Proc. Natl. Acad. Sci. USAVol. 90, pp. 3%3-3967, May 1993Cell Biology

Ligand-dependent transformation by the receptor for humangranulocyte/macrophage colony-stimulating factor andtyrosine phosphorylation of the receptor fJ subunit

(cytokine receptor/cell transformation/phosphotyrosine)

LILIANA B. ARECES*, MANFRED JUCKER*, JULIE A. SAN MIGUEL*, ALICE MUIt, ATSUSHI MIYAJIMAt,AND RICARDO A. FELDMAN**§*Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; *Medical Biotechnology Center, Universityof Maryland Biotechnology Institute, Baltimore, MD 21201; and TDNAX Research Institute of Molecular and Cellular Biology, Palo Alto, CA 94304

Communicated by Hidesaburo Hanafusa, January 27, 1993

ABSTRACT The receptor for human granulocyte/macro-phage colony-stimulating factor (hGMR) is composed of twosubunits, a and 3, which are both required for high-affinitybinding of the ligand. To examine the transforming potential ofhGMR, we have transfected cDNAs encoding the receptor a and(3 subunits into NIH 3T3 cells, which normally do not expressGMRs. Introduction of the receptor subunits into these cellsresulted in focal transformation, which was dependent on thepresence of human granulocyte/macrophage colony-stimulat-ing factor (hGM-CSF) in the culture medium. No transforma-tion was observed when hGM-CSF was replaced with othergrowth factors such as human epidermal growth factor orhuman interleukin 3 or when cells were transfected with the aor (3 subunit alone. Individual conditional transformants iso-lated after transfection expressed functional hGMRs, weresusceptible to transformation by picomolar levels of the ligand,and were capable of anchorage-independent growth in soft agarin the presence but not in the absence ofhGM-CSF. Biochemicalanalysis showed that treatment of these cells with hGM-CSFcaused a rapid phosphorylation of the (3 subunit and othercellular proteins on tyrosine residues, recapitulating some of theevents that take place duringGM-CSF signaling in myeloid cells.We conclude that coexpression ofthe a and 1B subunits ofhGMRin established murine fibroblasts is sufficient to reconstitute afunctional receptor, which is capable of causing ligand-dependent transformation. The oncogenic potential of hGMRlends support to the hypothesis that its deregulated or abnormalexpression may play a role in leukemogenesis.

Cytokines play important roles in the control of hematopoi-etic cell development and the coordination of host immuneresponses (1). The receptors for cytokines such as granulo-cyte/macrophage colony-stimulating factor (GM-CSF), in-terleukin (IL) 4, and erythropoietin belong to a large familyof structurally related molecules (2), which are responsiblefor the transduction and sorting of signals that are initiated atthe cell surface. Unlike the receptors for epidermal growthfactor (EGF) and platelet-derived growth factor, which aresingle polypeptide chains with intrinsic protein-tyrosine ki-nase activity (3), many cytokine receptors are composed ofat least two distinct subunits, with no recognizable kinasedomains (4). Nevertheless, a number of cytokines such asGM-CSF and IL-3 can induce the phosphorylation of cellularproteins on tyrosine (5-7), indicating that cytokine receptorshave a functional association with as yet unidentified cellulartyrosine kinases.GM-CSF is a hematopoietic growth factor that regulates

proliferation, differentiation, and effector functions of mono-

cyte-macrophage, granulocytic, and other cell types (1). Thereceptor for human (h) GM-CSF is composed of two sub-units, termed a and 1, which are transmembrane proteins of75 kDa and 120 kDa, respectively (8, 9). The a subunit bearsthe specificity for ligand recognition and can bind hGM-CSFwith low affinity, whereas the 13 subunit, which is believed tofunction as a signal transducer, cannot bind hGM-CSF byitself but is required for high-affinity binding (10). In humans,it has been shown that the same 1 subunit is utilized by thereceptors for GM-CSF, IL-3, and IL-5, providing an expla-nation for the overlapping biological activities of these cyto-kines (4).

