Proc. Natl. Acad. Sci. USAVol. 90, pp. 1746-1750, March 1993Biochemistry

Ligand-specifi'c activation of HER4/pl80erbB4, a fourth member ofthe epidermal growth factor receptor family

(receptor tyrosine kne/ERBB4 gene product)

GREGORY D. PLOWMAN, JEAN-MICHEL CULOUSCOU, GENA S. WHITNEY, JANELL M. GREEN,GARY W. CARLTON, LINDA FOY, MICHAEL G. NEUBAUER, AND MOHAMMED SHOYABBristol-Myers Squibb Pharmaceutical Research Institute, 3005 First Avenue, Seattle, WA 98121

Communicated by Hans Neurath, October 23, 1992

ABSTRACT This report describes the isolation and recom-bmiant expression of a cDNA clone encoding HER4, the fourthmember of the human epidermal growth factor receptor(EGFR) family. The HER4/erbB4 gene encodes a 180-kDatransmembrane tyrosine kinase (HER4/p18WbB) whose ex-traceilular domain is most similar to the orphan receptorHER3/pl6OerbD3, whereas its cytoplasmic kinase domain ex-hibits 79% and 77% identity withEGFR and HER2/pj85erbB2,respectively. HER4 is most predominaudy expressed in severalbreast carcinoma cell lines, and in normal skeletal muscle,heart, pituitary, brain, and cerebellum. In addition, we de-scribe the partial purification of a heparin-binding HER4-stimulatory factor from HepG2 cells. This protein was found tospecifically stimulate the intrinsic tyrosine kinase activity ofHER4/plW'rbB4 while having no direct effect on the phos-phorylation of EGFR, HER2, or HER3. Furthermore, thisheparin-binding protein induces phenotypic differentiation,and tyrosine phosphorylation, of a human mammary tumorcell line that overexpresses both HER4 and HER2. Thesefindings suggest that this ligand-receptor interaction may playa role in the growth and differentiation of some normal andtransformed cells.

Transmembrane receptors that contain a cytoplasmic tyro-sine kinase domain are ofparticular interest to developmentaland tumor biologists, since their activation often initiates acascade of events leading to cell growth and differentiation(1). Included in this group are the receptors for polypeptidegrowth factors such as epidermal growth factor (EGF),insulin, platelet-derived growth factor, fibroblast growthfactor, and neurotrophins. Recently, the ligands for several"orphan" receptors have been identified, including those forc-kit (steel factor), met (hepatocyte growth factor), trk (neu-rotrophins), and HER2 (heregulin) (1-3). However, numer-ous receptor tyrosine kinases (RTKs) have been isolated,including eph, eck, elk, ret, and HER3 (1, 4), for which aligand has not yet been identified.The EGF receptor (EGFR) family is one group of RTKs

that is frequently overexpressed in a variety of aggressiveepithelial carcinomas and comprises three members: EGFR,HER2/pe85rIbB2, and HER3/pl60erbB3 (4-6). Increased ex-pression of EGFR has been associated with more aggressivecancers of the breast, bladder, lung, and stomach (7); am-plification and overexpression of HER2 have been reportedfor breast and ovarian carcinoma, where high levels ofHER2directly correlate with a poor clinical prognosis (7, 8); andHER3 expression is amplified in a variety of human adeno-carcinomas (9). While several structurally related polypep-tides have been identified that specifically bind to the EGFR,including EGF, transforming growth factor a (TGF-a), am-

phiregulin, heparin-binding EGF, and vaccinia virus growthfactor (for review see refs. 7 and 10), none ofthese ligands hasbeen shown to interact with HER2 or HER3. However,several groups have recently reported the identification ofcandidate ligands for HER2 (2, 3, 11-13). Elucidation of theprimary structure of one of these molecules, heregulin/Neudifferentiation factor (NDF), reveals it to be an additionalmember of the EGF family (2, 3). Due to the biolbgicalimportance of this extended family of ligands and receptors,we have continued our search for additional homologues.This report describes the cloning and expression of a fourthmember of the EGFR family, HER4.*

