been shown that ARIA also increases the number of sodium channels in muscle (19Corfas G. Fischbach G.D. J. Neurosci. 1993; 13: 2118-2125Crossref PubMed Google Scholar) and causes a change in acetylcholine receptor subunit expression from embryonic to adult isoforms(20Martinou J.-C. Falls D.L. Fischbach G.D. Merlie J.P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7669-7673Crossref PubMed Scopus (121) Google Scholar). Thus, ARIA may be one of the important factors regulating the architecture of the developing and adult synapse. GGF was identified based on its ability to stimulate the growth of Schwann cells (21Lemke G.E. Brockes J.P. J. Neurosci. 1984; 4: 75-83Crossref PubMed Google Scholar, 22Goodearl A.D.J. Davis J.B. Mistry K. Minghetti L. Otsu M. Waterfield M.D. Stroobant P. J. Biol. Chem. 1993; 268: 18095-18102Abstract Full Text PDF PubMed Google Scholar) and was recently cloned(18Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonough O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (682) Google Scholar). The genomic cloning of GGF suggested that extensive alternative splicing of different functional domains within this single gene gives rise to all of the NDF/heregulin isoforms of this growing family of EGF-related growth/differentiation factors(18Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonough O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Crossref PubMed Scopus (682) Google Scholar). At least 15 variants of the NDF/heregulin family have been cloned from mesenchymal and neuronal sources, and many have been shown to display a tissue-specific pattern of expression (for review see (23Ben-Baruch N. Yarden Y. Soc. Exp. Biol. Med. 1994; 206: 221-227Crossref PubMed Scopus (71) Google Scholar)). Six different isoforms of rat NDF have been identified by exhaustive cDNA cloning from the ras-transformed Rat 1-EJ cells (24Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben-Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.-Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar). Based on their DNA sequences, all but one of the NDF cDNAs isolated from Rat 1-EJ were predicted to encode membrane-bound, glycosylated precursors of soluble NDF, henceforth referred to as proNDF. As a prerequisite to understanding the potential functional variation of this structurally diverse family, we have begun to characterize the biosynthetic processing of different NDF isoforms in stably transfected CHO cells expressing individual NDF isoforms, and in the native cell line, Rat 1-EJ. Because of the structural similarity of the proNDFs to proTGFα and other membrane-anchored growth factors(25Bringman T.S. Lindquist P.B. Derynck R. Cell. 1987; 48: 429-440Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 26Massagué J. Pandiella A. Annu. Rev. Biochem. 1993; 62: 515-541Crossref PubMed Scopus (600) Google Scholar), we have also explored the possibility that unprocessed proNDF might be transported intact to the cell surface, where it could undergo regulated proteolysis to control release, and/or be involved in juxtacrine signaling via cell-cell interactions with receptors on neighboring cells(27Bosenberg M.W. Massagué J. Curr. Opin. Cell Biol. 1993; 5: 832-838Crossref PubMed Scopus (66) Google Scholar). Using a combination of pulse-chase, deglycosylation and cell surface labeling techniques we provide evidence that proNDFs undergo typical glycosylation and trafficking. However, only a portion of the molecules undergo intracellular proteolysis, such that at steady state a significant level of full-length, membrane-bound proNDF is exposed at the cell surface in both native and transfected cells. Finally, we show that proNDF can undergo phorbol ester regulated cleavage from the membrane releasing the soluble growth factor into the medium. The biological implications of these results are considered in the discussion. Chinese hamster ovary cells deficient in dihydrofolate reductase activity (CHO d−, (28Urlaub G. Chasin L.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 4216-4620Crossref PubMed Scopus (665) Google Scholar)) were transfected with calcium phosphate using pDSRα2-derived expression vectors (29DeClerck 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) containing the rat NDFα2c or NDFβ4 cDNAs ((24Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben-Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.-Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar); see Fig. 1). Transfected colonies were cloned and analyzed by Western blot using an antibody directed against Escherichia coli-derived rat NDFα2c (see below). High expressing clones for NDFα2c and NDFβ4 were chosen for further analysis. The expression of NDFβ4 was gradually amplified with methotrexate(30Kaufman R.J. Sharp P.A. J. Mol. Biol. 1982; 159: 601-621Crossref PubMed Scopus (239) Google Scholar). CHO d− cells were grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, nonessential amino acids, glutamine, penicillin and streptomycin and hypoxanthine/thymidine. Transfected clones were grown in similar media using 5% dialyzed fetal bovine serum and lacking hypoxanthine/thymidine; in addition, methotrexate was added to 70 nM for the NDFβ4-expressing clone. Rat 1-EJ, a ras-transformed Rat 1 cell line(2Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Ben Levy R. Koski R.A. Lu H.S. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (526) Google Scholar), was grown in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum, glutamine, penicillin, and streptomycin. Recombinant E. coli-derived rat NDFα2c used in competition experiments, the generation of rabbit polyclonal antibody 1872 and the generation and purification of CHO d−-derived NDFα2c have been described(24Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben-Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.-Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar, 31Lu H.S. Hara S. Wong L.W.-I. Jones M.D. Katta V. Trail G. Zou A. Brankow D. Cole S. Hu S. Wen D. J. Biol. Chem. 1995; (in press)Google Scholar). The rabbit polyclonal antiserum 1219 was generated using CHO d−-derived NDFα2c as the immunogen. The antibody was purified by affinity chromatography with Actigel ALD as recommended by the manufacturer (Sterogene Bioseparation, Inc., Arcadia, CA). 1.3 mg of recombinant CHO d−-derived rat NDFα2c was coupled to 2 ml of Actigel ALD superflow resin. 20 ml of crude antiserum was bound, the column washed with PBS, and eluted with Immunopure gentle antigen/antibody elution buffer (Pierce). The eluted antibodies were pooled and dialyzed overnight against PBS and concentrated with an Amicon protein concentrator to approximately 1 mg/ml. The rabbit polyclonal antiserum 1310 is an anti-peptide antibody, generated using a peptide from the cytoplasmic domain of (pro)NDF (sequence: CNSFLRHARETPDSYRDS) covalently coupled to keyhole limpet hemacyanin as the immunogen. The affinity purification and characterization procedures were similar to antibody 1219 except that the peptide was coupled to the column matrix. We have determined that antibodies 1872, 1219, and 1310 react specifically with the α and β forms of (pro)NDF in Western blotting and quantitative immunoprecipitation. Cells were grown to 70-80% confluence and then starved in medium lacking methionine and cysteine (ICN, Costa Mesa, CA) for 1 h. Cells were labeled with [35S]methionine and -cysteine (Tran35S-label, ICN) at 0.5 mCi/ml. After a 30-min pulse, cells were either extracted in nonionic detergent (N-det) buffer (32Burgess T.L. Craik C.S. Kelly R.B. J. Cell Biol. 1985; 101: 639-645Crossref PubMed Scopus (44) Google Scholar) or chased with media containing excess unlabeled methionine and cysteine (Life Technologies, Inc.). Media and cell lysates were collected and protease inhibitors added immediately (aprotinin, leupeptin, pepstatin, and o-phenanthroline; Boehringer Mannheim). Samples were brought to 0.3% SDS and 1 × N-det and incubated at room temperature with preimmune serum and protein A-agarose (Boehringer Mannheim) for 5 h. Antibody 1219 (5 μg) or 1872 (25 μg) was prebound to protein A-agarose in immunoprecipitation buffer (N-det with 0.3% SDS and protease inhibitors) for 5 h at room temperature (immune beads). Preimmune treated samples were then added to washed immune beads and incubated overnight at 4°C. Immune complexes were washed, eluted in SDS-PAGE sample buffer, and analyzed by 10% reducing SDS-PAGE (Novex, San Diego, CA; (33Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207139) Google Scholar)). Quantitative depletion of NDF from the samples was shown by re-immunoprecipitation of the supernatants. To reduce the background, Rat 1-EJ samples were eluted and re-immunoprecipitated before SDS-PAGE analysis. Enhancement and fluorography was carried out as described(32Burgess T.L. Craik C.S. Kelly R.B. J. Cell Biol. 1985; 101: 639-645Crossref PubMed Scopus (44) Google Scholar). For competition, 10 μg of recombinant rat NDFα2c was added to the precleared samples prior to adding to the immune beads. Prestained protein molecular weight markers were from Bio-Rad and Amersham Corp. Radiolabeled pulse-chase samples from CHO d− clones were immunoprecipitated and NDF proteins eluted with 60-75 μl of elution buffer (10 mM Tris, 1 mM EDTA, 0.5% SDS, 1%β-mercaptoethanol). 