It has been proposed that the autocrine production ofcytokines by leukemic cells has a role in the generation ofleukemias (11-13). However, oncogenic activity by normalcytokine receptors has not been directly demonstrated. Toexamine this question, we have analyzed the ability of thehGM-CSF receptor (hGMR) to transform established murinefibroblasts. In this paper we show that cotransfection ofNIH3T3 cells with the a and 83 subunits of hGMR can lead tooncogenic transformation in the presence of hGM-CSF andthat ligand binding is accompanied by phosphorylation of thereceptor 13 subunit and other cellular proteins on tyrosineresidues.

MATERIALS AND METHODSCells. The stock of NIH 3T3 cells used in this study has

been described (14). NIH 3T3 cells were maintained inDulbecco's modified Eagle's medium (DMEM; GIBCO) sup-plemented with 10% (vol/vol) fetal calf serum.

Plasmid DNA. The plasmid pCEVGMR-a, which encodesthe a subunit of hGMR and has a hygromycin-resistancegene, has been described (8). pKH97 is a plasmid encodingthe 13 subunit of hGMR (9). pcDSR298 encodes a full-lengthhGM-CSF cDNA (15). pME18S is a control vector thatcontains no insert (10).DNA Transfection and Hygromycin Selection. NIH 3T3 cells

were transfected by the calcium phosphate method (16) using300 ng ofeach plasmidDNA per 35-mm plate as described (14).Twenty-four hours later cells were trypsinized and seeded in60-mm plates for the focus assay or in 100-mm dishes forhygromycin selection in the presence of hygromycin (Sigma)at 0.2 mg/ml. Individual colonies resistant to the drug wereisolated 2-3 weeks later.

Antibodies and Growth Factors. The rat monoclonal anti-body CRS1, directed against the 13 subunit of hGMR, has

Abbreviations: GM-CSF, granulocyte/macrophage colony-stimulat-ing factor; h, human; hGMR, hGM-CSF receptor; IL, interleukin;EGF, epidermal growth factor.§To whom reprint requests should be addressed at *.

3963

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

been described (17). The anti-phosphotyrosine mouse mono-clonal antibody 4G10 was purchased from Upstate Biotech-nology (Lake Placid, NY). Purified recombinant hGM-CSFand hIL-3 were produced in Escherichia coli (10). Recombi-nant hEGF was purchased from Promega.

Radioligands and Binding Assays. Recombinant hGM-CSFwas iodinated using the Bolton-Hunter reagent, and bindingassays and analysis of equilibrium binding data were per-formed as described (10).

Stimulation with hGM-CSF. For hGM-CSF stimulation,cells were preincubated for 2 hr inDMEM supplemented with0.1% (wt/vol) bovine serum albumin (fraction V; Sigma) afterwhich time, the cells were incubated with hGM-CSF asindicated in the figure legends. After incubation, cells wereextracted as described below.

Preparation of Cell Extracts and Protein Analysis. Prepa-ration of cell extracts for protein analysis was carried out asdescribed (18) in a buffer containing 50mM Hepes/KOH (pH7.4), 1% (vol/vol) Triton X-100, 150 mM NaCl, 1.5 mMMgCl2, 10% (vol/vol) glycerol, 10 mM sodium pyrophos-phate, 100 mM NaF, 1 mM sodium orthovanadate, and 2%Trasylol (FBA Pharmaceuticals, New York). The analysis ofproteins by immunoprecipitation, electrophoresis in SDS/8.5% PAGE gels, and Western blot analysis with antibodiesdirected against phosphotyrosine have been described (18,19).

RESULTSCoexpression of the a and 18 Subunits ofhGMR in NIH 3T3

Cells Causes Cell Transformation in the Presence of hGM-CSF. To determine if expression of hGMR in NIH 3T3 cellswould alter the growth properties of these cells, cDNAsencoding the a and P subunits of hGMR were cotransfectedby the calcium phosphate procedure, and the transfectantswere incubated in the presence or absence of recombinanthGM-CSF at 20 ng/ml (1 nM). In the presence of hGM-CSF,foci of morphologically transformed cells were detected 6days after transfection (Fig. 1A, plate h), but in its absence,no focal changes were observed (Fig. 1A, plate d). Untrans-fected cells or cells transfected with the a or ,B subunit alonedid not develop foci in the absence or presence of hGM-CSF(Fig. 1A). The efficiency oftransformation measured 2 weeksafter transfection was 180-300 foci per ,g of plasmid DNA(Table 1). This transforming activity is 5-10 times lower thanthat obtained with v-fps/fes, a transforming retroviral gene,but 2 orders of magnitude higher than that of c-fps/fes (19),its normal cellular homolog (data not shown). The hGM-CSF-