MATERIALS AND METHODSMolecular Cloning. Several pools of degenerate oligonu-

cleotides were synthesized on the basis of conserved se-quences from EGFR family members. Total genomic DNAwas isolated from murine K1735 melanoma cells and used asa template with these oligonucleotide primers in a 40-cyclepolymerase chain reaction (PCR) amplification (4). Using thedegenerate oligonucleotides H4TVWELM and H4VYMIIL,we identified one clone (MER4-85) that contained a 144-ntinsert corresponding to murine erbB4. This 32P-labeled insertwas used to isolate a 17-kb fragment from a murine T-cellgenomic library (Stratagene) that was found to contain twoexons of the murine erbB4 gene. A specific oligonucleotide(4M3070) was synthesized on the basis of the DNA sequenceof an erbB4 exon and used in a PCR protocol with adegenerate 5'-oligonucleotide (H4PIKWMA) on a templateof single-stranded MDA-MB-453 cDNA. This reaction gen-erated a 260-nt fragment (pMDAPIK) corresponding to hu-man HER4. cDNA libraries were constructed in A ZAP II(Stratagene) from oligo(dT)- and specific-primed MDA-MB-453 and human heart RNA (4, 10). HER4-specific clones wereisolated by probing the libraries with the 32P-labeled insertfrom pMDAPIK. To complete the cloning ofthe 5' portion ofHER4, we used a PCR strategy to allow for rapid amplifica-tion of cDNA ends (4). All cDNA clones and several PCR-generated clones were sequenced on both strands, using T7polymerase with oligonucleotide primers.The oligonucleotide mixtures (including their degeneracy,

corresponding amino acid residues, and orientation) whichwere used for PCR are as follows:H4TVWELM 5'-ACNGTNTGGGARYTNAYHAC-3'

(256-fold, TVWELMT, sense)H4VYMIIL 5'-ACAYTTNARDATDATCATRTANAC-3'

(576-fold, VYMIILK, antisense)

Abbreviations: EGF, epidermal growth factor; EGFR, EGF recep-tor; RTK, receptor tyrosine kinase; mAb, monoclonal antibody;TGF-a, transforming growth factor a.*The sequence reported in this paper has been deposited in theGenBank data base (accession no. L07868).

1746

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 90 (1993) 1747

H4PIKWMA 5'-GACGAATTCCNATHAARTGGATGGC(48-fold, PIKWMA, sense)

4M3070 5'-CTGCTGTCAGCATCGATCAT-3' (antisense).Degenerate residues are: D = A, G, or T; H = A, C, or T; N= A, C, G, or T; R = A or G; and Y = C or T.Assays of Tyrosine Kinase Stimulatory Activity. A panel of

four recombinant cell lines, each of which expresses only asingle member of the human EGFR family, was generated.The complete 4-kb HER4 coding sequence was reconstructedand inserted into a glutamine synthetase expression vector,pEE14 (14). The resulting construct (pEE14HER4) wastransfected into CHO-KI cells, and a stable cell line (CHO/HER4 #3) expressing high levels of recombinant humanp180erbB4 was selected by immunoblotting solubilized cellextracts with a sheep polyclonal antipeptide antibody againstHER2 residues 929-947 (Cambridge Research Biochemicals,Valley Stream, NY). CHO/HER2 cells (clone 1-2500) wereselected to express high levels of recombinant humanp185eItB2 by dihydrofolate reductase-induced gene amplifi-cation in dhfr-deficient CHO cells. NRHER5 cells (15) ex-pressing =106 human EGFRs per cell were obtained fromHsing-Jien Kung (Case Western Reserve University, Cleve-land). 293/HER3 cells were selected to express high levels ofhuman p160erbB3 (16).

Cells were allowed to attach in six-well tissue culture plates(Falcon) at 37°C for 18-24 hr and then incubated in serum-free medium for at least 1 hr prior to addition of the indicatedamounts of ligand preparations. After treatment with ligandfor 10 min at room temperature, cells were solubilized (17)and the cleared supernatants were allowed to react with 1 iLgof murine monoclonal antibody (mAb) to phosphotyrosine(PY20, ICN) for CHO/HER4 and 293/HER3 cells, or 1 ,ug ofmurine mAb to HER2 (Neu-Ab3, Oncogene Sciences, Man-hasset, NY) for CHO/HER2 cells, or 1 ,ug of mouse mAb Rlto the human EGFR (Amersham) for NRHER5 cells. Anti-mouse IgG-agarose (for PY20 and Neu-Ab3 mAbs) or staph-ylococcal protein A-Sepharose (for EGFR-R1 mAb) wasused to precipitate the immune complexes. Proteins wereseparated by SDS/7% PAGE and analyzed by immunoblot-ting with PY20 mAb essentially as described (12), exceptphosphoproteins were detected with 125I-labeled goat anti-mouse IgG F(ab')2 and exposure on a Molecular Dynamicsphosphorimager.

Cell Differentiation Assay. MDA-MB-453 cells (ATCC)were grown in Dulbecco's modified Eagle's medium(DMEM) supplemented with 5% fetal bovine serum and 1 xessential amino acids. Cells (7500 per well) were allowed toadhere to 96-well plates for 24 hr. Samples were diluted in theabove medium and added to the cell monolayer in 50 ul finalvolume, and the incubation was continued for an additional3 days. Cells were then examined by inverted light micros-copy for morphologic changes.

