Release of the Neuregulin Functional Polypeptide Requires Its Cytoplasmic Tail

Based on both in vivo and in vitro studies, we have shown previously that the intracellular domain of a membrane-bound isoform of the growth factor, neuregulin, regulates proteolytic release of its extracellular domain ErbB receptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfection in COS-7 cells. These studies revealed that regulation of extracellular domain cleavage by the intracellular domain is sequence-specific and involves three distinct 30–60-residue segments. The presence of any two of these three segments is both necessary and sufficient for proteolytic processing, and resistance to proteolysis is not due to an alteration in cellular localization or transport. Evidence was also obtained that regulation of extracellular domain processing involves initial intracellular domain dimerization. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Furthermore, resistance to proteolytic processing of a construct lacking a large portion of the intracellular domain was rescued with a chimera, in which the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain interactions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms. Based on both in vivo and in vitro studies, we have shown previously that the intracellular domain of a membrane-bound isoform of the growth factor, neuregulin, regulates proteolytic release of its extracellular domain ErbB receptor-activating ligand. To investigate the mechanism(s) involved in this regulation, a series of intracellular domain mutants were constructed and tested for susceptibility to proteolytic processing after transient transfection in COS-7 cells. These studies revealed that regulation of extracellular domain cleavage by the intracellular domain is sequence-specific and involves three distinct 30–60-residue segments. The presence of any two of these three segments is both necessary and sufficient for proteolytic processing, and resistance to proteolysis is not due to an alteration in cellular localization or transport. Evidence was also obtained that regulation of extracellular domain processing involves initial intracellular domain dimerization. Thus, with expression of a construct encoding only the intracellular domain, dimerization could be demonstrated in cross-linking experiments. Furthermore, resistance to proteolytic processing of a construct lacking a large portion of the intracellular domain was rescued with a chimera, in which the intracellular domain was replaced with a spontaneously dimerizing Fc fragment. Taken together these studies indicate that intracellular domain interactions are critically involved in the spacial and temporal control of growth and development by membrane-bound neuregulin isoforms. epidermal growth factor neu differentiation factor transforming growth factor polyacrylamide gel electrophoresis glutathione S-transferase neuregulin phosphate-buffered saline amino acids. Four members of the ErbB superfamily of receptor tyrosine kinases (also called HER) have been characterized extensively. erbB-1 (receptor for epidermal growth factor or EGFR)1 binds several distinct ligands containing an EGF-like domain and mediates proliferation and differentiation of normal cells (1Ullrich A. Coussens L. Hayflick J.S. Dull T.J. Gray A. Tam A.W. Lee J. Yarden Y. Libermann T.A. Schlessinger J. Downward J. Mayes E.L.V. Whittle N. Waterfield M.D. Seeberg P.H. Nature. 1984; 309: 418-425Crossref PubMed Scopus (1969) Google Scholar). The second member, ErbB-2, initially characterized as the protooncogene, neu, encodes a 185-kDa protein. This protooncogene is frequently overexpressed in various carcinomas and is associated with a poor prognosis (2Yamamoto T. Ikawa S. Akiyama T. Semba K. Nomura N. Miyajima N. Saito T. Toyoshima K. Nature. 1986; 319: 230-234Crossref PubMed Scopus (1053) Google Scholar). The other two members, ErbB-3 and ErbB-4, have been cloned subsequently and identified based on consensus protein-tyrosine kinase domains and homology with the other ErbB members (3Kraus 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 (660) Google Scholar, 4Plowman G.D. Culouscou J.M. Whitney G.S. Green J.M. Carlton G.W. Foy L. Neubauer M.G. Shoyab M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 1746-1750Crossref PubMed Scopus (674) Google Scholar). Neuregulins (NRGs) isolated from ras-transformed mouse fibroblasts and human breast cancer cells, originally called neu differentiation factor (NDF) or heregulin, were initially identified as putative ErbB receptor ligands based on their ability to stimulate tyrosine phosphorylation of ErbB-2 (5Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Levy R.B. Kiski R.A. Lu Hsieng S.L. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 6Holmes 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. et al.Science. 