A Cell Type-specific Constitutive Point Mutant of the Common beta -Subunit of the Human Granulocyte-Macrophage Colony-stimulating Factor (GM-CSF), Interleukin (IL)-3, and IL-5 Receptors Requires the GM-CSF Receptor alpha -Subunit for Activation*

Brendan J. JenkinsDagger , Fei Le, and Thomas J. Gonda§

From the Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia

    ABSTRACT
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INTRODUCTION
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The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha -subunit (hGMRalpha ) and a common signal-transducing beta -subunit (hbeta c) that is shared with the interleukin-3 and -5 receptors. We have previously identified a constitutively active extracellular point mutant of hbeta c, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287). This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMRalpha (mGMRalpha ) subunit, since introduction of mGMRalpha , but not hGMRalpha , into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence. Experiments utilizing mouse/human chimeric GMRalpha subunits indicated that the species specificity lies in the extracellular domain of GMRalpha . Importantly, the requirement for mGMRalpha correlated with the ability of I374N (but not wild-type hbeta c) to constitutively associate with mGMRalpha . Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMRalpha surface expression. Taken together, these findings suggest a critical role for association with GMRalpha in the constitutive activity of I374N.

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GM-CSF1 is a potent cytokine that promotes the survival, proliferation, differentiation, and functional activity of a wide variety of hemopoietic cell types including monocytes/macrophages, granulocytes, and myeloid progenitor cells (reviewed in Ref. 1). Like other cytokines, GM-CSF exerts its biological activities through binding to specific receptors on the surface of target cells. The high affinity receptor for human GM-CSF (hGMR) is composed of a cytokine-specific alpha -subunit (hGMRalpha ) associated with a common signal-transducing beta -subunit (hbeta c) that is also utilized by the IL-3 and IL-5 receptors (2-6), all of which belong to the cytokine receptor family (reviewed in Ref. 7). Members of this family are characterized by a structurally conserved extracellular cytokine receptor module (CRM) of about 200 amino acids that consists of two fibronectin type III-like domains (8). The beta -subunit has two CRMs, whereas the alpha -subunits contain one CRM and an additional N-terminal domain of about 100 amino acids.

Although the stoichiometry of subunits in active hGMR, hIL-3R, and hIL-5R complexes remains unresolved, it has become clear that ligand-induced alpha -beta -subunit heterodimerization is a key step in the formation of these complexes (9, 10). More recently, it has been shown that beta -subunit homodimers are found in active hGMR (11) and human IL-3R (12) complexes and that the functional hGMR complex may contain at least two alpha -subunits (13). Taken together, these results suggest that the alpha - and beta -subunits may form higher order receptor complexes, and indeed it has been proposed that the GMR/IL-3R/IL-5R normally functions as an alpha 2beta 2 tetramer (10, 12, 13).

The isolation of constitutively active cytokine receptor mutants has provided a useful tool for examining the normal activation process of some receptors (e.g. erythropoietin receptor and c-Mpl (14, 15)), since these mutant receptors most likely mimic the structure of the normal cytokine-activated receptors. With regard to the GMR/IL-3R/IL-5R system, we have previously combined random mutagenesis with retroviral expression cloning to identify constitutively activating point mutations in hbeta c by virtue of their ability to confer factor-independent proliferation on mouse factor-dependent FDC-P1 cells (16, 17). One of these mutations, V449E, is located in the transmembrane domain of hbeta c and is similar to an activating mutation in the neu/c-erbB-2 oncogene (18, 19). By analogy, this mutant most likely acts by inducing hbeta c homodimerization. Another group of activating point mutations, exemplified by I374N, lies in the extracellular region of hbeta c; however, it is unclear precisely how this group might affect receptor function. Interestingly, only certain transmembrane mutants, such as V449E, were able to confer factor independence on mouse factor-dependent BAF-B03 cells, suggesting that the I374N mutation activates hbeta c in a cell type-specific manner.

One possible explanation for the cell type specificity of the I374N mutant is that a molecule that is present in FDC-P1 (and other myeloid) cells is required for its constitutive activity. We report here the use of retroviral expression cloning to identify the mouse GMRalpha (mGMRalpha ) subunit as one such molecule and show that one effect of the I374N mutation is to induce constitutive association with mGMRalpha .