Protein Purification. HepG2 cells (ATCC) were cultured inDMEM containing 10% fetal bovine serum. Ten liters ofserum-free conditioned medium (HepG2-CM) was cleared bycentrifugation, concentrated 16-fold by using an Amiconultrafiltration unit (10,000 molecular weight cutoff), andprecipitated with 2 M ammonium sulfate. The supernatantwas dialyzed against phosphate-buffered saline (PBS) andpassed through a DEAE-Sepharose (Pharmacia) column. Theflow-through fraction was then applied onto a 4-ml heparin-acrylic (Bio-Rad) column. The MDA-MB-453 cell differenti-ation assay was used throughout the purification procedure tomonitor the column fractions. Active fractions, which elutedfrom the heparin column between 0.4 and 0.8 M NaCl, were

precipitated with 2.0 M ammonium sulfate, and the resultingsupernatant was loaded onto a phenyl-5PW column (8 x 75mm, Waters). Bound proteins were eluted by using a 2.0-0.0

M ammonium sulfate gradient in 0.1 M Na2HPO4, pH 7.4.Dialyzed fractions were assayed for tyrosine phosphoryla-tion of MDA-MB-453 cells, essentially as described (2).

RESULTSIsolation ofcDNA Clones Encoding a Fourth Member of the

Human EGFR Gene Family. EGFR and the related proteinsHER2, HER3, and Xmrk exhibit extensive amino acid ho-mology in their tyrosine kinase domains (4-6, 18). In addi-tion, there is strict conservation of the exon-intron bound-aries within the genomic regions that encode these catalyticdomains (ref. 5 and unpublished observations). Degenerateoligonucleotide primers were designed on the basis of con-served amino acids encoded by a single exon or by adjacentexons from the kinase domains of these four proteins. Theseprimers were used in a PCR protocol to isolate genomicfagments corresponding to murine EGFR, erbB2 and erbB3.In addition, a highly related DNA fragment (designatedMER4) was identified as distinct from these other genes. Asimilar strategy was used to obtain a cDNA clone corre-sponding to the human homologue of MER4 from the breastcancer cell line MDA-MB-453. Using this fragment as aprobe, we found several breast cancer cell lines and humanheart to be an abundant source of the EGFR-related tran-script. cDNA libraries were constructed by using RNA fromhuman heart and MDA-MB-453 cells, and overlapping clonesspanning the complete open reading frame of HER4/erbB4were isolated.The assembled HER4 cDNA sequence contains a single

open reading frame that encodes 1308 amino acids. Thecoding region is flanked by a 33-nt 5'-untranslated region anda 1517-nt 3'-untranslated region ending with a poly(A) tail. A25-amino acid hydrophobic signal sequence follows a con-sensus initiating methionine at position 34. Removal of thissignal sequence would give a mature protein beginning atGln-26 followed by 1283 amino acids with a calculated Mr of144,260 (Fig. 1).

Structural Analysis of HER4. HER4 has all the structuralfeatures of the EGFR family of RTKs (19). Excluding theN-terminal signal sequence, the HER4 protein contains asingle hydrophobic stretch of 26 amino acids that is charac-teristic of a transmembrane region and bisects the proteininto a 625-residue extracellular ligand binding domain and a633-residue C-terminal cytoplasmic domain. The ligand bind-ing domain can be further divided into four subdomains(I-IV), including two cysteine-rich regions (II, residues 186-334; and IV, residues 496-633) and two flanking domains (I,residues 29-185; and III, residues 335-495). This organiza-tion is similar to that of EGFR, where domains I and III havebeen implicated to define specificity for ligand binding (ref.20; Fig. 1). The extracellular domain of HER4 is most similarto that of HER3: domains II-IV of HER4 share 56-67%identity to the respective domains of HER3, while the sameregions of EGFR and HER2 exhibit 43-51% and 34-46%identity to HER4, respectively (Fig. 1). In contrast, the fourextracellular subdomains of EGFR and HER2 share 39-50%identity. HER4 also conserves all 50 cysteines present in theextracellular portion of EGFR, HER2, and HER3, exceptthat the HER2 protein lacks the fourth cysteine in domain IV(4-6). There are 11 potential N-linked glycosylation sites inHER4, conserving 4 of 12 potential sites in EGFR, 3 of 8 sitesin HER2, and 4 of 10 sites in HER3.