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Table 1. Focus-forming activity of hGMRconditional transformants

FFU/,ug of DNA

Exp. Plasmid - hGM-CSF + hGM-CSF

1 ta + vector 0 03+ vector 0 0a + ,8 0 177

2 a + vector 0 0,8 + vector 0 0a + 8 0 302

The indicated plasmid DNAs were transfected into NIH 3T3 cellsas described in the Materials and Methods, and foci of transformedcells were scored 2 weeks after transfection. FFU, focus-formingunits. The transfected a, /3, and vector plasmids were PCEVGMR-a,pKH97, and pME18S, respectively.

dependent foci consisted ofhighly refractile cells that had lostcontact inhibition (Fig. 1B). Similar results were obtainedwhen hGM-CSF was supplied by an autocrine mechanism.Cotransfection of cDNAs encoding hGM-CSF and the re-ceptor a and ,3 subunits resulted in focal transformation in theabsence of added hGM-CSF, at a slightly higher frequencythan in the paracrine system described above (data notshown).We conclude from these results that coexpression of the a

and ,B subunits ofhGMR in NIH 3T3 cells can reconstitute abiologically active receptor, which is capable of causingefficient transformation in the presence of hGM-CSF.

Biological Properties of the hGMR Transfectants. To ex-amine the properties of cells that express the hGMR, indi-vidual transfectants were isolated. Since the a subunit ofhGMR was cloned into a vector that confers resistance to theantibiotic hygromycin, individual colonies were isolated bygrowing cells cotransfected with the a and A3 subunits in thepresence of this drug. Hygromycin-resistant colonies werecylinder cloned and expanded, and sister plates of eachcolony were incubated in the presence or absence of hGM-CSF. Without hGM-CSF, no transformation was observedwith any of the isolated clones. By contrast, when hGM-CSFwas present in the growth medium, 12% of the individualclones examined became fully transformed 3-4 days afteraddition of the ligand. Two of these clones were kept forfurther analysis as described below.

Quantitative binding analysis with 1251-labeled hGM-CSFshowed that the selected clones expressed between 1500 and3800 high-affinity receptors per cell, confirming that theconditional hGM-CSF transformants expressed functional

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FIG. 1. hGM-CSF induces focus formation in NIH 3T3 cells transfected with the a and ,/ subunits ofhGMR. (A) Control NIH 3T3 cells (platesa and e) and NIH 3T3 cells cotransfected with pME18S vector and a (plates b and f), pME18S and /3 (plates c and g), and a and /8 (plates dand h) plasmid DNAs were grown in the absence (plates a-d) or the presence (plates e-h) of hGM-CSF at 20 ng/ml. The transfected cultureswere stained with Giemsa and photographed 2 weeks after transfection. (B) Microscopic appearance of a hGM-CSF-induced focus in culturescotransfected with the hGMR a and ( subunits.

3964 Cell Biology: Areces et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 3965

hGM-CSF receptors (data not shown). This number of re-ceptors is similar to that found in hematopoietic cells, whichnormally express from a few hundred to several thousandhigh-affinity receptors per cell (20). Thus, transformation inthis system did not require the expression of abnormally highnumbers of hGM-CSF receptors.hGM-CSF did not significantly affect the growth rate of the

transfectants before they reached confluency, but after con-fluency the treated cells lost contact inhibition and exhibiteda highly refringent phenotype. After 7 days ofincubation withhGM-CSF, the number of cells in the transformed plates was3-6 times higher than that in the untreated sister cultures. Agrowth curve showed that without hGM-CSF, the conditionaltransformants had a similar growth rate and reached the samesaturation density as control NIH 3T3 cells, which wereunaffected by hGM-CSF (data not shown). Thus, in theabsence of a ligand, the receptor subunits had no measurablebiological activity, probably reflecting their inability to as-semble into a functional signaling complex.Appearance of the transformed phenotype in the presence

