granulocyte/macrophage colony-stimulating factor and tyrosine phosphorylation of the receptor fJ subunit. (cytokine receptor/cell transformation/phosphotyrosine).
Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3%3-3967, May 1993 Cell Biology
Ligand-dependent transformation by the receptor for human granulocyte/macrophage colony-stimulating factor and tyrosine phosphorylation of the receptor fJ subunit (cytokine receptor/cell transformation/phosphotyrosine)
LILIANA B. ARECES*, MANFRED JUCKER*, JULIE A. SAN MIGUEL*, ALICE MUIt, ATSUSHI MIYAJIMAt, RICARDO A. FELDMAN**§
AND
*Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201; *Medical Biotechnology Center, University of 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/macrophage colony-stimulating factor (hGMR) is composed of two subunits, a and 3, which are both required for high-affinity binding of the ligand. To examine the transforming potential of hGMR, we have transfected cDNAs encoding the receptor a and (3 subunits into NIH 3T3 cells, which normally do not express GMRs. Introduction of the receptor subunits into these cells resulted in focal transformation, which was dependent on the presence of human granulocyte/macrophage colony-stimulating factor (hGM-CSF) in the culture medium. No transformation was observed when hGM-CSF was replaced with other growth factors such as human epidermal growth factor or human interleukin 3 or when cells were transfected with the a or (3 subunit alone. Individual conditional transformants isolated after transfection expressed functional hGMRs, were susceptible to transformation by picomolar levels of the ligand, and were capable of anchorage-independent growth in soft agar in the presence but not in the absence of hGM-CSF. Biochemical analysis showed that treatment of these cells with hGM-CSF caused a rapid phosphorylation of the (3 subunit and other cellular proteins on tyrosine residues, recapitulating some of the events that take place during GM-CSF signaling in myeloid cells. We conclude that coexpression of the a and 1B subunits ofhGMR in established murine fibroblasts is sufficient to reconstitute a functional receptor, which is capable of causing liganddependent transformation. The oncogenic potential of hGMR lends support to the hypothesis that its deregulated or abnormal expression may play a role in leukemogenesis.
cyte-macrophage, granulocytic, and other cell types (1). The receptor for human (h) GM-CSF is composed of two subunits, termed a and 1, which are transmembrane proteins of 75 kDa and 120 kDa, respectively (8, 9). The a subunit bears the specificity for ligand recognition and can bind hGM-CSF with low affinity, whereas the 13 subunit, which is believed to function as a signal transducer, cannot bind hGM-CSF by itself but is required for high-affinity binding (10). In humans, it has been shown that the same 1 subunit is utilized by the receptors for GM-CSF, IL-3, and IL-5, providing an explanation for the overlapping biological activities of these cytokines (4). It has been proposed that the autocrine production of cytokines by leukemic cells has a role in the generation of leukemias (11-13). However, oncogenic activity by normal cytokine receptors has not been directly demonstrated. To examine this question, we have analyzed the ability of the hGM-CSF receptor (hGMR) to transform established murine fibroblasts. In this paper we show that cotransfection of NIH 3T3 cells with the a and 83 subunits of hGMR can lead to oncogenic transformation in the presence of hGM-CSF and that ligand binding is accompanied by phosphorylation of the receptor 13 subunit and other cellular proteins on tyrosine residues.
MATERIALS AND METHODS Cells. The stock of NIH 3T3 cells used in this study has been described (14). NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with 10% (vol/vol) fetal calf serum. Plasmid DNA. The plasmid pCEVGMR-a, which encodes the a subunit of hGMR and has a hygromycin-resistance gene, has been described (8). pKH97 is a plasmid encoding the 13 subunit of hGMR (9). pcDSR298 encodes a full-length hGM-CSF cDNA (15). pME18S is a control vector that contains no insert (10). DNA Transfection and Hygromycin Selection. NIH 3T3 cells were transfected by the calcium phosphate method (16) using 300 ng of each plasmid DNA per 35-mm plate as described (14). Twenty-four hours later cells were trypsinized and seeded in 60-mm plates for the focus assay or in 100-mm dishes for hygromycin selection in the presence of hygromycin (Sigma) at 0.2 mg/ml. Individual colonies resistant to the drug were isolated 2-3 weeks later. Antibodies and Growth Factors. The rat monoclonal antibody CRS1, directed against the 13 subunit of hGMR, has
Cytokines play important roles in the control of hematopoietic cell development and the coordination of host immune responses (1). The receptors for cytokines such as granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin (IL) 4, and erythropoietin belong to a large family of structurally related molecules (2), which are responsible for the transduction and sorting of signals that are initiated at the cell surface. Unlike the receptors for epidermal growth factor (EGF) and platelet-derived growth factor, which are single polypeptide chains with intrinsic protein-tyrosine kinase activity (3), many cytokine receptors are composed of at least two distinct subunits, with no recognizable kinase domains (4). Nevertheless, a number of cytokines such as GM-CSF and IL-3 can induce the phosphorylation of cellular proteins on tyrosine (5-7), indicating that cytokine receptors have a functional association with as yet unidentified cellular tyrosine kinases. GM-CSF is a hematopoietic growth factor that regulates proliferation, differentiation, and effector functions of mono-
Abbreviations: GM-CSF, granulocyte/macrophage colony-stimulating factor; h, human; hGMR, hGM-CSF receptor; IL, interleukin; EGF, epidermal growth factor. §To whom reprint requests should be addressed at *.