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Cell Lines-- BOSC 23 (20) and Psi 2 (21) ecotropic retroviral packaging cell lines were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. The BING amphotropic retroviral packaging cell line was kindly provided by Prof. Suzanne Cory (Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) with permission from Dr. Warren Pear (MIT, Cambridge, MA) and was maintained as described above. The CTL-EN subline of the mouse IL-2-dependent cell line, CTLL-2 (22), was kindly provided by Dr. John Norton (Paterson Institute for Cancer Research, Manchester) and was maintained as described previously for CTLL-2 cells (16). Mouse IL-3-dependent BAF-B03 cells (23) were maintained as described previously (16). Human factor-dependent UT7 cells (24) were maintained in Dulbecco's modified Eagle's medium plus 10% fetal calf serum supplemented with 2 ng/ml human GM-CSF.

Construction of the FDC-P1 cDNA Library-- cDNA library construction was performed essentially as described by Rayner and Gonda (25). Briefly, cDNA was synthesized from the mouse IL-3/GM-CSF-dependent myeloid cell line FDC-P1 (26) and size-selected for cDNA fragments greater than 500 base pairs. Following digestion with BamHI and XhoI, the size-selected cDNA was ligated directionally into the pRUFNeo retroviral expression vector (25). The library was amplified in Escherichia coli by electroporation of aliquots of the ligated FDC-P1 cDNA. The resultant colonies from each electroporation were harvested, and plasmid DNA was prepared from each pool.

Infection of Target Cells with the FDC-P1 cDNA Library-- Retroviral DNA was used to generate a library of retroviruses by a modification of the method described by Rayner and Gonda (25). Briefly, amphotropic BING packaging cells were transiently transfected using the procedure described by Jenkins et al. (27) with 10 µg of retroviral plasmid per 60-mm culture dish (seeded 18 h previously with 2 × 106 cells). At 48 h post-transfection, virus-containing supernatants were filtered and used to infect ecotropic Psi 2 packaging cells. Infected Psi 2 cells were harvested and selected in medium containing G418 (400 µg/ml) to generate the stable G418-resistant Psi 2 retroviral library. BAF-B03 cells expressing the I374N hbeta c mutant were infected with the Psi 2 retroviral library by co-cultivating 3.75 × 105 BAF/I374N cells with 1.2 × 106 irradiated (30 grays) Psi 2 cells for 48 h in each of eight 25-cm2 culture flasks. The BAF/I374N cells were then harvested, washed, and selected for factor-independent growth in 24-well multidishes (204 wells, each seeded with 105 cells) in liquid culture medium without factor.

PCR Recovery and Sequencing of cDNAs from Factor-independent Cells-- PCR was performed on 100 ng of genomic DNA (prepared essentially as described by Hughes et al. (28)) with an XL PCR kit (Perkin-Elmer) under conditions recommended by the manufacturer. The primers used for amplification were RCF1 (25), which corresponds to the vector gag sequence approximately 80 base pairs 5' of the polylinker in the pRUFNeo vector and RCR2 (5'-ATAGCCTCTCCACCCAAGCG-3'), which corresponds to the MC1Neo sequence 364 base pairs 3' of the polylinker. PCR products were agarose gel-purified, and the 5'- and 3'-ends were sequenced with PCR primers. Internal primers corresponding to cDNA sequences obtained from initial sequencing with PCR primers were subsequently used to fully sequence PCR products. Sequencing reactions were performed using a Taq DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer), and sequence data were obtained by running reactions on an ABI Prism 377 DNA Sequencer.

Receptor Expression Constructs-- The pRUFNeo/mGMRalpha expression construct was generated by subcloning the full-length mGMRalpha cDNA recovered from factor-independent BAF/I374N infectants into the BamHI and HindIII restriction sites of pRUFNeo. The pRUFNeo/hGMRalpha expression construct was generated by inserting the cDNA for hGMRalpha into the XhoI site of pRUFNeo.