1992; 256: 1205-1210Crossref PubMed Scopus (921) Google Scholar). Three other neuregulin homologues were subsequently isolated from neural sources. One is acetylcholine receptor-inducing activity from chicken brain, which induces the synthesis of acetylcholine receptors in skeletal muscle (7Falls D.L. Rosen K.M. Corfas G. Lane W.S. Fischbach G.D. Cell. 1993; 72: 801-815Abstract Full Text PDF PubMed Scopus (544) Google Scholar). The other is glial growth factor from the bovine brain, which stimulates Schwann cell growth (8Marchionni M.A. Goodearl A.D. Chen M.S. Bermingham-McDonogh 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 (674) Google Scholar). The third is sensory and motor neuron-derived factor (9Ho W.H. Armanini M.P. Nuijens A. Phillips H.S. Osheroff P.L. J. Biol. Chem. 1995; 270: 14523-14532Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Despite their varied functions, all these homologues are derived from a single gene by alternate splicing or by use of several cell type-specific transcription initiation sites, and all contain an EGF-like domain required for receptor activation (8Marchionni M.A. Goodearl A.D. Chen M.S. Bermingham-McDonogh 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 (674) Google Scholar). Recent studies demonstrated that neuregulins bind directly to ErbB-3 and ErbB-4 and activate receptors through heterodimerization with ErbB-2 (10Pinkas-Kramarski R. Soussan L. Waterman H. Levkowitz G. Alroy I. Klapper L. Lavi S. Seger R. Ratzkin B.J. Sela M. Yarden Y. EMBO J. 1996; 15: 2452-2467Crossref PubMed Scopus (690) Google Scholar, 11Tzahar E. Waterman H. Chen X. Levkowitz G. Karunagaran D. Lavi S. Ratzkin B.J. Yarden Y. Mol. Cell. Biol. 1996; 16: 5276-5287Crossref PubMed Scopus (853) Google Scholar, 12Carraway III., K. Cantley L.C. Cell. 1994; 78: 5-8Abstract Full Text PDF PubMed Scopus (582) Google Scholar, 13Plowman G.D. Green J.M. Culouscou J.M. Carlton G.W. Rothwell V.M. Buckley S. Nature. 1993; 366: 473-475Crossref PubMed Scopus (435) Google Scholar, 14Carraway 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; 296: 14303-14306Google Scholar, 15Kita Y.A. Barf J. Luo Y. Wen D. Brankow D. Hu S. Liu N. Prigent S.A. Gullick W.J. Nelson M. FEBS Lett. 1994; 349: 139-143Crossref PubMed Scopus (68) Google Scholar). Gene inactivation experiments have demonstrated that neuregulins are essential for early heart and central nervous system development. Inactivation of both neuregulin alleles results in embryonic lethality due to maldevelopment of the heart. In these animals, the cardiac trabeculae fail to form normally, and there is a severe defect in endocardial cushion development. In addition, neuregulin null mice display defects of the nervous system including abnormal development of both Schwann cell precursors and cranial ganglia (16Meyer D. Birchmeier C. Nature. 1995; 378: 386-390Crossref PubMed Scopus (1036) Google Scholar, 17Kramer R. Bucay N. Kane D.J. Martin L.E. Tarpley J.E. Theill L.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4833-4838Crossref PubMed Scopus (188) Google Scholar). The varied biological functions mediated by neuregulins are likely related to differential tissue expression and structural diversity. At least 15 distinct isoforms are expressed in different tissues (18Ben-Baruch N. Yarden Y. Proc. Soc. Exp. Biol. Med. 1994; 206: 221-227Crossref PubMed Scopus (71) Google Scholar). They can be classified into two major groups based on their domain structures. One group is comprised of proteins that have an N-terminal signal peptide, but lack transmembrane and intracellular domains. Thus, these isoforms, which are largely expressed only in neural cells, are packaged presumably and subject to regulated release. The other group, expressed in both neural and mesenchymal cells, are membrane-associated. In addition to an extracellular segment (consisting of an Ig-like domain, a glycosylated region, and an EGF-like domain), these isoforms contain a transmembrane and intracellular domain. The extracellular domain of the membrane-associated forms can be proteolytically cleaved to release their ErbB receptor-activating functional peptide (5Wen D. Peles E. Cupples R. Suggs S.V. Bacus S.S. Luo Y. Trail G. Hu S. Silbiger S.M. Levy R.B. Kiski R.A. Lu Hsieng S.L. Yarden Y. Cell. 1992; 69: 559-572Abstract Full Text PDF PubMed Scopus (521) Google Scholar). Although some NRG isoforms, such as NDF-β4a, are more localized in the plasma membrane, others, such as NDF-α2c, show minimal plasma membrane localization. Both of these are processed predominantly in intracellular organelles (19Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Pulse-chase labeling studies indicate that processing occurs within 30 min of translation. Since the cleavage process is regulated in cultured cells by phorbol esters, cleavage may require a signaling event (19Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). In addition, it has been suggested that the efficiency of proteolytic cleavage is directly proportional to the length of the intracellular domain (20Wen 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 (231) Google Scholar), which varies among the different isoforms. Membrane-bound NRG isoforms are expressed in mesenchymal cells that are in juxtaposition to cells expressing ErbB receptors (21Marchionni M.A. Nature. 1995; 378: 334-335Crossref PubMed Scopus (60) Google Scholar). For example, in the early stage of the developing heart, the membrane-bound forms are expressed in the endocardial lining, whereas their cognate receptors are expressed in cardiac myocytes and in cells forming the endocardial cushion. This suggests that neuregulins function in a paracrine fashion during the early stages of heart development. We have found recently, in both in vivo and in vitro studies, that the NRG intracellular domain is essential for proteolytic release of the NRG extracellular domain and for determination of its subcellular localization (22Liu X. Hwang H. Cao L. Buchland M. Cunningham A. Chen J. Chien K.R. Graham R.M. Zhou M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13024-13029Crossref PubMed Scopus (81) Google Scholar). In this study genetic disruption of only the intracellular domain of the membrane-bound NRG isoforms resulted