15-μl aliquots were diluted in water, and CHAPS (Sigma) was added to 19 μM. Glycosidase enzymes were added (endoglycosidase H, 2 milliunits; N-glycanase, 500 milliunits; neuraminidase, 20 milliunits; O-glyconase, 2 milliunits) (Genzyme, Boston, MA) in a final volume of 30 μl. After overnight digestion at 37°C, samples were analyzed by 10% SDS-PAGE. For partial digestion of secreted NDFα2c and NDFβ4a with N-glycanase, the enzyme was used at 62.5, 250, and 1000 milliunits/reaction, then incubated at 37°C for 4 h. Western blotting was carried out essentially as described(1Peles E. Bacus S.S. Koski R.A. Lu H.S. Wen D. Ogden S.G. Ben Levy R. Yarden Y. Cell. 1992; 69: 205-216Abstract Full Text PDF PubMed Scopus (478) Google Scholar), except that blots were blocked, then incubated with affinity-purified 1219 at 2 μg/ml in 5% nonfat dry milk for 2 h, washed three times for 5 min each in 1% nonfat dry milk, and reacted with horseradish peroxidase-conjugated anti-rabbit IgG (Amersham Corp.) in 1% nonfat dry milk for 30 min. Transfected and untransfected CHO d− cells were grown on Permanox chamber slides (Nunc, Naperville, IL). PBS-rinsed, live cells were incubated at 0°C with antibody 1219 at 1 μg/ml for 1 h. Cells were washed three times with PBS and then incubated with fluorescein-labeled goat anti-rabbit secondary antibody for 30 min at 0°C. Cells were washed and fixed with 4% formaldehyde for 20 min at room temperature. Slides were mounted with FITC-Guard (Testog, Chicago, IL) and then viewed and photographed on a Zeiss Axiovert 10 microscope. Cell surface proteins were biotinylated with sulfosuccinimidyl-6-(biotinamido)hexanoate (NHS-LC-biotin, Pierce) following procedures recommended by the manufacturer. Following cell lysis, immunoprecipitation was performed as described above. Cell surface NDF was identified by a subsequent streptavidin Western blot. For cell surface immunoprecipitation, transfected CHO d− cells were grown to confluence. Cells were removed from the culture dishes with PBS containing 5 mM EDTA and 5 mM EGTA at room temperature for 10 min. The intact, suspended cells were washed with PBS three times and then incubated with 5-10 μg/ml antibody 1219 on ice for 1 h. After extensive washing with PBS, cells were lysed with N-det buffer. The nuclei were removed by centrifugation, and supernatants were subjected to immunoprecipitation with protein A-agarose beads as described above. CHO d− clones expressing NDFα2c and NDFβ4 were grown to 70-80% confluence. Growth medium was replaced by serum-free medium for 1 h, followed by addition of fresh serum-free medium containing either 1 μM phorbol 12-myristate 13-acetate (PMA, Sigma) and 0.1% Me2SO or 0.1% Me2SO alone as a control. Medium and lysate samples were collected at 10 and 30 min. Medium samples were concentrated 10-fold by trichloroacetic acid precipitation. Samples were separated by SDS-PAGE and analyzed by Western blot. The domain structure of the NDF isoforms expressed in Rat 1-EJ cells is shown schematically in Fig. 1(see also (24Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben-Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.-Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar)). The extracellular portion of all NDF isoforms consists of a common N terminus, which contains an immunoglobulin domain, a spacer region containing multiple N- and O-glycosylation sites, and an EGF-like repeat. The two major classes of NDF molecules diverge in the C terminus of the EGF domain giving rise to the α and β isoforms. Additional variation is seen in the juxtamembrane region following the EGF domain by the insertion of one of three different sequences (numbered 2, 3, or 4). Isoform β3 terminates prior to the transmembrane domain and is predicted to encode a cytoplasmic version of NDF. The other isoforms all contain a common transmembrane domain. Finally, the α isoforms show further variation in the length of their cytoplasmic domains (Fig. 1; i.e. α2a, α2b, or α2c), whereas the β isoforms share the same cytoplasmic tail (Fig. 1; i.e. β2a and β4a). Additional NDF isoforms are expressed in other tissues (for review see (23Ben-Baruch N. Yarden Y. Soc. Exp. Biol. Med. 1994; 206: 221-227Crossref PubMed Scopus (71) Google Scholar)); for simplicity, only those identified in Rat 1-EJ cells are illustrated here (Fig. 1). Quantitative immunoprecipitates of pulse-chase samples from transfected CHO cells expressing proNDFα2c were analyzed by reducing SDS-PAGE and are shown in Fig. 2a (see “Experimental Procedures”). proNDFα2c is initially synthesized as a precursor of ~63 kDa (Fig. 2a, lane1, openarrowhead). Within 30 min the majority of the precursor shows a reduced mobility of ~66 kDa consistent with maturation and addition of carbohydrate moieties (Fig. 2a, lane2, solidarrowhead; see also Fig. 3). After 30 min of chase, and continuing for several hours, this glycosylated species is proteolytically cleaved to yield the mature ~44-kDa NDFα2c molecule, a clear precursor-product relationship is seen (Fig. 