of hGM-CSF was not the consequence of secondary domi-nant events that took place during incubation with the ligandbecause transformation required the continued presence ofhGM-CSF. Subculturing of hGM-CSF-transformed cells intofresh medium lacking hGM-CSF resulted in reversion to theuntransformed parental phenotype, and these cells remainedfully susceptible to retransformation by hGM-CSF.The hGM-CSF-responsive clones were very sensitive to

transformation by its ligand. When these cells were platedtogether with control NIH 3T3 cells at a ratio of 1.5 x 103transfectants per 6 x 105 control cells, followed by incubationwith increasing concentrations of hGM-CSF, focal changeswere already detectable at a dose of50 pg/ml, and the numberand size of the foci increased up to a concentration of 20ng/ml (Fig. 2A). At these two hGM-CSF concentrations, thenumber of foci counted under the microscope were 18 and203, respectively. No foci were observed in the absence ofhGM-CSF or in control NIH 3T3 cells treated in the samemanner (Fig. 2A). Similarly, other growth factors such ashEGF and hIL-3 (0-100 ng/ml) were not able to elicit anymorphological changes in the conditional transformants (datanot shown). The concentration ofhGM-CSF required to elicittransformation was in the same range as that which is used topropagate hGM-CSF-dependent hematopoietic cell linessuch as TF-1 (2-5 ng/ml) (21), indicating that transformationwas obtained with physiological concentrations of hGM-CSF.

The hGMR transfectants were also tested for anotherproperty of transformed cells, anchorage-independentgrowth in soft agar. These cells were able to grow in soft agaronly in the presence of hGM-CSF, whereas under the sameconditions, control NIH 3T3 cells did not grow in soft agar(Fig. 2B).We conclude that the ligand-activated hGMR can deliver a

full oncogenic signal in NIH 3T3 cells and that, therefore, thenormal receptor subunits can function in concert as onco-genes.hGM-CSF-Dependent Tyrosine Phosphorylation of the P

Subunit. In hematopoietic cells, binding of GM-CSF andother cytokines to their receptors causes rapid phosphory-lation of cellular proteins on tyrosine residues, and this isbelieved to be an essential step in signaling by these receptors(5-7). To determine if hGM-CSF can induce tyrosine phos-phorylation in NIH 3T3 cells expressing hGM-CSF recep-tors, we carried out Western blot analysis of total cellularproteins from cells stimulated with hGM-CSF, using anti-bodies directed against phosphotyrosine. In these cells,hGM-CSF induced the phosphorylation of proteins of 130,120, 78, 73, 70, 57, 54, and 47 kDa (Fig. 3A, lane B).Preincubation of the anti-phosphotyrosine antibodies with 30mM phosphotyrosine completely blocked detection of theseproteins in the immunoblots (Fig. 3A, lane C), indicating thatthe antibody specifically recognized phosphotyrosine-containing proteins.

In hematopoietic cells, the p subunit ofGMR is one of theproteins that is phosphorylated on tyrosine in response toGM-CSF or IL-3 (6, 7, 22). To determine if /3 subunitphosphorylation also occurred duringhGMR activation in theNIH 3T3 transfectants, extracts from untreated and hGM-CSF-treated cells were immunoprecipitated with a monoclo-nal antibody specific for the P subunit, followed by Westernblot analysis using anti-phosphotyrosine antibodies. Thisanalysis showed that after hGM-CSF treatment, the /3 subunitbecame phosphorylated on tyrosine (Fig. 3B, lane G). Bycontrast, no /8 subunit phosphorylation was detected inuntreated cells (Fig. 3B, lane F) or in control NIH 3T3 cellstreated in the same way (Fig. 3B, lanes D and E). Tyrosinephosphorylation of the 3subunit was detectable within 2 minof hGM-CSF treatment, and maximum phosphorylation wasreached 30 min after stimulation (Fig. 3C). In other experi-ments, this maximum varied between 10 and 30 min.We conclude that in NIH 3T3 cells the ectopically ex-

pressed hGMR can reproduce some of the biochemicalevents that take place during GM-CSF signaling in myeloidcells, and we postulate that tyrosine phosphorylation may be