in a similar phenotype of embryo maldevelopment, to that observed previously with disruption of the entire gene. This indicates that the NRG intracellular domain is critical for NRG signaling and raises the following questions: (i) how do cells cleave the signaling peptide, and (ii) what processes regulate and trigger To the involved in these we have the of the in proteolytic series of mutants of the isoform were in which the intracellular domain was from the or segments of the intracellular domain were The were to membrane expression and release of the functional peptide into the after transient expression in COS-7 cells. The results of these studies indicate that segments of the intracellular domain of the NRG NDF-α2c, regulate through a process that likely involves initial intracellular (i) To construct mutants encoding proteins in which 30 were from the of the intracellular domain, the following were were based on the the of the intracellular domain segments and of (20Wen 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 (231) Google Scholar). Since various amino the of have been in the regulation of its proteolytic processing, all mutants were constructed with a for their of the intracellular of was with the encoding the of and of the as as the in the as a mutants were constructed (ii) were by the of the encoding the of its intracellular domain with a amino of or by the of the intracellular domain with a human Fc gene The gene was with with a and with as the The Fc gene was with with a and an encoding the was an of to or to the N-terminal of the intracellular domain of to expression of a intracellular domain The was for expression of the intracellular domain and the The was to the other were cloned and in the of the expression and by to COS-7 cells were obtained from the and were cultured in containing bovine were on and for 15 were with of the as previously J. T. Scholar). containing was to the bovine and the cells were for a breast cancer cells were cultured in with bovine by the the cells were for in cells were by with and and by 15 with a the The was for and the was in the and to a of this was with of in and for 30 distinct to the of the were These were and in of and the were in the was as previously J. T. plasma as previously Graham R.M. Mol. Google or that was initially through a and to by through a was the and 30 of were into extracellular domain or was as the a of and or with was as the second proteins were and the system The from COS-7 cells was through a and The was to of a containing human breast cancer cells in a the cells were with that and the and The cell were to with a with and were in and cultured for the cells with were with for 30 The cells were with in for 30 by with intracellular domain or for min with and was and for cells were with in and and were a and with a The and were into and expressed by with for of the for 15 the was in with and and for The was for 30 min and the The was to cross-linking with for and with for 15 min to the COS-7 cells, with were with into of with and for min to the for The was to The intracellular domain of is comprised of We have demonstrated previously that a which the entire intracellular domain the amino is to extracellular domain proteolytic To extracellular domain cleavage a of the domain or is on the length of the an construct was in which the were replaced by an of the This the construct NDF-α2c, and the previously construct were for cleavage after expression of construct in COS-7 cells. shown in cleavage of the resulted in the release of a extracellular domain into the Despite a of only for this peptide, as previously (19Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 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 (231) Google Scholar), results in its as a by the release of the peptide was not observed with expression of the two other This was not due to poor transfection or of the their expression could be by of cell In with these release of the ErbB receptor-activating from the construct could also not be in a functional release of the neuregulin from the construct was with this These indicate that a of the intracellular domain amino is involved in the proteolytic the of the expressed by the is with the of its peptide In studies with the of this was shown to be due to a lack of of the not The the NDF-α2c, was glycosylated as by the that it a that for its peptide Although expression of the mutants was in the cell studies were to their subcellular localization in more membrane expression of the and was from of plasma membrane labeling studies of cells that both the and proteins predominantly to the intracellular organelles in the plasma membrane intracellular localization was observed for all not Since the proteins expressed by the and are their intracellular localization is likely in the some expression in the be These are in with by et (19Burgess T.L. Ross S.L. Qian Y. Brankow D. Hu S. J. Biol. Chem. 1995; 270: 19188-19196Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), which membrane expression of with To the within the domain that are involved in a series of intracellular domain of was as shown in with the construct these mutants were by 30 amino acids. Thus, construct 30 and construct the of the intracellular domain. shown in the proteins expressed in cells with of the two and to the release of the functional peptide was In of the peptide by the or was only or Since expression of these proteins was by of cell it is likely that the various not transcription or or transfection Taken these that a