Following the transmembrane domain of HER4 is a cyto-plasmicjuxtamembrane region of 37 amino acids. This regionshares the highest degree of homology with EGFR (73%amino acid identity) and contains one potential protein kinaseC phosphorylation site, Ser-679, that is not likely to be useddue to its close proximity to the transmembrane domain.Notably, HER4 lacks a site analogous to Thr-654 in the

Biochemistry: Plowman et al.

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1748 Biochemistry: Plowman et aL Proc. Nati. Acad. Sci. USA 90 (1993)

HER4 1 MKPA----TGLW4VWVSLLVAAGTVQPSDSQS CGTENKLSSLSDLEQQYRALRKYYENCEVVMGN4LEITSIEHNRDLSFLRSVREVTGYVLVALNQFRYEGFR -24 MR.SGTAGAA.LA.LLAA.CP.S--RALEEKK. .Q.. S...TQ.GTF.DH8LS.QRMFN....L....YVQR.Y....KTIQ.A.A...I.2...TVERHER2 1 M---ELAALCR.GLLLA.LPP.AA----.TQ. .T..GM. .RLPASP.THLGM. .HL.QG.Q.. .0... .L.YLPT.AS.... .QDI0.. .0... I.H.4..V.QHER3 -19 MRAND--ALQVLGLLFS.ARGSE.--GN. .A. .P. .L.G. .VTG.A.N. .QT.Y.L. .R......VLTG. .A...01.I......M.E.ST

HER4 97 LPLENLRIIRGTKLYEDRYALAIFLN------YRKDGNFGLQELGLKNLTEILN4GGVYVDQN4KFLCYADTIHWQDIVRNPWPSNLTLVSTNGSS~EGFR 75 I.....Q.... MY..NS. .... VLS.-------DANKT-. .K..PMR.Q.0...H.A.RFSN4.PA. .NVES.Q.R....SSDFL. .MSMDF-Q.HLG .Q14ER2 94 V..QRR... V... Q.F..N.N. .. VLD.GDPLNNTTPVTGASPG. .R. .Q.RS....K....LIQR.PQ .. Q.0...L.K. .FHKNN141A. .. .ID. .R. .HHER3 78 .. P....VV .. .QV.DGKF.IFVM.------- NTNSSH1A.RQ.R.TQ...S.....IEK.DK..HM4M.. .D.R. .....DR---DAEI.VKDN4GR .P

188165194166

287265293265

364393361

462491461

582560589559

680649680651

778748780750

8781DI GmiWLCHRFHSVSGTWLTVKYGPRIDLKELOPCIVMMKWDDRKKLAFR848880850

K.DP. .PN.S.. .AG.EN. .K..KII..Q.SS...R.KSP....NQ. .A. .T..RES. .LV.RK.R.EAT.KDT. .PLM4L....Y.MIDV.PEG. .SF.P.SPM.KGS. ... ESSED..S.... GGA-. .K..LPT. ... EQ..A..T. .. HS..L..LH.1.14..1..I.ELH..ALVT. .TD. .ESMP.PEGR. .F.P. .EV.K-.....OSED....K.I..P..N4.H.F..NPNQ.. .D.... ...Q....RH.....PR....PL... KL ...P.PHT. .Q..

.T....R.Y. .T.HG.G...ADSY.M. -.D.VRK. .K.EGP.R.V4. ...I.EEKDSLSI14AT. .K.K....S.S.D.HILPVAFR. .SF'THTPPL.S..TA. .Y.YLST.VG. .TLV. .LHNQ.VTA.D.TQR.EK.SKP.ARV]. L.MEH4.REVRA.T.A. .QE.AG.KK.F.S.A. .PESFD. .. .ASNTAPLGV..AS... -.Q..T.....PD.....D-K..L. .. .E.CGOL.....T.S. .--RF......G.V ...L....D. .I.1.1N...WHK.P.L

.QE.DILK. .K....L..A. .E.R. .LHA.E. .EI.R. .TKQH.QFS.-AVVSLN4 .... GLR....D.DVI.SG.K ... AN.A1....KK. .G.S-GQ. .Q.Q. .E.LE .....Y.Y.SA. .DSLP.L. .. .Q..QV.R. .I.HN4.AYS.-T.QGL. .S98.GLR. .R.LGS.LAL.HH.TH. .EV..VP.DQ. .RNP-H4.........Y.....H.HN.....T....S. .NRGFS.L.M.NLN4V .. .G.R.....R....SA.RQ. .. .HHSL. .. .KVLRGPTE