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FIG. 2. Dose dependence of hGM-CSF-induced transformation and anchorage-independent growth in soft agar. (A) NIH 3T3 cells (6 x 105)were plated alone (plates a-f) or mixed with 1.5 x 103 cells from a hygromycin-selected conditional transformant cell line (plates g-l) in 60-mmdishes. Two days later, both sets of plates (a-f and g-l) received the indicated concentrations of hGM-CSF, and the cultures were stained withGiemsa 9 days after hGM-CSF addition. (B) Conditional transformants and control NIH 3T3 cells were plated in soft agar at a cell density of105 cells per 60-mm dish in the presence or absence of hGM-CSF at 100 ng/ml. Soft agar cultures were photographed 3 weeks after plating.

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Cell Biology: Areces et al.

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

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FIG. 3. Tyrosine phosphorylation of the (8 subunit of hGMR. (A) a- and 13subunit transfectants were incubated in the absence (lane A) or

in the presence (lanes B and C) of hGM-CSF at 20 ng/ml for 10 min at 37°C as described in the Materials and Methods. After incubation, cellswere extracted in 1% Triton X-100 buffer, and cell extracts (50 ,ug of protein) were analyzed by SDS/PAGE, followed by Western blot analysisusing anti-phosphotyrosine antibodies as described in the Materials and Methods. In lane C, the anti-phosphotyrosine antibody was incubatedwith 30 mM phosphotyrosine (P-Y) prior to immunoblotting. (B) Control NIH 3T3 cells (lanes D and E) or a- and t3-subunit transfectants (lanesF and G) were incubated in the absence (lanes D and F) or presence (lanes E and G) of hGM-CSF at 20 ng/ml for 10 min at 37°C. Cells werethen extracted as in A, and the extracts (250 lAg of protein) were immunoprecipitated with monoclonal antibody CRS1 directed against thesubunit ofhGMR as described in the Materials and Methods. Immunoprecipitates were then analyzed by SDS/PAGE followed by Western blotanalysis using anti-phosphotyrosine antibodies. (C) a- and (3-subunit transfectants were incubated with hGM-CSF at 20 ng/ml for the indicatedtimes at 37°C. Cells were then extracted, and the cell extracts were immunoprecipitated with anti-1B subunit antibody, followed by SDS/PAGEand Western blot analysis using anti-phosphotyrosine antibodies.

essential to the mechanism of ligand-dependent transforma-tion by hGMR.

DISCUSSIONIn this paper we show that coexpression of the a andsubunits of hGMR in established murine fibroblasts is suffi-cient to reconstitute a functional receptor, which is capableof causing oncogenic transformation in the presence ofhGM-CSF.hGM-CSF-dependent transformation was observed only in

cells transfected with both the a and subunits, suggestingthat reconstitution of a biologically active receptor requiredthe assembly of a heterodimeric or multisubunit complex.Several observations suggest that formation of the hGMRcomplex in NIH 3T3 cells was very efficient and that theectopically expressed receptor behaved as a potent trans-forming agent. The transforming activity of the activatedreceptor was moderately high, and transformation in thissystem required only picomolar levels of hGM-CSF. In thepresence of ligand, the conditional transformants acquiredthe properties of transformed cells. These cells were veryrefractile, lost contact inhibition, and were capable of an-

chorage-independent growth in soft agar.In the absence of hGM-CSF, the transfectants were indis-

tinguishable from parental NIH 3T3 cells in their morphologyand growth properties, indicating that without ligand, thereceptor subunits had no detectable biological activity. Fur-thermore, the presence of the hGMR a and 8 subunits in thetransfectants did not increase the sensitivity of the cells toother growth factors such as hEGF and hIL-3. Although thehIL-3 receptor utilizes the same (3 subunit as hGMR (4),

hIL-3 was unable to elicit foci even at a concentration1000-fold higher than that required for hGM-CSF-dependenttransformation. Since the hGMR transfectants expressed a

subunits for hGMR but not a subunits for the IL-3 receptor,these results are consistent with the idea that the specificityfor ligand recognition lies with the a subunit and that thesubunit functions primarily as a signaling subunit.

