Proc. Nati. Acad. Sci. USAVol. 89, pp. 2287-2291, March 1992Medical Sciences

Characterization of a growth factor that binds exclusively to theerbB-2 receptor and induces cellular responses

(oncogene/growth factor/breast carcinomas)

RUTH Lupu*, RAMON COLOMER, BHANU KANNAN, AND MARC E. LIPPMANVincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, 3800 Reservoir Road, Washington, DC 20007

Communicated by Erminio Costa, November 26, 1991

ABSTRACT The erbB-2 oncogene encodes a 185-kDatransmembrane protein that has been suggested to be a growthfactor receptor. We have previously identified and purified a30-kDa growth factor (gp3O) that is a ligand for the pl85wrbZ2protein that at high concentrations induces growth inhibition ofcells with erbB-2 amplification. We now report the purificationand characterization of a protein from SKBr-3 human breastcancer cells with a molecular mass of 75 kDa (p75) that is apl85erb-2 ligand. An affinity column coupled to the extracel-lular domain of pj8g5rbB2 was used for the purification. Wefound that p75 induced tyrosine phosphorylation of the erbB-2oncoprotein, as determined by in vivo and in vitro phospho-rylation and phosphoamino acid analysis. p75, as well as gp3O,stimulated cell proliferation and colony formation of cellsoverexpressing erbB-2. The specificity of this effect was con-firmed by showing that the antiproliferative effects of solubleerbB-2 extracellular domain were reversed by either p75 orgp3O. p75 did not show binding to the epidermal growth factorreceptor and had no growth effects on cells overexpressingepidermal growth factor receptor. These data show thatSKBR-3 cells, which exhibit erbB-2 amplification and overex-pression, secrete a growth factor that binds and activatesp185erbB-2 specifically.

The erbB-2 protooncogene encodes a tyrosine kinase protein(p185erbB-2) that is associated with poor prognosis in breastcancer patients (1). pl85erbB-2 shows structural similarity withthe p170 epidermal growth factor receptor (EGFR) and waspostulated to be a growth factor receptor (2-6). The erbB-2protooncogene also has considerable homology to the re-cently described human c-erbB-3 gene (7). erbB-2 protoon-cogene amplification has been found in carcinomas of thebreast, ovary, stomach, and salivary gland and in non-smallcell carcinomas of the lung (3, 8-12). Amplification and/oroverexpression of the erbB-2 protooncogene has been foundto correlate with poor prognosis in breast, ovarian, andnon-small cell lung carcinomas (1, 8, 13-15). In addition tothese clinical studies, in vitro studies strongly suggest thatoverexpression of p185c&bB-2 may have an important role inmalignant progression (15, 16).To understand the function of pl85erbB-2 and to evaluate its

potential as a therapeutic target for neoplasia, it is necessaryto identify specific pl85erbB-2 ligands. We recently reportedthe identification and purification of an apparent growthfactor secreted by MDA-MB-231 human breast cancer cells(gp3O) (17). The gp3O was purified to apparent homogeneityby sequential low-affinity heparin-Sepharose chromatogra-phy and by reversed-phase chromatography (17). Purifiedgp3O stimulated phosphorylation of p185erb-2 in cells thatoverexpress erbB-2 (18). gp3O also binds to EGFR and hastransforming growth factor a-related properties (17). We

studied the effects of gp3O in human breast cancer cellsspontaneously expressing different levels of erbB-2 and incells transfected with erbB-2. We found that gp3O inhibitedcell growth in all cells that overexpressed erbB-2 (11). Usingradioiodinated 4D5 antibody (19) we developed an erbB-2radioreceptor assay (RRA) (18). In this report we describe thesuccessful utilization of this technique to identify and char-acterize'a ligand for erbB-2, with a molecular mass of75 kDa,which interacts exclusively with pl85erbB-2 and shows nobinding or activation of EGFR. This ligand is growth stimu-latory (17).

MATERIALS AND METHODSCell Lines. Cells from the following sources were used in

these studies: MCF-7, MDA-MB-453, MDA-MB-468, andSKBr-3 breast cancer cells were obtained from the AmericanType Culture Collection. All cell lines were propagated inimproved modified Eagle's medium (IMEM; GIBCO) sup-plemented with 10o fetal bovine serum (GIBCO).