2a, lanes 2-6). The prominence of the ~44-kDa species in the cell extracts at 30 min, 2 h, and 4 h of chase (Fig. 2a, lanes2, 3, and 5) and the later appearance of mature NDFα2c in the media samples (Fig. 2a, lanes4 and 6) are consistent with intracellular cleavage and subsequent secretion of the mature ~44-kDa NDFα2c (secreted NDF is not detected at 30 min, but is seen in the media after 1 h of chase; data not shown). Note that by 4 h, most, but not all, of the pulse-labeled ~66-kDa proNDFα2c has been converted to the mature protein. The prominent, long-lived species at ~60 kDa is immunologically related to proNDFα2c (as demonstrated by competition with excess cold NDFα2c, data not shown) and may be an unglycoslyated and/or improperly folded proNDFα2c remaining in the endoplasmic reticulum.Figure 3:Deglycosylation analysis of intracellular and secreted NDF. Anti-NDF 1872 immunoprecipitated 35S-labeled NDFα2c samples were digested with endoglycosidase H (Endo H), N-glycanase (N-glyc.), and O-glycanase (O-glyc.), as indicated, and analyzed by reducing SDS-PAGE. a, pro-NDFα2c. Lanes 1-4, 30-min pulse extract; lanes 5-9, 30-min chase extract. b, mature intracellular and secreted NDFα2c. Lanes 1-4, 1-h chase extract; lanes5-9, 4-h chase medium. c, partial digestion of mature, secreted NDFα2c (lanes 1-4) and NDFβ4 (lanes 5-9) with N-glycanase, showing that one or two of the three potential N-glycosylation sites are utilized. Lanes1 and 5, mock-digested; lanes2 and 6, 62.5 milliunits of N-glycanase; lanes3 and 7, 250 milliunits of N-glycanase; lanes4 and 8, 1000 milliunits of N-glycanase was added to the digest. A schematic representation of all forms of NDFα2c is shown at the right, and the region of each gel shown is indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT) An identical experiment was performed with CHO cells expressing proNDFβ4 (Fig. 2b). Similar to proNDFα2c, proNDFβ4 is synthesized as a large, glycosylated precursor, which is proteolytically processed to release the ~44-kDa mature NDFβ4. Due to the extended cytoplasmic domain, proNDFβ4 has a significantly larger apparent molecular weight than the proNDFα2c (Fig. 1; (24Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben-Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.-Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koski R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar)). Immediately following the 30-min pulse, most of the precursor migrates at about 105 kDa (Fig. 2b, lane1, openarrowhead). Following the chase, proNDFβ4 migrates at about 110 kDa (Fig. 2b, lanes2, 3, and 5, solidarrowhead). Proteolytic processing of proNDFβ4 begins at a similar time as proNDFα2c; after 30 min" @default.
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• W1606112682 title "Biosynthetic Processing of neu Differentiation Factor" @default.
• W1606112682 cites W1488828742 @default.
• W1606112682 cites W1530138845 @default.
• W1606112682 cites W1562926557 @default.
• W1606112682 cites W1563725780 @default.
• W1606112682 cites W1797949464 @default.
• W1606112682 cites W1947994527 @default.
• W1606112682 cites W1966603679 @default.
• W1606112682 cites W1972561134 @default.
• W1606112682 cites W1973188134 @default.
• W1606112682 cites W1976492411 @default.
• W1606112682 cites W1981108516 @default.
• W1606112682 cites W1986841892 @default.
• W1606112682 cites W1987242362 @default.
• W1606112682 cites W1987657791 @default.
• W1606112682 cites W1990654868 @default.
• W1606112682 cites W1991050149 @default.
• W1606112682 cites W1997914540 @default.
• W1606112682 cites W2001124278 @default.
• W1606112682 cites W2007206244 @default.
• W1606112682 cites W2016142807 @default.
• W1606112682 cites W2019647633 @default.
• W1606112682 cites W2020180059 @default.
• W1606112682 cites W2025561101 @default.
• W1606112682 cites W2032525309 @default.
• W1606112682 cites W2037010084 @default.
• W1606112682 cites W2037224058 @default.
• W1606112682 cites W2043471865 @default.
• W1606112682 cites W2047559880 @default.
• W1606112682 cites W2048444775 @default.
• W1606112682 cites W2048905477 @default.
• W1606112682 cites W2052398986 @default.
• W1606112682 cites W2069233295 @default.
• W1606112682 cites W2093735302 @default.
• W1606112682 cites W2095156669 @default.
• W1606112682 cites W2095796144 @default.
• W1606112682 cites W2100837269 @default.
• W1606112682 cites W2101862196 @default.
• W1606112682 cites W2108325176 @default.
• W1606112682 cites W2126943028 @default.
• W1606112682 cites W2133084600 @default.
• W1606112682 cites W2141393790 @default.
• W1606112682 cites W2143536256 @default.
• W1606112682 cites W2147334994 @default.
• W1606112682 cites W2151127067 @default.
• W1606112682 cites W30485879 @default.
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