.KTK.IS. .GEH Kw.T.Q6. HA... PE....E.RD.V. .. .NVS. .. .E.VDK.K.LE.P.P...VEN.E.IQ.H.E.--LPQANNI. .T.R....IQ.A.ALLHTA. .PED .VG. .LA.HQ..ARRALL.S. .T. .VN4.SQ.L. .QE.V.E.RVLQ.LP. .YV.ARH.LP.H.E.Q--P01NGSV. .F..EA.Q.VA.AE.LD.KH. .PRRV.V...K..DP. .....G.....G....NY.. .GV.VTH. .FLN.1.P. .. .AHE.AE.FS.H.E.QP.--.GTA. .N.S.S.T.AQ.A

.YI. .. .H..KT. .A.VM.E.NTL-VW. .....AHV.L......Y..T. .GLEG.P------TNGPKI.S. .T.MV.A.LL.LV.A.GIGL8..RH

.Y. .P.F..AR. .S.VKPDL.YMP.WKFP.EEGA.Q..PP... .HS.VDLDDKG.P-----AEQRkASPLTS.VSA.V.-ILLV.VL.VV.GILIK.RQ.R. . .4.H.SS. .H.VL. .KG--P.Y. .P.VN..R...E.E....K..ELQ.L.-----QTLVLIGKTHLTM4.LT. .A. .VVI9M-.GGTPLYW.GR

1 e00O

.V-R. .T....L.Q.R ......E. 1.L.. .KI........L.I.....K.....ELR.A.S.....K.IL. .. .YV .. .V.NQ.IRYTM. .L.Q .......AM.......M......RK..........I.D..N.....V.R.N4.S .... K.IL. .. .YV. GVGSRIQN. ..4.M.Y.ERG.SI. .. .D..-EKA.KV.A.. ....RKL....V.... H.1.V.I ... SI .... C. .VIEDKS.RQSFQAVT.HM.AIG.L..

..VC.... .1..TS.V.I....F.....D..R......Y..........D.........T.Q......K..GAE.... H.1.)VS. .. ..I.TS.V......Y ... DH.R.NRGRL....D.....M...S... DV....... ..........DI. .T..14.A.I....L.PGSSL....YL.L.S. .DH.RQ.RGAL.P....GV....Y.... H1GM... N.......L...SQ.QVA....V.D. .PP.D.QLLY

E.. .V.....S.LH.IY .......V.....S.....AS. .SSI...........I.R.......R..II...K...V.....S.LR.R........V.....A.....A..............I.....SEC. .R.R..VS...

SPAT.....S.. MGY .......V.....E..A.LRLA.V ......A...0.........ENIR.T.....N.T..V V V V

948 ...... . .14...-.. .T. .N.YRA.M. .....MD.VV..D. ..I.QG.8SS.S--..---------------------980 .... FV... E.-LGP-A. .L...T.YRS. .EDD.M4G.LV......QG.FC.D.APGAGGK4VHHRHRKSSSTRSGGGDL ---------TL950 ... ... SGPGIA.G.EPHGLTNKK.E.VE. .PEL. LDLD.EAEEDN4LKTTTLGSALSLPVGTLNRKPRGSQSLLSPSSGYMPMN4QGN4LGGSCQE

V V V ~~~~~~~~~~~~VL076 GVSVPYRAPTSTIPEAPVA--QGATAEIF8DDSCCNGTLRKPVAPH4VQEDSSTQRYSADPTVFAPERSPRGELDEEGYMTPM4RDKPKQEYL14PVEEN4PEVSL008---------T.LLSSLS. .SN--NSTVACIDRNGLQSCPIK. .. FL1.... S....GALT.D.I----DDTL-----VP. .I.QS----L062 ----GLEPSEEEA.RS.L.PSE. .GSDV. .GDLGM.AAKGLQSLPTHDP.PL .... E.... PL.S-----ETD. .VA.LTCS.QP. .V.QPDVR.QPPL050 SAVSGSSERCPRPVSLHPMPRGCLASESSEGHVTGSEAELQEKVSMCRSRSRSRSPRPRGDSAYHSQRHSLLTPVTPLSPPGLEEEDVN4GYVMPDTHLKG

V V VVVv V VV

1075 K.PA.SV---Q.VV...QPLN.APS----RD.H4.QDPHSTAV.N4P... .NT---VQPTCVN4STFDSP ----H.AQKGSHQISLDN4 .. .Q.DFFP.E.A-1151 SPRE. P. P.AKPAGATLERAKTLSPGKNG.VKD3VF--A. GGAVENP....TPQGGAAPQPHP.PAFSP.... LY. .DQDP.EP. GAPPST------1150 TSRGLSGSVGEEEEYYNRRSPPRSLEGEMVSLALSQCLPPMTGTDDEMRR