Conditioned Medium (CM) Preparation, Collection, andConcentration. CM collections were carried out as described(17).

Affinity Chromatography. erbB-2 extracellular domain(ECD) was coupled to polyacrylamide hydrazido-Sepharosebeads (Sigma). After extensive washes of the beads withice-cold 1.0 M HCI, the beads were activated with 0.5 MNaNO2. The temperature was maintained at 0C for 20 min;this was followed by filtration and washing with ice-cold 0.1M HC1. After activation of the beads ECD was coupled witha percentage of binding of 90-98%. When the column waspacked, 500 ml ofCM was run through the beads by gravity.The column was then washed and eluted stepwise with 1.0Mcitric acid at pH values from 4.0 to 2.0. All fractions weredesalted on PD10 columns (Pharmacia).

Phosphorylation Assay as Determined by Western Blot Anal-ysis. Cells were growth to 90% confluence in 24-well plates(Costar). Assays were performed as described (18). SDS/PAGE and electroblotting (20) were performed as described(18).In Vivo Phosphorylation Assay. Cells were grown to 80o

confluence in a 35-mm dish (Costar). Fetal calf serum (FCS)was removed 16 hr prior to labeling. Cells were rinsed withphosphate-free Dulbecco's modified Eagle's medium(DMEM; GIBCO) and then incubated for 3 hr at 370C with 1.0mCi (1 Ci = 37 GBq) of 32Pi per ml per dish ([32P]orthophos-phate; Amersham). After 3 hr, cells were treated for 20 minat 370C with samples to be tested. Lysates were preparedwith a modified Ripa buffer (21-23). After preincubation with10 .1A of normal mouse IgG, the nonspecific complexes wereclarified using protein A-Sepharose (Sigma). The supernatantwas incubated with a monoclonal anti-phosphotyrosine an-

Abbreviations: EGF, epidermal growth factor; EGFR, EGF recep-tor; CM, conditioned medium(ia); ECD, extracellular domain; FCS,fetal calf serum; RRA, radioreceptor assay.*To whom reprint requests should be addressed.

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2288 Medical Sciences: Lupu et al.

tibody IG2 (kindly provided by A. R. Frackelton) and spe-cifically eluted using 1 mM phenyl phosphate. A secondimmunoprecipitation was then performed using a polyclonalantibody against the erbB-2 C-terminal sequence or with apolyclonal antibody against the EGFR (Oncogene Sciences,Mineola, NY). After precipitation with protein A-Sepharosethe pellets were washed with buffer and the pellet was thenresuspended and loaded onto a 7.5% SDS/polyacrylamidegel.Phosphoamino Acid Analysis. Phosphoproteins in individ-

ual bands were extracted from polyacrylamide gels and thensubjected to partial acid hydrolysis and two-dimensionalthin-layer electrophoresis using HTLE-7000 (C.B.S. Scien-tific, Del Mar, CA) as described (24, 25).Anchorage-Independent and -Dependent Growth Assay.

Soft agar cloning assays were carried out as described (18).For growth assay cells were grown in IMEM containing 5%FCS. Cells were seeded in 24-well plates at 20,000 cells perwell in the presence of 5% FCS as described (18).

RESULTSScreening for erbB-2 Putative Ligands: RRA. We screened

CM derived from different human cell lines for the presenceof pl85erbB-2 binding activity using the 4D5 RRA (18). CMwere prepared, concentrated, and tested as described (17).Another monoclonal antibody against pl85erbB-2 (6E9) thatdoes not compete with gp3O for p185crbB-2 binding (18) wasiodinated and used as a control. It is known that SKBr-3 cellsrelease pl85erbB-2 ECD into the culture medium (26). ShederbB-2 ECD would compete with the binding of4D5 and 6E9to p185er*B2, whereas erbB-2 ligands would compete exclu-sively with 4D5. Binding assays were performed as described(18). In brief, growing SKBr-3 cells were incubated withiodinated anti-erbB-2 antibodies (4D5 or 6E9) at a concen-tration of 1.0 nM in the presence of several concentrations ofCM. We evaluated media from several breast cancer cells aswell as several nonmalignant and transformed breast epithe-lial cells, which included nonmalignant breast epithelial 184cells, immortalized 184A1N4 cells, and oncogene-trans-formed 184A1N4T [simian virus 40 (SV-40), ras, myc, SV-40-myc, and myc-ras] cells (kindly provided by MarthaStampher, Berkeley, CA). Only 1 of the 15 different mediatested, that from SKBr-3 cells, showed ability to competeexclusively with 4D5 for pl85erIB-2 binding. CM derived fromMDA-MB-157 and 184A1N4 cells showed ability to stimulateerbB-2 tyrosine phosphorylation, but no competition with4D5 was observed.