To introduce the 8-amino acid DYKDDDDK FLAG polypeptide (Eastman Kodak Co.) at the N terminus of mGMRalpha (FmGMRalpha ), a 5' BamHI/NaeI fragment encoding the signal sequence and first 8 structural residues of mGMRalpha was excised from pRUFNeo/mGMRalpha and replaced in frame with a PCR-generated BamHI/NaeI fragment from pcDNA1Neo/FhIL-3Ralpha (kindly provided by Richard D'Andrea, Hanson Center for Cancer Research, Adelaide, South Australia, Australia) encoding the hIL-3Ralpha signal sequence, FLAG octapeptide, and first 6 structural residues of hIL-3Ralpha . The sense primer corresponded to the T7 promoter sequence and included a BamHI site, and the antisense primer corresponded to codons 19-24 (as numbered by Kitamura et al. (5)) of hIL-3Ralpha and included a NaeI site. The pRUFPuro/FmGMRalpha expression vector was constructed by inserting the BamHI/EcoRI FmGMRalpha cDNA from pRUFNeo/FmGMRalpha into the BamHI and EcoRI sites of the pRUFPuro retroviral expression vector (16).

The HSV-derived 11-amino acid QPELAPEDPED polypeptide (Novagen) was inserted after the signal sequence of the wild-type and I374N mutant beta -subunits (between residues Cys16 and Trp17 as numbered by Hayashida et al. (4)) by site-directed mutagenesis using the pAlter-1 system (Promega) in accordance with the manufacturer's instructions. The modified beta -subunit cDNAs were subcloned into the BamHI and HindIII restriction sites of pRUFNeo.

The following GMRalpha chimeras were generated by PCR amplification and ligation of the relevant portions of human and mouse GMRalpha : (i) the pRUFNeo/halpha malpha 1 chimera encoding the extracellular and transmembrane domains of hGMRalpha (346 amino acids) and the cytoplasmic domain of mGMRalpha (38 amino acids); (ii) the pRUFNeo/halpha malpha 2 chimera encoding the extracellular N-terminal domain of hGMRalpha (117 amino acids) and the extracellular CRM, transmembrane, and cytoplasmic domains of mGMRalpha (262 amino acids); (iii) the pRUFNeo/Fmalpha halpha 1 chimera encoding the extracellular and transmembrane domains of FmGMRalpha (335 amino acids) and the cytoplasmic domain of hGMRalpha (54 amino acids); and (iv) the pRUFNeo/Fmalpha halpha 2 chimera encoding the extracellular FLAG-tagged N-terminal domain of FmGMRalpha (111 amino acids) and the extracellular CRM, transmembrane, and cytoplasmic domains of hGMRalpha (283 amino acids). A full description of the templates and primers used is available upon request.

Extracellular truncations of mGMRalpha were generated by PCR on the pRUFNeo/FmGMRalpha construct with primers designed to amplify the entire construct except for the desired extracellular sequence to be removed while leaving the N-terminal signal sequence and FLAG octapeptide intact. Each PCR was performed with different sense primers corresponding to codons 97-102 (for malpha D1) and codons 195-200 (for malpha D2) and the same antisense primer corresponding to codons 9-14 of mGMRalpha . The blunt ends of each PCR fragment were then ligated together in frame.

The cytoplasmic truncation mutant of mGMRalpha was generated by PCR on the pRUFNeo/FmGMRalpha construct with RCF1 as the sense primer and an antisense primer that contained codons 344-339 of the mGMRalpha cytoplasmic domain together with a HindIII restriction site and termination codon. The PCR products were subcloned into the BamHI and HindIII restriction sites of pRUFNeo.

All PCRs were performed on 20 ng of plasmid DNA with Pfu DNA polymerase (Stratagene) under conditions recommended by the manufacturer. The structures of all mutated or chimeric cDNAs were verified by sequencing.

Infection of Hemopoietic Cells-- Retroviral infection of mouse BAF-B03 cells and CTL-EN cells was performed using either stably transfected Psi 2 packaging cells (16) or transiently transfected BOSC 23 packaging cells as described previously (27). Infected BAF-B03 cells were selected in liquid culture medium containing growth factor and either G418 (1.5 mg/ml) or puromycin (2 µg/ml). Infected CTL-EN cells were selected as described previously for CTLL-2 cells (16).

