Biosynthetic Processing of neu Differentiation Factor

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 Vandlen R.L. R.A. J. Full Text PDF PubMed Google Scholar, M.X. G. Akita R.W. Nuijens R.A. Vandlen R.L. III, K.L. J. Full Text PDF PubMed Google Scholar, E. G. D. Peles E. S. Chang D. Liu N. Wen D. Yarden Y. J. Full Text PDF PubMed Google Scholar, J. Luo Y. Wen D. D. Hu S. Liu N. W.J. PubMed Scopus Google Scholar); for III, K.L. Cell. Full Text PDF PubMed Scopus Google oncogene of to in this receptor tyrosine to the of and in In it shown that of neu/HER-2 in of and HER-2 in breast and cancer is for S.G. W.J. Science. PubMed Scopus Google Scholar, S.G. W.J. S.G. J. Science. PubMed Scopus Google Scholar, G.W. S. W.J. Google Scholar); E. Yarden Y. 1993; PubMed Scopus Google for is known the of the members of this family in human and but that these may be in S. PubMed Scopus Google Scholar). the for these NDF/heregulin, in and cell growth is of and is of III, K.L. Cell. Full Text PDF PubMed Scopus Google and E. Yarden Y. 1993; PubMed Scopus Google other of NDF identified from neuronal one from ARIA acetylcholine receptor inducing G. G.D. Cell. 1993; Full Text PDF PubMed Scopus Google and one from GGF glial growth D. J. D. D. Nature. 1993; PubMed Scopus Google ARIA was its ability to the of acetylcholine in It shown that ARIA also the of in G. G.D. J. 1993; PubMed Google and causes a in acetylcholine receptor from to G.D. S. PubMed Scopus Google Scholar). ARIA may be one of the the of the and GGF was identified based on its ability to stimulate the growth of cells J. PubMed Google Scholar, J. 1993; Full Text PDF PubMed Google and was D. J. D. D. Nature. 1993; PubMed Scopus Google Scholar). cloning of GGF suggested that splicing of different this single gene gives rise to of the NDF/heregulin isoforms of this family of D. J. D. D. Nature. 1993; PubMed Scopus Google Scholar). least 15 variants of the NDF/heregulin family from and neuronal and shown to a of N. Yarden Y. PubMed Scopus Google different isoforms of NDF identified cloning from the Rat 1-EJ cells D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google Scholar). on but one of the NDF cDNAs from Rat 1-EJ predicted to encode glycosylated precursors of soluble NDF, to as a to the of this structurally family, we to the biosynthetic processing of different NDF isoforms in stably transfected cells expressing individual NDF isoforms, and in the native cell Rat of the structural of the to and other growth R. Cell. Full Text PDF PubMed Scopus Google Scholar, J. 1993; PubMed Scopus Google we also the that be transported to the cell where it undergo regulated to be in on J. 1993; PubMed Scopus Google Scholar). a of and cell surface we that undergo typical glycosylation and trafficking. However, a portion of the molecules undergo intracellular that at a significant of full-length, membrane-bound is at the cell surface in both native and transfected we show that can undergo phorbol ester regulated cleavage from the releasing the soluble growth factor into the medium. of these are in the Chinese hamster ovary cells in activity G. S. PubMed Scopus Google transfected Lu H.S. J. J. Full Text PDF PubMed Google the cDNAs D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google Scholar); and expressing for and for of was J. PubMed Scopus Google Scholar). cells in medium and and in similar and in was to for the Rat 1-EJ, a Rat cell 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 was in medium and E. used in the of and the and of D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google Scholar, H.S. S. Trail G. D. S. Hu S. Wen D. J. 1995; Scholar). was as the was purified as the of was to of of was the and and and protein to is a from the of to as the and similar to that the was to the We that and the and of in and to and in medium and for and at a cells in J. PubMed Scopus Google and and cell and and to and and at and protein for was to protein in and for at to and at in and Nature. PubMed Scopus Google of NDF from the was shown of the the Rat 1-EJ and and was as J. PubMed Scopus Google Scholar). of was to the to to the protein from and from and NDF of in and was to in a of at of secreted and the was used at and at for was as 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 that at in for for in and in for and cells on cells at at for and for at and for at and and on a surface the cell was as surface NDF was identified a cell surface transfected cells to from the culture and at for cells and on for cells and to protein as expressing and to medium was medium for addition of medium phorbol 12-myristate and as a and at and acid and of the NDF isoforms in Rat 1-EJ cells is shown in also D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. 