Purification of a Putative erbB-2 Ligand Secreted fromSKBr-3 Breast Cancer Cells. Our next step was to determinewhether the erbB-2 ligand activity secreted from SKBr-3 cellsshowed heparin binding, which might indicate that this ac-tivity was similar to gp3O. After medium from SKBr-3 cellswas processed by heparin chromatography, we were able torecover only part of the pl85erbB-2 binding and phosphoryla-tion activity, which suggested that the binding affinity of p75to heparin was low (0.4 M NaCl). ECD, however, mightinterfere with heparin binding or might be washed off thecolumn bound to the erbB-2 ECD. Our next step was todevelop a purification procedure with which pl85ertB-2 ECDwould not interfere. We obtained a 94-kDa recombinantpl85erbB-2 ECD (kindly provided by M. Shepard, Genentech)and coupled it to a polyacrylamide hydrazido-Sepharoseaffinity chromatography matrix (27). Concentrated CM(x 100) from SKBr-3 cells (50 ml) was loaded onto the columnand elution was performed stepwise with 1.0 M citric acidacross a pH range from 4.0 to 2.0, to allow the dissociationof the erbB-2 ECD and putative ligand. The resulting frac-tions were tested for their p185erbB-2 binding properties in the4D5 binding assay. A single purification yielded an appar-

ently homogeneous polypeptide of 75 kDa at 3.0-3.5 elutionpH. The homogeneity of the sample was confirmed byanalysis on SDS/PAGE (28) by silver staining (29) (Fig. 1).This preparation was the source used for further experi-ments.

Biochemical Characterization of p75. Binding of p75 topJ85erbB-2. To assess whether the 75-kDa polypeptide (p75)obtained from SKBr-3 CM was indeed a ligand for the erbB-2oncoprotein, we used two independent binding assays, inaddition to activity assays to be described later. We first usedthe 4D5 RRA to find out whether p75 showed specific bindingto the erbB-2 oncoprotein in SKBr-3 cells. We found that thep75 exhibited binding activity, whereas material from otherchromatography fractions did not show such activity (datanot shown). The flow-through material showed some bindingactivity. This might be due to the presence of shed erbB-2ECD. We next performed a binding assay using the 6E9antibody (pl85erbB-2), which has the same affinity forpl85erbB-2 as 4D5 but does not compete with gp3O. 6E9antibody binding to pl85erbB-2 was not altered after p75treatment, further suggesting that the eluted material was notpl85erbB-2 ECD. Second, we used an anti-erbB-2 monoclonalantibody raised against the ECD of pl85erbB-2 (OncogeneSciences) to assess the presence of shed ECD in the purifi-cation process. In the input material (CM derived fromSKBr-3 cells) and in the flow through, we observed a bandwith a molecular mass of -95 kDa, which is consistent withthe molecular mass of erbB-2 ECD. The 95-kDa band was notpresent in the fractions eluting at pH 3.0-3.5.Binding ofp75 to EGFR. Since gp3O had been identified as

a ligand common to the EGFR and pl85erb 2, we tested theactivity of all of the eluted fractions in an EGFR binding assayusing MDA-MB-468 cells and iodinated EGF (10). In thisassay EGF and gp3O, used as controls, displaced the bindingof iodinated EGF in a dose-dependent manner. In contrast,none of the eluted fractions derived from SKBr-3 CM showedEGFR binding activity, indicating that the pl85erbB-2 bindingactivity from this medium does not bind to the EGFR (Fig. 2).Phosphorylation ofpJ85erbB-2 by p75. To explore whether

p75 activated pl85erbB-2, we studied the ability of p75 tophosphorylate pl85erbB-2 on MDA-MB-453 human breastcancer cells, which overexpress the erbB-2 oncoprotein butdo not express detectable levels of EGFR (30). In a Westernblot analysis using phosphotyrosine antibody (20), we found