Retroviral infection of human UT7 cells was performed using amphotropic BING packaging cells based on the method for infecting mouse hemopoietic cells with BOSC 23-derived retroviruses (27). Briefly, BING cells were transiently transfected with 10 µg of retroviral DNA, following which infections were performed by co-cultivating 3 × 105 UT7 cells with the BING cells for 48 h in growth medium supplemented with 4 µg/ml polybrene. Cells were harvested and selected in liquid culture medium containing growth factor and G418 at 1.5 mg/ml.

Analysis of Receptor Subunit Expression by Flow Cytometry-- Expression of receptor subunits on the surface of infected cells was detected by high sensitivity immunofluorescence followed by flow cytometry on an Epics-Profile II analyzer (Coulter). High sensitivity immunofluorescence was performed by incubating cells with primary antibody followed by biotinylated anti-mouse IgG (Vector Laboratories) and streptavidin-phycoerythrin (Caltag Laboratories). Expression of FLAG epitope-tagged mGMRalpha subunits was detected by staining with the anti-FLAG monoclonal antibody M2 (Kodak), and expression of hGMRalpha subunits was detected by staining with the anti-hGMRalpha monoclonal antibody 8G6 (29). Expression of wild-type and I374N mutant beta -subunits on the surface of infected BAF-B03 cells was detected by staining with the anti-hbeta c monoclonal antibody 1C1 (10), whereas HSV epitope-tagged wild-type and I374N mutant beta -subunits expressed on the surface of human UT7 cells were detected by staining with an HSV tag monoclonal antibody (Novagen).

Cell Proliferation Assays-- Infected cells were washed twice, and triplicate samples of equal cell number (5 × 103) were cultured in a 96-well microtiter plate with or without appropriate growth factor for 72 h. Cell proliferation was measured by the CellTiter 96 nonradioactive cell proliferation assay (Promega).

Immunoprecipitation and Immunoblotting-- Cells (2 × 107) were cultured overnight in the absence of growth factor and left unstimulated. Cells were washed with cold PBS containing 20 mM sodium orthovanadate and lysed on ice in lysis buffer (50 mM Hepes (pH 7.5), 150 mM NaCl, 10% glycerol, 1% Nonidet P-40, 2 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA, 1 mM EGTA, 2 mg/ml iodoacetamide, 0.2 mg/ml trypsin inhibitor (Boehringer Mannheim), and CompleteTM protease inhibitor (Boehringer Mannheim)) for 15 min. Insoluble material was removed by centrifugation, and cell lysates were incubated with primary antibody for 2 h at 4 °C. Antibodies used for immunoprecipitation were the anti-hbeta c antibody 8E4 (30) and the anti-FLAG antibody M2 (Kodak). Immune complexes were precipitated with 75 µl of protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C, washed three times with lysis buffer, and boiled in 1× reducing SDS sample buffer. In the case of whole cell protein analyses, samples were lysed in buffer without 10% glycerol, and insoluble material was removed and boiled in 1× reducing SDS sample buffer.

Immunoprecipitates were separated by SDS-polyacrylamide gel electrophoresis on 10% gels and electrophoretically transferred to PolyScreenR polyvinylidene difluoride membranes (NEN Life Science Products). Membranes were then incubated with the anti-hbeta c antibody 1C1 (10), the anti-hGMRalpha antibody 8D10 (29), or the biotinylated anti-FLAG antibody BIOM2 (Kodak), as indicated, following which the membranes were washed and incubated with either an alkaline phosphatase-conjugated anti-mouse antibody (Amersham Pharmacia Biotech) or a streptavidin-conjugated alkaline phosphatase antibody (Molecular Probes, Inc., Eugene, OR), as appropriate. Membranes were washed and subjected to enhanced chemifluorescence detection (Amersham Pharmacia Biotech) as per the manufacturer's instructions, following which they were scanned on a FluorImager (Molecular Dynamics, Inc., Sunnyvale, CA). For reprobing, membranes were stripped in 50 mM Tris (pH 7.4), 2% SDS, 100 mM beta -mercaptoethanol at 55 °C for 20 min; washed; and subsequently probed with the indicated antibodies.