HER4 1269

EG8K 1158

HER2 1237

HER3 1250

v v

KQNGRIRPI-VAENPEYLSEFSLKPGTVLPPPPYRHRNTVV.P. .IFKGS-T. ...A... RVAPQSSEFIGA

FKGTPT.....GLDVPV

1308

1186

1255

1323

FiG. 1. Deducedamino acid se-quence of human HER4 and align-ment with other human EGFRfamily members. Sequences aredisplayed in the single-letter codeand are numbered at left. Identicalresidues are denoted with dots,gaps (introduced for optialaign-ment) are represented by hyphens,cysteine residues are marked withasterisks, and N-linked glycosyla-

IVtion sites are denoted with plus

IV signs. Potential sites of phosphor-ylaton by protein kinase C (Ser-679) or mitogen-activated protein

Tm kinase (Thr-699) are shown witharrows. An additional arrow atAla-685 marks the location of themajor site of protein kinase C-in-duced phosphorylation of EGFR.The predicted ATP-binding site isshown with four circled crosses,

TK C-terminal tyrosines are denotedwith open triangles, and tyrosinesin HER4 that are conserved withthe four major autophosphoryla-tion sites in the EGFR are indi-cated with filled triangles. The pre-dicted extraceilular domain ex-tends fr-om the boundary of thesignal sequence marked by an ar-row at position 25 to the hydropho-bic transmembrane domain whichis overlined from position 650 toposition 675. Various subdomainsare labeled on the right: 1,11, mI,and IV = extraceilular subdomainswith II and IV being cysteine-rich;TM = transmembrane domain;and TK = tyrosine kinase domain.Domains I, HI, and TK are boxed.

EGFR, which is its major -site of protein kinase C-induced

phosphorylation. Phosphorylation at this residue in the

EGFR appears to block ligand-induced internaliztion and

plays an important role in its transmembrane signaling (21).

HER4 also conserves Thr-699 with Thr-669 in EGFR, which

is its major EGF-stimulated mitogen-activated protein kinase

phosphorylation site (22).

The remaining cytoplasmic portion of HER4 consists of

three domains: a 276-amino acid tyrosine kinase domain, an

acidic helical structure of 38 amino acids that is homologous

to a domain required for ligand-induced internalization of the

EGER (23) and contains a single conserved ty'rosine residue,

and a 282-amino acid region containin~g 18 tyrosine residues

char'acteristic of the autophosphorylation domains of other

EGFR-related proteins (Fig. 1). The 276-amino acid domain

conserves all the diagnostic structural motifs of a tyros'inekinase (19) and is most related to the catalytic domains of

EGFR (79% identity) and HER2 (77% identity), and to a

lesser degree, HER3 (63% identity). In this same region,

EGER and HER2 share 83% identity. The C-terminal 282

amino acids ofHER4 has limited homology with HER2 (27%o)and EGFR (19%). However, the C-terminal domain of each

receptor is proline-rich and conserves stretches of 2-7 amino

acids that are generally centered on a tyros'ine residue. These

residues include the major tyrosine autophosphorylation

sites of EGFR at residues 1068, 1086, 1148, and 1173 (Fig. 1,

filled triangles; ref. 24).

The HER4 sequence was unique compared (October 1992)

with sequences the available DNA sequence data bases,

but a search of the protein data bases revealed a stretch of

60/64 amino acid identity with HER2 and a 54/54 amino acid

identity with an entry termed tyro-2. This entry was deduced

from the nucleotide sequence of a small PCR-generated

fr-agment of a rat protein-tyrosine kinase gene (25).

Several related cDNAs were also discovered from the

MDA-MB-453 library that were identical to the consensus

HER4 sequence but diverged at the 5' or 3' ends. These

cDNA variants comprised two forms: one encodes a tras-

membrane HER4 with the deletion of its C-terminal auto-

phosphorylation domain and the second encodes only a

portion of the HER4 cytoplasmic domain.

Tissue Distribution of HER4 Transcripts. Northern blots of

poly(A)+ mRNA from human tissues were hybridized with

antisense RNA probe to the 3' end of HER4. An --6-kb

mRNA transcript was most abundant in the heart and skeletal

muscle (Fig. 2), while an mRNA of greater than 15 kb was

detected in the braini, with lower levels in heart, skeletal

muscle, kidney, and pancreas. To confirm and extend the

HER4 ex'pression profile, we performed quantitative reverse

transcriptase PCR, using primers from sequences in the

HER4EG8KHER2HER3

14ER4EGFRHER2HER3

HER4EGFKHER2HER3

HER4EGFR14ER2HER3

HER4EG8RHER2HER3

HER4EG8RHER2HER3

HER4EGFRHER2HER3

HER4EG8RHER2HER3

HER4EGFRHER2HER3

HER4EGFRHER2HER3

HER4EG8RHER2HER3

1111

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Proc. Natl. Acad. Sci. USA 90 (1993) 1749

1 2 3 4 5 6 7 8

9.5-75- FIG. 2. Northern blot analysis

IP* ~~~of mRNA from human tissues hy-

4r.; 4- ii bridized to a 3' HER4-specific

24- [a-~~Zi 32p]UTP-labeled antisenseRNA probe. RNA size markers (in

1.35- 1:8arkb)ae shown on the left. Lanes1-8 represent 2 ;g of poly(A)+mRNA from pancreas, kidney,skeletal muscle, liver, lung, pla-

centa, brain, and heart, respec-tively.