La 0cvi c-i

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FIG. 1. SDS/PAGE of sam-ples eluted from ECD affinitychromatography. Aliquots fromthe input medium and from thefractions containing activitywere analyzed by 10-20%o SDS/PAGE, which was followed bysilver staining. Lanes (from rightto left) show unconcentratedCM from SKBr-3 cells, elutionat pH 3.0, and elution at pH 3.5.

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 2289

4000 -

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FIG. 2. EGFR and ERBb-2 binding assays. A431 and SKBR-3cells were plated (100,000 cells per well) in 24-well plates in 5% FCS.All of the fractions after the ECD affinity chromatography weretested for p185erbB-2 competition binding assays using iodinated 4D5.The EGFR competition assay was performed using iodinated EGF(Amersham). The control represents the total amount of binding (nocompetitor was added). The pH gradient represents all fractionscollected during the elution step. *, EGFR binding; o, erbB-2binding.

that p75 activated tyrosine phosphorylation ofMDA-MB453cells. As can be seen in Fig. 3A, treatment of cells withincreasing concentrations of p75 (2-8 ng/ml) resulted in a 10-to 20-fold increase in phosphorylated tyrosine content.To determine that the tyrosine phosphorylation observed

was indeed 185erbB-2, we used metabolic labeling of MDA-MB-453 cells with 32p, in the presence of increasing concen-trations of p75. Tyrosine phosphorylated proteins were iso-lated from the extracts by microbatch affinity chromatogra-phy using a highly specific monoclonal antibody tophosphotyrosine as described (21-23) and then were specif-ically eluted from the immunosorbent with hapten phenylphosphate and resolved by reducing SDS/PAGE (Fig. 3B). Insome cases the tyrosine phosphorylated erbB-2 oncoproteinwas purified further by an additional immunoprecipitationwith a polyclonal antibody that reacts with the C-terminaldomain of pl85erbB-2 but does not show cross-reactivity withEGFR. In this assay we found that the tyrosine phosphory-lation that is induced by p75 in MDA-MB453 cells wasindeed pl85erbB-2 and that the phosphorylating ability of p75occurred in a dose-dependent manner.To assess any potential EGFR binding activity of p75 we

used MDA-MB-468 cells, which overexpress EGFR. In theWestern blot using phosphotyrosine antibodies shown in Fig.3C it is clear that p75 did not induce tyrosine phosphorylationin these cells, whereas gp3O and EGF, used as controls,exhibited phosphorylating activity as expected.

Finally, we performed phosphoamino acid analysis of thephosphoproteins after phosphate incorporation. After se-quential immunoprecipitation with an phosphotyrosine anti-body and further immunoprecipitation with an anti-erbB-2antibody, individual bands of 185 kDa were extracted frompolyacrylamide gels and then subjected to partial acid hy-drolysis and two-dimensional thin-layer electrophoresis asdescribed (24, 25). An increase in tyrosine phosphorylationwas observed after p75 (4 ng/ml) treatment of MDA-MB-453cell induction, as shown in Fig. 4. Taken together, theseobservations show that p75 activates pl85erbB-2 by means oftyrosine phosphorylation.

Biological Characterization of p75. Effect of p75 on ananchorage-dependent cell proliferation assay. We next ex-amined the biological effects of p75 in breast cancer cellsusing anchorage-dependent and anchorage-independentgrowth assays. p75 inhibited the cellular proliferation of the