In NIH 3T3 cells, hGMR activation recapitulated some ofthe GM-CSF-dependent signaling events that take place inmyeloid cells. In the transfectants, hGM-CSF induced thephosphorylation of cellular proteins on tyrosine, indicatingthat the activated receptor has a functional interaction withan unidentified cellular tyrosine kinase(s) present in fibro-blasts. Based on our current knowledge about the role oftyrosine phosphorylation in mitogenic signaling, it is likelythat tyrosine phosphorylation is essential for the normal andthe transforming activity of the GMR. Identification of thetyrosine kinases and substrates involved will be an importantstep in understanding the mechanism of action of GMR andthe molecular basis of its oncogenic potential.The hGM-CSF-dependent phosphorylation of the 8 sub-

unit on tyrosine residues is reminiscent of the ligand-dependent autophosphorylation of tyrosine kinase receptors(3). The fact that this biochemical modification has beenconserved in receptors that do not encode tyrosine kinasesargues that tyrosine phosphorylation of the subunit isimportant for some aspect ofhGMR function. With receptortyrosine kinases, autophosphorylation is followed by thespecific binding of src homology 2 (SH2) regions of signalingmolecules such as the ras GTPase-activating protein, phos-pholipase Cy, and the 85-kDa subunit of phosphatidylinositol3-kinase to the phosphorylated sites (23-25). Whether the

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3966 Cell Biology: Areces et al.

Proc. Natl. Acad. Sci. USA 90 (1993) 3967

phosphorylated ,3 subunit of hGMR also binds SH2 proteinsremains to be determined. In the present study, this questionwas not examined further.

Autocrine and paracrine mechanisms involving cytokinesand their receptors are believed to contribute to the devel-opment of leukemias (11-13). However, unlike the case oftyrosine kinase receptors such as those for EGF and themononuclear phagocyte growth factor (CSF-1) (26-28), li-gand-dependent oncogenic activity by a normal cytokinereceptor has not been directly demonstrated before. It hasbeen reported that altered versions of this class of receptorscan cause neoplastic transformation in hematopoietic cells.v-mpl, the transforming gene of myeloproliferative leukemiavirus, encodes a protein consisting of viral env sequencesfused to a cell-derived sequence that resembles a truncatedcytokine receptor (29). v-mpl can immortalize multipotenthematopoietic cells and give rise to factor-independent linesof various hematopoietic lineages. In another example, aretrovirus encoding a modified erythropoietin receptor,which contained a point mutation in the exoplasmic domain,was shown to cause erythroleukemia in mice (30). Presum-ably, these two modified receptors were constitutively acti-vated and caused transformation by interacting with down-stream effectors in the absence of ligand. By contrast, ourstudy shows that structural alterations are not required tounmask the oncogenic potential of hGMR and that in theproper cellular environment the normal receptor can generatea ligand-dependent oncogenic signal. These results providedirect experimental support to paracrine and autocrine mod-els of leukemogenesis involving the normal alleles ofhGMR.The conditional system of transformation described in this

paper should be very useful to identify the tyrosine kinase(s)involved and to define the structural requirements of thereceptor subunits and the ligand, for their functional inter-action during GM-CSF signaling.

Excellent technical assistance by Margaret A. Tate is greatlyappreciated. We also thank Robert Freund and Yen Li for criticalreading of the manuscript. This work was supported by NationalCancer Institute Grant R29 CA 55293-02, by the National LeukemiaAssociation, and by University of Maryland at Baltimore "SpecialResearch Initiative Support" award to R.A.F. M.J. is a recipient ofa Deutsche Forschungsgemeinschaft Fellowship from F.R.G.DNAX Research Institute is supported by Schering-Plough.

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Cell Biology: Areces et al.