HER4 kinase domain. As observed by Northern analysis,brain, heart, and kidney express the highest levels ofHER4transcripts, in addition to parathyroid, cerebellum, pituitary,spleen, testis, and breast. Lower levels were found in thy-mus, lung, salivary gland, and pancreas, and low or unde-tectable expression was found in liver, prostate, ovary,

adrenal, colon, duodenum, epidermis, and bone marrow.Various human cell lines were also examined by PCR anal-ysis, revealing the highest expression ofHER4 RNA in fourmammary adenocarcinoma cell lines (T-47D, MDA-MB-453,BT-474, and H3396) and in neuroblastoma (SK-N-MC) andpancreatic carcinoma (Hs766T) cell lines. Intermediate ex-pression was detected in three additional mammary carci-noma cell lines (MCF-7, MDA-MB-330, and MDA-MB-361).Low or undetectable expression was found in other cell linesderived from carcinomas ofthe breast (MDB-MB-231, MDA-MB-157, MDA-MB-468, and SK-BR-3), kidney (Caki-1,Caki-2, and G-401), liver (SK-HEP-1 and HepG2), pancreas(PANC-1, AsPC-1, and Capan-1), colon (HT-29), cervix(CaSki), vulva (A-431), ovary (PA-1 and Caov-3), melanoma(SK-MEL-28), or in a variety of leukemic cell lines.Recombinant Expression of the HER4 Kinase. To determine

the binding characteristics of HER4, we stably overex-pressed its complete coding sequence in CHO-KI cells.These cells lack any detectable EGFR, HER2, or HER3 byimmunoblot, tyrosine phosphorylation, and immunoprecipi-tation analysis of 35S-labeled proteins (data not shown). TheHER4 protein was detected by immunoblot analysis onsolubilized cells or membrane preparations by using anantiserum generated to a 19-amino acid region of the HER2kinase domain, which coincidentally is identical to the HER4sequence (residues 927-945). The recombinant HER4 mi-grated with an apparent Mr of 180,000, slightly faster thanHER2, while the parental CHO cells showed no cross-reactive bands (data not shown). These cells were then usedto assess ligand-specific binding and autophosphorylation ofthe HER4 tyrosine kinase. EGF, TGF-a, and amphiregulinare three related ligands whose growth-regulatory signals aremediated in part by their interaction with EGFR (7, 10, 17).All three ligands stimulated tyrosine phosphorylation ofEGFR (for EGF, see Fig. 3C, lane 2), but not of HER4,HER2, or HER3 (Fig. 3 A, B, and D, lanes 2 and 3). On thebasis of the structural homologies between EGFR familymembers and EGFR, we presumed these orphan receptorsmay bind yet-unidentified ligands.

Identification and Partial Purification of a Ligand ThatSpecifically Activates the HER4 Kinase. In an effort to identifyligands specific for HER2, HER3, or HER4, we took advan-tage ofthe receptor expression profile ofMDA-MB-453 cells.This cell line expresses HER2 and HER3 but contains nodetectable EGFR (4, 26). In addition, HER4 cDNA was firstisolated from this cell line. Serum-free conditioned mediafrom numerous human cancer cells were screened for

growth-regulatory activity on MDA-MB-453 cells, andHepG2 human hepatocarcinoma cells were found to secrete

a 1 2345221 w*S.,.4HER 41 2 3 4 S

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221- i - -EGFR

D 1 245221-ekt It--HER3

FIG. 3. Specific activation of HER4 tyrosine kinase by a breastcancer differentiation factor. Four recombinant cell lines were de-veloped that each overexpress a single member of the EGFR familyof tyrosine kinase receptors (EGFR, HER2, HER3, and HER4).These cells were then stimulated with various ligand preparationsand assayed for receptor tyrosine phosphorylation. The cell linesinclude the following: A, CHO/HER4 #3 cells; B, CHO/HER2 cells;C, NRHER5 cells; and D, 293/HER3 cells. Lane 1, buffer control;lane 2, EGF at 100 ng/ml; lane 3, amphiregulin at 200 ng/ml; lane 4,10 ,ul of phenyl-5PW column fraction 17; lane 5, 10 dL1 of phenyl-5PWcolumn fraction 14. The sizes (in kDa) of the prestained molecularweight markers are indicated on the left of each gel. The phosphor-ylated receptor in each series migrates just below the 221-kDamarker.