p75 (ng/ml)E

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FIG. 3. Detection of phosphorylated proteins in MDA-MB-453cells. (A) MDA-MB-453 cells were grown to 90% confluence in a24-well plate. Cells were treated for 20 min with control medium (lane1), control medium containing 5 ng of gp30 per ml (lane 2), controlmedium containing 2 ng of p75 per ml (lane 3), control mediumcontaining 8 ng of p75 per ml (lane 4), and control medium containing4 ng of p75 per ml (lane 5). The media were removed and cells werelysed in 100 ,ul of sample buffer and loaded in a 7.5% SDS/polyacrylamide gel. Proteins were then transferred for immunoblot-ting. The filter was incubated with an anti-phosphotyrosine antibody.Immune complexes were detected by a goat anti-mouse antibodyconjugated to alkaline phosphatase. (B) MDA-MB-453 cells weregrown to 80%o confluence in a 24-well plate (Costar). Cells weretreated for 20 min with control medium (lane 1), control mediumcontaining different concentrations ofp75 (0.25-10 ng/ml; lanes 2-6,respectively), and control medium containing 2.0 ng of gp30 per mlas control (lane 7). Following incubation the cultures were lysed. Thespecific complexes with an anti-erbB-2 C-terminal polyclonal anti-body were precipitated with protein A-Sepharose. The samples wereloaded in a 7.5% SDS/polyacrylamide gel. (C) MDA-MB-468 cellswere grown to 80%o confluence in a 24-well plate (Costar). Cells weretreated for 20 min with control medium (lane 1), control mediumcontaining 10.0 ng of p75 per ml (lane 2), control medium containing4.0 ng of transforming growth factor a per ml (lane 3), controlmedium containing 4.0 ng ofEGF per ml (lane 4), and control mediumcontaining 2.0 ng of gp30 per ml (lane 5). The media were removedand cells were lysed in 100 ,ul of sample buffer and loaded in a 7.5%SDS/polyacrylamide gel. Immune complexes were detected as de-scribed for A.

erbB-2-overexpressing cells SKBr-3, BT-474, and MDA-MB-453 by 70-80% at a concentration of 4 ng/ml. No inhibitionwas observed in MDA-MB-468 cells, which overexpress theEGFR, or in MCF-7 cells, which do not overexpressp185erbB-2 or EGFR. gp3O, used as control, inhibited theproliferation of SKBr-3 and MDA-MB-468 cells at a concen-tration of 2.0 ng/ml (Fig. 5).

Effect of p75 on an anchorage-independent soft agarcolonyformation assay. In an anchorage-independent assay,p75, used at a concentration of4 ng/ml, inhibited the soft agarcolony formation of SKBr-3 and MDA-MB-453 cells by60-70o and did not have any effect on MDA-MB-468 cells,consistent with the anchorage-dependent growth studies. p75treatment at concentrations 10- to 20-fold lower than inhib-itory concentrations (0.04-10 ng/ml) resulted in up to a 3-foldincrease in the number of SKBr-3 colonies. We used gp30 asa control in this experiments and found that concentrationsranging from 0.02 to 0.5 ng/ml also stimulated colony for-mation of SKBr-3 cells. To compare the results of the twogrowth factors, we plotted the results using approximatemolar concentrations. As can been seen in Fig. 6, p75 and

Medical Sciences: Lupu et al.

2290 Medical Sciences: Lupu et al.

2400

9S

yT

Control

9S. T

p75

Ye'

FIG. 4. Detection of phosphorylated proteins by phosphoaminoanalysis. MDA-MB-453 cells were grown to 80% confluence. Cellswere treated for 20 min at 37°C with control medium and mediumcontaining p75 (4 ng/ml). Following incubation the cultures werelysed. The specific complexes were precipitated and, after acidhydrolysis, the specific phosphoamino acids were subjected totwo-dimensional thin-layer chromatography. Spots for serine (S),threonine (T), and tyrosine (Y) are indicated.

gp30 had superimposable effects in anchorage-independentassays, showing a biphasic stimulation followed by inhibitionof proliferation.

Specificity of p75. To determine the specificity of p75 weperformed growth assays in the presence of erbB-2 ECD. Theaddition of soluble pl85erlB-2 ECD to SKBr-3 cell culturesinhibited their soft agar colony formation. ECD had similarinhibitory effects in other cells overexpressing pl85erbB-2, andthey occurred in a dose-dependent manner. The optimal inhib-itory dose was determined to be 12 ,ug/ml. No inhibition wasobserved in MDA-MB-468 and MCF-7 cells (Fig. 7). We nextused growth-stimulatory concentrations of p75 (0.2 ng/ml) incombination with ECD to treat SKBr-3 and MDA-MB453 cellsand found that the inhibitory effect ofECD was reversed by theaddition of p75. gp3O was used as a positive control.