The publication costs of this article were defrayed in part by page charge payment. 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)
been described (17). The anti-phosphotyrosine mouse monoclonal antibody 4G10 was purchased from Upstate Biotechnology (Lake Placid, NY). Purified recombinant hGM-CSF and hIL-3 were produced in Escherichia coli (10). Recombinant hEGF was purchased from Promega. Radioligands and Binding Assays. Recombinant hGM-CSF was iodinated using the Bolton-Hunter reagent, and binding assays and analysis of equilibrium binding data were performed as described (10). Stimulation with hGM-CSF. For hGM-CSF stimulation, cells were preincubated for 2 hr in DMEM supplemented with 0.1% (wt/vol) bovine serum albumin (fraction V; Sigma) after which time, the cells were incubated with hGM-CSF as indicated in the figure legends. After incubation, cells were extracted as described below. Preparation of Cell Extracts and Protein Analysis. Preparation of cell extracts for protein analysis was carried out as described (18) in a buffer containing 50 mM Hepes/KOH (pH 7.4), 1% (vol/vol) Triton X-100, 150 mM NaCl, 1.5 mM MgCl2, 10% (vol/vol) glycerol, 10 mM sodium pyrophosphate, 100 mM NaF, 1 mM sodium orthovanadate, and 2% Trasylol (FBA Pharmaceuticals, New York). The analysis of proteins by immunoprecipitation, electrophoresis in SDS/ 8.5% PAGE gels, and Western blot analysis with antibodies directed against phosphotyrosine have been described (18, 19).
Table 1. Focus-forming activity of hGMR conditional transformants
FFU/,ug of DNA - hGM-CSF + hGM-CSF 0 0 0 0 0 177 2 0 0 0 0 ,8 + vector a+ 8 0 302 The indicated plasmid DNAs were transfected into NIH 3T3 cells as described in the Materials and Methods, and foci of transformed cells were scored 2 weeks after transfection. FFU, focus-forming units. The transfected a, /3, and vector plasmids were PCEVGMR-a, pKH97, and pME18S, respectively. Exp. 1
Plasmid ta + vector 3 + vector a + ,8 a + vector
dependent foci consisted of highly refractile cells that had lost contact inhibition (Fig. 1B). Similar results were obtained when hGM-CSF was supplied by an autocrine mechanism. Cotransfection of cDNAs encoding hGM-CSF and the receptor a and ,3 subunits resulted in focal transformation in the absence of added hGM-CSF, at a slightly higher frequency than in the paracrine system described above (data not shown). We conclude from these results that coexpression of the a and ,B subunits of hGMR in NIH 3T3 cells can reconstitute a biologically active receptor, which is capable of causing efficient transformation in the presence of hGM-CSF. Biological Properties of the hGMR Transfectants. To examine the properties of cells that express the hGMR, individual transfectants were isolated. Since the a subunit of hGMR was cloned into a vector that confers resistance to the antibiotic hygromycin, individual colonies were isolated by growing cells cotransfected with the a and A3 subunits in the presence of this drug. Hygromycin-resistant colonies were cylinder cloned and expanded, and sister plates of each colony were incubated in the presence or absence of hGMCSF. Without hGM-CSF, no transformation was observed with any of the isolated clones. By contrast, when hGM-CSF was present in the growth medium, 12% of the individual clones examined became fully transformed 3-4 days after addition of the ligand. Two of these clones were kept for further analysis as described below. Quantitative binding analysis with 1251-labeled hGM-CSF showed that the selected clones expressed between 1500 and 3800 high-affinity receptors per cell, confirming that the conditional hGM-CSF transformants expressed functional
RESULTS Coexpression of the a and 18 Subunits of hGMR in NIH 3T3 Cells Causes Cell Transformation in the Presence of hGMCSF. To determine if expression of hGMR in NIH 3T3 cells would alter the growth properties of these cells, cDNAs encoding the a and P subunits of hGMR were cotransfected by the calcium phosphate procedure, and the transfectants were incubated in the presence or absence of recombinant hGM-CSF at 20 ng/ml (1 nM). In the presence of hGM-CSF, foci of morphologically transformed cells were detected 6 days after transfection (Fig. 1A, plate h), but in its absence, no focal changes were observed (Fig. 1A, plate d). Untransfected cells or cells transfected with the a or ,B subunit alone did not develop foci in the absence or presence of hGM-CSF (Fig. 1A). The efficiency of transformation measured 2 weeks after transfection was 180-300 foci per ,g of plasmid DNA (Table 1). This transforming activity is 5-10 times lower than that 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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