segment is required for To more the segments of the involved in proteolytic processing, were in which the 30 the mutants and were but the of the was proteolytic Thus, the segment amino is required for cleavage the of the is also lacking as is cleavage was not observed with two other or based on in The only the amino whereas the the but the segment Thus, the and the are necessary for is an is also This is also from studies with two and In these the segment or the segment were to a construct Since cleavage was with both these the as required for cleavage can be to a segment only of only these 30 not Taken these studies indicate that two of the three and are both necessary and sufficient to extracellular domain To these segments regulate cleavage by cross-linking experiments were a construct encoding only the intracellular domain following expression in E. shown in after with a cross-linking a with the of the intracellular domain was by this was in not with the cross-linking The results were observed after expression of the in COS-7 cells and was observed with cross-linking cells were with of the that resulted in expression of the not These indicate that regulation of extracellular domain cleavage may initial dimerization. Although studies demonstrated that forms in vitro X. P. R.N. 1992; PubMed Scopus Google Scholar), this may not be the for the in the was not observed with expression of this construct To this a construct or the in the construct containing an N-terminal was expressed in E. and the expressed proteins to and and after shown in dimerization was with the the was to dimerization. To dimerization regulates NRG an construct was In this an Fc from human was to the of the which in the of the Fc not proteolytic cleavage of extracellular domain. Fc spontaneously through and dimerization can be due to shown in cleavage was observed with expression of the construct but not with the and in vitro by could be demonstrated only for the of the and growth J. Biol. PubMed Scopus Google Scholar). These are as proteins of an N-terminal signal peptide, a EGF-like domain, a transmembrane domain, and a domain. that activate their cognate receptors can be from the extracellular domains of these proteins by proteolytic Since can be by the activation of cellular signaling (20Wen 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 (231) Google Scholar, T. K. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. J. J. Biol. 1995; PubMed Scopus Google Scholar, A. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar), it is likely that and temporal control of proteolytic cleavage is an and for growth and development. In the of there is that the proteolytic cleavage from its membrane-bound is regulated by the in by the of the amino A. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, M.A. Lee Mol. Cell. Biol. Scopus (68) Google Scholar). In this we the of the intracellular domain of neuregulins in the regulation of extracellular domain cleavage by a series of which were characterized after transient expression in a cell Despite the of this it proteolytic release of the functional peptide from the neuregulin and has been to the of other proteins T. K. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, J. J. J. Biol. 1995; PubMed Scopus Google Scholar, A. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar, M.A. Lee Mol. Cell. Biol. Scopus (68) Google Scholar). Evidence that the domain of regulates proteolytic cleavage in a sequence-specific is the that the which an intracellular domain of the length as the was to proteolytic Furthermore, studies with mutants indicate that of the 30 or intracellular domain amino is of more proteolytic Thus, a of the intracellular domain amino and to be critical for proteolytic processing of membrane-bound neuregulin all a of the intracellular domain of in the release of its functional peptide from that of the presence of an amino to be critical for proteolytic cleavage A. J. Cell. 1992; Full Text PDF PubMed Scopus Google Scholar). This is not there are other and have a which is In addition to the two other and were subsequently shown to also be involved in any two of these three functional were shown to be sufficient for intracellular we also demonstrated that to proteolysis is not due to intracellular transport. Thus, extracellular proteolytic cleavage of NRG is regulated by its intracellular domain in a sequence-specific and may for the various NRG isoforms to of to different biological the results is membrane or intracellular is with any of the Since a signal signal is within the transmembrane domain), membrane or of is by the transmembrane domain and by a of the transmembrane domain. Since the to the it is that membrane and were with this or other In addition, the subcellular localization of both the and proteins suggests that for the of these We show that regulation of extracellular domain cleavage may initial intracellular domain dimerization. Thus, expressed the intracellular domains can be by with a cross-linking In addition, susceptibility to cleavage can be by a intracellular domain that Taken that the and temporal control of growth and development by membrane-bound neuregulin isoforms involves proteolytic cleavage of the extracellular domain and that this cleavage is regulated by initial intracellular domain dimerization. intracellular domain interactions may be by activation of cellular signaling to a signaling system to regulate proteolytic cleavage activation of ErbB We and for the human gene and human EGF-like domain protein. We also Martin and for