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PubMed Scopus Google for identified in Rat 1-EJ cells are of from transfected cells expressing and are shown in is synthesized as a precursor of the of the precursor a of and addition of also of and for several this glycosylated is proteolytically cleaved to the mature a is of the in the cell at and of and and the of mature in the and are intracellular cleavage and secretion of the mature NDF is not at but is in the of not that but not of the to the mature at is related to not and may be in the of intracellular and secreted NDF. and as and mature intracellular and secreted medium. of secreted and that one two of the are and and of and of and of was to the A of of is shown at the and the of gel shown is was cells expressing to is synthesized as a glycosylated which is proteolytically to the mature to the a the D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google the of the precursor at the at and processing of at a similar as of mature is in the cell to and is in the and However, the of is that of of is a portion of that is the that may be transported to and as a full-length, transmembrane cell surface protein the that the precursors of and undergo at least intracellular cleavage constitutive secretion of mature NDF a and A similar was on Rat 1-EJ cells, which multiple isoforms of 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, D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google Scholar). to the of the precursor protein molecules in the cell is However, NDF to for NDF we for the of multiple and based on cloning of NDF from Rat 1-EJ cells, we several different of which may to the and isoforms in the and also are also these not predicted from the known and not the and isoforms the used well both a NDF is in cell of precursor a the of the is NDF to the for both the precursor and mature of NDF and NDF is of but in the of cells for not in conditioned to in Rat 1-EJ cells, appears to be to in transfected a portion of the precursor molecules are proteolytically cleaved secretion of mature NDF. However, of the the a to escape intracellular and may be as transmembrane, cell surface molecules We to the and of on intracellular and secreted NDF its cells transfected and expressing and as the and the and the two are at is to and that it one and as the protein not transported to the and to In at this it appears that is not to in is and at not to be to and may be that in the also of of the precursor at is to and is to both and is glycosylated at both and and is transported through where of the and addition of of the intracellular and secreted mature is shown in intracellular and secreted mature are and and and All mature NDF is to the and that the of secreted is derived from membrane-bound precursor molecules intracellular and of from and of cells expressing was in to shown for and glycosylation of and not the biosynthetic found these two isoforms was the of intracellular of the cDNAs and for D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. 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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, 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). recent however, that HER-2 to the related tyrosine HER-3 and 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 Vandlen R.L. R.A. J. Full Text PDF PubMed Google Scholar, M.X. G. Akita R.W. Nuijens R.A. Vandlen R.L. III, K.L. J. Full Text PDF PubMed Google Scholar, E. G. D. Peles E. S. Chang D. Liu N. Wen D. Yarden Y. J. Full Text PDF PubMed Google Scholar, J. Luo Y. Wen D. D. Hu S. Liu N. W.J. PubMed Scopus Google Scholar). least 15 different isoforms are in both and neuronal E. Yarden Y. 1993; PubMed Scopus Google for but are derived from a single D. J. D. D. Nature. 