a factor(s) that induced a morphologic differentiation in thesebreast cancer cells (Fig. 4). Whereas untreated MDA-MB-453cells are moderately adherent, with a rounded morphology(Fig. 4A), the addition of semipurified material induced thecells to display a flat morphology with larger nuclei andincreased cytoplasm (Fig. 4B and C) and had minimal effectson cell growth. This differentiation factor(s) binds to heparin(data not shown) and elutes from a phenyl-5PW column at 1.0M ammonium sulfate (fractions 16-18, Fig. 4D). Activefractions were also found to stimulate tyrosine phosphory-lation of a 185-kDa protein in MDA-MB-453 cells (Fig. 4E);fraction 16 induced a 4.5-fold increase compared with theunstimulated control (Fig. 4F). This activity was then testedagainst a panel of cell lines that each overexpress a singlemember of the EGFR-family. Fraction 17 induced a signifi-cant and specific activation ofthe 180-kDaHER4 kinase (Fig.3A, lane 4) without directly affecting the phosphorylation ofHER2, EGFR, or HER3 (Fig. 3 B-D, lane 4). An adjacentfraction from the phenyl-5PW column (fraction 14) was usedas a control and had no effect on the phosphorylation of anyof these receptors (Fig. 3, lane 5).

DISCUSSIONThis report describes the cloning and expression ofHER4, anadditional member of the EGFR family whose extracellulardomain is most similar to HER3, whereas its cytoplasmickinase domain is strikingly related to EGFR and HER2. Wealso describe the partial purification of a heparin-bindingfactor that specifically stimulates the intrinsic tyrosine kinaseactivity of this receptor while having no direct effect onEGFR, HER2, or HER3. In contrast, several ligands thatbind the EGFR fail to interact with HER4. Owing to ubiq-uitous expression ofEGFR, the presence ofnumerous EGFRligands, and the recognition that crosstalk exists betweenrelated RTKs, we elected to set up assay systems that wouldpermit us to look at specific interactions with each of thesereceptors in absence of crosstalk. For screening potentialligands, we have generated recombinant cell lines that eachexpress only one member of the EGFR family. The heparin-binding factor described in this report activates only the cellline containing HER4 and not the parental CHO cells or the

Biochemistry: Plowman et al.

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1750 Biochemistry: Plowman et al.

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FIG. 4. Biological and biochemical properties of the MDA-MB-453-cell differentiation activity. (A-C) Induction of MDA-MB-453differentiation. Protein in conditioned medium from HepG2 cells wascleared by precipitation with ammonium sulfate and the supernatantwas dialyzed prior to being added to cells in the following amounts:A, control; B, 80 ng per well; C, 2.0 ,ug per well. (D) Phenyl-5PWcolumn elution profile. (E) Phosphotyrosine immunoblot analysis ofMDA-MB-453 cells stimulated with the following ligand prepara-tions: None, media control; TGF-a, 50 ng/ml; CM, 16-fold-concentrated HepG2 conditioned medium tested at 2 ,ul and 10 Al perwell; fraction, phenyl-SPW column fractions 13-20 (10 ,l per well).(F) Densitometry analysis of the phosphorylated bands shown in E.

CHO cells expressing HER2, strongly suggesting that this isa direct ligand-receptor interaction. Further confirmationwill require crosslinking and binding studies with homoge-neous purified material.

Recently, several groups have reported the identificationof specific ligands for HER2 (2, 3, 11-13). Some of theseligands, such as gp3O (11), interact with both EGFR andHER2, while others are reported to bind specifically to HER2(2, 3). In contrast to these molecules, the cell differentiatingactivity described in this report shows no direct activation ofHER2 and appears to transduce its signal by interaction withthe highly related receptor HER4.The identification of this member of the EGFR family and

the availability of a ligand that specifically activates it willexpedite the characterization of the biological function ofHER4. Since EGFR and HER2 have been shown to actsynergistically (27-29) it is conceivable that HER4 may alsointeract with other EGFR family members either by het-erodimer formation or receptor crosstalk. Understanding therole of HER4 and its ligand in the process of growth and

differentiation as well as in neoplasia might contribute to thedevelopment of new anticancer therapies.

We thank Hsing-Jien Kung and Susan Radka for the NRHER5 andHepG2 cells, respectively.

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