DISCUSSIONWe have previously described a 30-kDa erbB-2 ligand. gp3Obinds tightly to heparin, eluting from a heparin chromatog-

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FIG. 5. Effect of p75 on the growth of human breast cancer cells.SKBr-3 and MDA-468 cells were plated (30,000 cells per well) in24-well plates in IMEM plus 5% FCS. After 24 hr cells were treatedwith purified p75 (4 ng/ml), purified gp3O (2.0 ng/ml), or EGF (10ng/ml). Ab, antibody. Cells were grown to 90% confluence ofcontroland counted. Each group was assayed in triplicate. SEM are indi-cated.

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FIG. 6. Effect of p75 on the soft agar colony formation of humanbreast cancer cells. SKBr-3 cells were plated in 35-mm tissue culturedishes (Costar). After the bottom layer was solidified, 10,000 cellsper dish were added in a 0.8-ml top layer containing the sample. Thesamples were gp3O (0-330 pM) and p75 (0-132 pM). All samples wererun in triplicate. SEM are indicated.

raphy column at a concentration of 0.5-0.7 M NaCl. Inaddition to binding to pl85erbB-2, gp30 also showed binding tothe EGFR and induced cellular responses in cells expressingelevated levels of erbB-2 or EGFR.We have now identified a polypeptide of 75 kDa that binds

to the pl8serbB-2 oncoprotein, activates it, and induces cel-lular proliferation. A chromatographic method using solubleECD coupled to polyacrylamide agarose was used for itspurification.The heparin binding ability of p75 is significantly lower

than that of gp30. p75 stimulated tyrosine phosphorylation ofp185erbB-2 by 10- to 20-fold, as assessed by Western blotanalysis and in vivo metabolic labeling. Phosphoamino acidanalysis showed an increase in pl85erbB-2 tyrosine phosphor-ylation after p75 treatment. p75, however, did not inducetyrosine phosphorylation of the EGFR.

p75 modulates the proliferation of cells with very highlevels of erbB-2. We and others (31, 32) have previouslyreported that EGF is growth stimulatory at low concentra-tions in the EGFR-overexpressing cells MDA-MB-468,whereas a strong growth inhibition is observed when elevatedconcentrations of EGF are used. It has also been reported

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FIG. 7. Effect of soluble p185erbB-2 ECD on the soft agar colonyformation of human breast cancer cells. SKBr-3 cells were plated in35-mm tissue culture dishes (Costar). The top layer contained thesample, 0.4% Bacto agar, and 10% FCS. The samples were p75 (2.0pM = 0.15 ng/ml), gp3O (2.0 pM = 0.3 ng/ml), soluble recombinantECD (12 ,g/ml), and p75 or gp3O (2.0 pM) in the presence of ECD(12 Zg/ml). Cells were incubated 7-9 days at 37°C in 5% CO2.Colonies larger than 60 ,m were counted in a colony counter. Theexperiments were performed three times and the results were repro-ducible. SEM are indicated.

Proc. Natl. Acad. Sci. USA 89 (1992)

Proc. Natl. Acad. Sci. USA 89 (1992) 2291

that cells overexpressing the estrogen receptor after trans-fection are growth inhibited by physiological doses of estro-gen (33). We have shown here that the erbB-2 ligand p75, aswell as gp3O, has dual growth effects in cells with erbB-2overexpression. In anchorage-independent assays p75 stim-ulated the colony formation of cells overexpressing erbB-2when used at low concentrations but inhibited the formationof colonies at concentrations higher than 0.5 ng/ml. Thisdose-response pattern in soft agar assays was paralleled bygp3O when used at equimolar concentrations. In similarassays performed with MDA-MB-468 cells, p75 had nosignificant proliferative effects.