1993; PubMed Scopus Google Scholar). of these isoforms identified cloning based on D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google in ARIA and G. G.D. Cell. 1993; Full Text PDF PubMed Scopus Google and J. 1993; Full Text PDF PubMed Google tissue and multiple in that the of structural may as In this study we shown that isoforms undergo typical glycosylation and in Rat 1-EJ cells, which at least six different isoforms of and in transfected cells expressing individual isoforms two and multiple are to H.S. S. Trail G. D. S. Hu S. Wen D. J. 1995; of the and of the molecules undergo intracellular processing and mature NDF is secreted into the culture medium in Rat 1-EJ cells to a similar biosynthetic a significant portion of the molecules in transfected and Rat 1-EJ cells escape intracellular cleavage and are transported to and at the cell surface we found that cells expressing can be to proteolytically soluble mature NDF NDF/heregulin to a family of growth not a of other EGF family members but also of and J. 1993; PubMed Scopus Google transmembrane of members of this shown to to and through H.S. J. J. Lee Cell. Full Text PDF PubMed Scopus Google Scholar, R. R. Cell. Full Text PDF PubMed Scopus Google and factor N. Cell. Full Text PDF PubMed Scopus Google direct in of direct J. 1993; PubMed Scopus Google as it not a growth for a in is however, several lines of that these membrane-bound are and that may in of the of a growth factor is that of the in a of it the and transmembrane a in and activity in Cell. Full Text PDF PubMed Scopus Google Scholar, J. D.M. D. S. PubMed Scopus Google Scholar, J. Cell. 1992; PubMed Scopus Google Scholar, D. Hu S. D. Suggs S.V. S. 1992; PubMed Scopus Google Scholar). However, as the of the from and of the of the transmembrane of is for the of is a of other of the transmembrane as of intracellular cell surface may be for the of in from several in J. 1993; PubMed Google for the of the cell of the differentiation of the cell is the of the receptor tyrosine in and the of its transmembrane of in the cell and that of the of ligand and receptor We that is at the cell surface of transfected and native cells, the that may be in as is the for and of structural of the transmembrane of the NDF/heregulin family from to N. Yarden Y. PubMed Scopus Google also function for of this growth and differentiation are to direct for a for It is to that recent to the of that a transmembrane of is at the cell surface in the at on and G. function for the transmembrane isoforms of growth and differentiation as NDF/heregulin be to the of the soluble factor its from the cell We shown that can be cleaved and mature NDF a to that for several other growth J. 1993; PubMed Scopus Google processing of and a recent report that at the of the of is for regulated J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). of for the the of it however, that and function as the a of a and the a the however, a D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google but processing the of the to it is to that the least for the of processing may from that of acid the cleavage of a of transmembrane growth are and they show to J. 1993; PubMed Scopus Google Scholar). Lu H.S. S. Trail G. D. S. Hu S. Wen D. J. 1995; identified the cleavage for and and both to the cleavage of the other membrane-bound growth However, the NDF Lu H.S. S. Trail G. D. S. Hu S. Wen D. J. 1995; was not cleavage but was purified of NDF into the cell medium intracellular we on this the cleavage be shown that one processing is regulated a protein and J. J. 1993; PubMed Scopus Google Scholar). the of this are now a for direct phosphorylation of is phosphorylation of not be J. S. PubMed Scopus Google and of of the and in the of not J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). the of processing direct phosphorylation of protein be All of the different isoforms from Rat 1-EJ cells and in two protein phosphorylation D. Suggs S.V. D. Liu N. Cupples R.L. Luo Y. N. Lu H.S. Hu S. Chang D. D. Koski R.A. Yarden Y. Cell. PubMed Scopus Google Scholar, K.L. J. PubMed Scopus Google Scholar). is direct in of growth factor the in and that of a protein a in for the of growth for the of growth factor to its may and of the growth 1993; Google Scholar). of the NDF/heregulin family are also growth 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, G. G.D. Cell. 1993; Full Text PDF PubMed Scopus Google and a recent study shown that secreted ARIA is in to the its in and G. of the of NDF from the of the of cell growth to the of the of the growth factor be in intracellular of the be to its through a soluble is however, a recent report R. J. 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