Since the potential cross-talk between EGFR and erbB-2has been reported (34), we also performed all of the phos-phorylation experiments in a human breast cancer cell linethat does not express EGFR (MDA-MB-453) to show that p75does not activate EGFR in cells that do not express erbB-2(MDA-MB-468 cells). Therefore p75 appears to be a ligandthat interacts exclusively with the erbB-2 oncoprotein, unlikegp3O, which showed dual binding to pl85erbB-2 and EGFR(although with substantial lower affinity for EGFR).The specific effect of p75 on pl85,ibB-2 was further estab-

lished by showing that p75 reversed the growth-inhibitoryeffect of soluble erbB-2 ECD. This effect of ECD may be dueeither to dimerization of the soluble domain with the cellularp185erlB-2 oncoprotein or to binding and neutralization ofp75,which is essential for their growth, therefore leading toinhibition of cell proliferation. The inhibitory effect of theECD was reversed by the addition of growth-stimulatingdoses of p75, suggesting that complex regulatory pathwaysmay exist for pl8erbB-2. Given the fact that these cellsspontaneously release ECD into the CM in addition to the p75ligand, the affinity of the binding of p75 to the soluble erbB-2ECD is very low as compared with the affinity of p75 to thecell membrane receptor, and therefore the amount of solubleerbB-2 ECD required is extremely high. Stimulatory doses ofgp3O served as a positive control for the reversed inhibitoryeffects of ECD in our experiments.

In the course of this work and our previous report (18), wehad to determine criteria for putative ligands to growth factorreceptors. Recently Yarden and Peles (35) have shown thata putative EGFR/erbB-2 ligand is secreted from Rat-1 cells.This growth factor could have similar activity to our previ-ously described gp3O (17). Since p75 does not bind to theEGFR, we can exclude the possibility that they are identicalligands.To our knowledge, purification of an erbB-2-specific ligand

has not been reported previously. Certainly the ultimateassignment of a growth factor to a receptor should rely oncovalent cross-linking of a radiolabeled ligand to the respec-tive receptor. This experiment, however, is hampered by thevery low concentration of the molecule. The observed highapparent molecular mass of the p185erbB-2 ligand may beattributed to its glycoprotein nature and possibly to anoligomeric structure.We have provided evidence that cells that overexpress the

erbB-2 oncoprotein may also secrete one of its ligands, whichis required for their proliferation, therefore implying anautocrine loop. We believe that manipulation of this andother erbB-2 ligands may turn out to have an importantbiological effect on growth of human neoplasia.We thank Karen Ku for her excellent technical help and Michael

Shepard for providing the monoclonal anti-erbB-2 antibodies and therecombinant erbB-2 ECD polypeptide and also for his constructivescientific discussions. This work was supported by National Insti-tutes of Health Grant CA55406-01, The Susan G. Komen Breast

Cancer Foundation, the G. Harold and Leila Y. Mathers Foundation,and the Concern Foundation for Cancer Research.

1. Slamon, D. J., Clark, G. M., Wong, S. G., Levin, W. J.,Ullrich, A. & McGuire, W. L. (1986) Science 235, 177-182.

2. Yarden, Y., Kwang, W. J., Yang-Feng, T., Coussens, L.,Munemitzu, S., Dull, T. J., Chem, E., Schlessinger, J.,Froncke, U. & Ullrich, A. (1987) EMBO J. 6, 3341-3351.

3. Bargmann, C. I. & Weinberg, R. A. (1988) EMBO J. 7, 2043-2050.

4. van Zoelen, E. J. J., van Rooijen, M. A., van Oostwaard,T. M. J. & de Laat, S. W. (1987) Cancer Res. 47, 1582-1587.

5. King, C. R., Kraus, M. H. & Aaronson, S. A. (1985) Science229, 974-976.

6. Kraus, M., Issing, W., Miki, T., Popescu, N. & Aaronson, S.(1989) Proc. Nat!. Acad. Sci. USA 86, 9193-9197.

7. Slamon, D., Godolphin, W., Jones, L. A., Holt, J. A., Wong,S. G., Keith, D. E., Levin, W. J., Stuart, S. G., Udove, J.,Ulirich, A. & Press, M. F. (1989) Science 244, 707-712.

8. Yokota, J., Yamamoto, T., Toyoshima, K., Terada, M., Sug-imura, T., Battifora, H. & Cline, M. J. (1986) Lancet i, 765-767.

9. Fukushige, S. I. (1986) Mol. Cell. Biol. 6, 955-958.10. Semba, K., Kamata, N., Toyoshima, K. & Yamamoto, T.

(1985) Proc. Natd. Acad. Sci. USA 82, 6497-6501.11. Weiner, D. B., Nordberg, J. E., Robinson, R. A., Nowell,

P. C., Gazdar, A., Greene, M. I., Williams, W. V., Cohen,J. A. & Kerns, J. A. (1990) Cancer Res. 50, 421-425.

12. Guerin, M., Barrois, M., Terrier, M. J., Spielmann, M. & Riou,G. (1988) Oncogene Res. 3, 21-31.

13. Wright, C., Angus, B., Nicholson, S., Sainsbury, J. R., Cairns,J., Gullic, W. J., Kelly, P. & Harris, A. (1989) Cancer Res. 49,2087-2090.

14. Kerns, J. A., Schwartz, D. A., Nordberg, J. E., Weiner,D. B., Greene, M. I., Torney, L. & Robinson, R. A. (1990)Cancer Res. 50, 5184.

15. DiFiore, P. P., Pierce, J. H., Kraus, M. H., Segatto, O., King,C. R. & Aaronson, S. A. (1987) Science 237, 178-182.

16. Hudziak, R. M., Schlessinger, J. & Ullrich, A. (1987) Proc.Nat!. Acad. Sci. USA 84, 7159-7163.

17. Lupu, R., Wellstein, A., Sheridan, J., Ennis, B., Zugmaier, G.,Katz, D., Lippman, M. E. & Dickson, R. B. (1991) Biochem-istry, in press.

18. Lupu, R., Colomer, R., Zugmaier, G., Shepard, M., Slamon, D.& Lippman, M. E. (1990) Science 249, 1552-1555.

19. Hudziak, R. M., Lewis, G. D., Winget, M., Fendly, B. M.,Shepard, M. H. & Ullrich, A. (1989) Mol. Cell. Biol. 9, 1165-1172.

20. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Nat!.Acad. Sci. USA 76, 4350-4354.

21. Beitz, J. G. (1991) Proc. Nat!. Acad. Sci. USA 88, 2021-2025.22. Frackelton, A. R., Jr., Posne, M., Kannan, B. & Mermelstein,

F. (1991) Methods Enzymol. 201, 79-92.23. Frackelton, A. R., Jr., Tremble, P. M. & Williams, L. T. (1991)

J. Biol. Chem. 259, 7909-7915.24. Cooper, J. A., Sefton, B. & Hunter, T. (1983) Methods En-

zymol. 99, 387-402.25. Frackelton, A. R., Jr., Ross, A. N. & Eisen, H. N. (1983) Mol.

Cell. Biol. 3, 1343-1352.26. Langton, B. C., Crenshaw, M. C., Chao, L. A., Stuart, S. G.,

Akita, R. W. & Jackson, E. (1991) Cancer Res. 51, 2593-2598.27. Avromeas, S., Ternynck, T. & Guesdon, J. (1978) Scand. J.

Immunol. 8, 7.28. Laemmli, U. K. (1970) Nature (London) 227, 680-685.29. Morissey, J. H. (1981) Anal. Biochem. 177, 307-310.30. Yarden, Y. & Weinberg, R. A. (1989) Proc. Nat!. Acad. Sci.

USA 86, 3179-3183.31. Ennis, B. W., Valverius, E, M., Bates, S. E., Lippman, M. E.,

Bellot, F., Kris, R., Schlessinger, J., Masui, H., Goldenberg,A., Mendelsohn, J. & Dickson, R. B. (1989) Mol. Endocrinol.3, 1830-1838.

32. Kawamoto, T., Mendelsohn, J., Le, A., Sato, G. H., Lazar,C. S. & Gill, G. N. (1984) J. Biol. Chem. 259, 7761-7766.

33. Kushner, P. J., Hort, E., Baxter, J. D. & Greene, G. (1990)Mol. Endocrinol. 4, 1465-1469.

34. Wada, T., Qian, X. & Greene, M. (1990) Cell 61, 1339-1347.35. Yarden, I. & Peles, E. (1991) Biochemistry 30, 3543-3550.

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