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Isolation of Factor-independent BAF/I374N Cells Infected with an FDC-P1 cDNA Retroviral Expression Library-- We have previously identified a constitutively activating point mutation, I374N, in the extracellular region of hbeta c by virtue of its ability to confer factor-independent growth on FDC-P1 cells (16). Surprisingly, this mutant was unable to confer factor independence on mouse IL-3-dependent BAF-B03 cells, leading us to suggest that the cell type-specific activity of this mutant may reflect the presence of a beta -subunit-associated signaling molecule in FDC-P1 cells, but not in BAF-B03 cells, that is required by this mutant for constitutive activation (16). We therefore reasoned that the introduction of such a molecule from FDC-P1 cells into BAF-B03 cells expressing the I374N mutant should lead to its constitutive activity and thus render these cells factor-independent.

Using procedures described previously (25), an FDC-P1 cDNA library (~8.5 × 105 independent plasmid clones, with an average insert size of 1.1 kb) was generated in the pRUFNeo retroviral expression vector. As described under "Experimental Procedures," the plasmid DNA was used to generate a stable Psi 2 retroviral library estimated to contain ~3.5 × 106 independent viral producer clones, which should adequately represent all cDNA species present in the plasmid library.

BAF-B03 cells expressing I374N (BAF/I374N) were infected by co-cultivation with the virus-producing Psi 2 cells at an infection frequency of 18% (estimated by colony assays in the presence of G418). As a control, parallel infections were also performed on uninfected BAF-B03 cells and BAF-B03 cells expressing wild-type hbeta c. Cells were then selected for factor-independent growth in 24-well multidishes. After 1 week in the absence of factor, 37 of 204 wells seeded with 105 infected BAF/I374N cells contained viable, proliferating cells, while no such cells were present in control cultures. Factor independence was not the result of autocrine growth factor production, since conditioned medium from the factor-independent cell cultures did not support the growth of uninfected BAF-B03 cells (data not shown).

PCR Recovery of Mouse GMRalpha cDNA from Factor-independent BAF/I374N Infectants-- To identify the cDNA sequence carried by the provirus in the factor-independent BAF/I374N infectants, long range PCR was performed with retroviral primers on genomic DNA samples from 17 of the 37 factor-independent cell populations. This revealed a common fragment of approximately 2.3 kb that was amplified from all 17 genomic DNA samples (data not shown); considering the positions of the PCR primers relative to the cloning sites in pRUFNeo, the size of the cDNA insert was estimated to be 1.9 kb. For 8 of the 17 samples, the 2.3-kb fragment was the only PCR product generated, suggesting that these factor-independent cell populations contained only one retroviral insertion and that its presence was responsible for factor independence. Sequence analysis of the 1.9-kb cDNA insert recovered from two of the factor-independent cell populations revealed that it corresponded to the full-length cDNA for the mGMRalpha subunit (31).

Expression of mGMRalpha with I374N in BAF-B03 and CTL-EN Cells Results in Factor Independence-- To confirm that mGMRalpha would allow the constitutive activation of I374N, we expressed the recovered mGMRalpha subunit in BAF/I374N cells and then tested these cells for factor independence. In order to monitor cell surface expression of mGMRalpha , a FLAG epitope-tagged mGMRalpha (FmGMRalpha ) was generated in the pRUFNeo vector (see "Experimental Procedures"). This was introduced into puromycin-resistant BAF/I374N cells as well as wild-type hbeta c-expressing and uninfected BAF-B03 cells. Following selection for G418 resistance, flow cytometric analysis with a FLAG-specific monoclonal antibody indicated that the FmGMRalpha subunit was efficiently expressed on the surface of these cells (Fig. 1A). Upon selection for growth in medium without factor, only BAF-B03 cells co-expressing FmGMRalpha and I374N exhibited factor-independent growth (Fig. 1B). The ability of FmGMRalpha to behave as wild-type mGMRalpha was demonstrated by the proliferation of all FmGMRalpha -infected BAF-B03 cells in response to mGM-CSF (Fig. 1B).


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Fig. 1.   Co-expression of FmGMRalpha with I374N confers factor independence on BAF-B03 cells. A, flow cytometric analysis of FmGMRalpha and beta -subunit expression on G418-selected BAF-B03 cells. Uninfected BAF-B03 cells (uninf) or cells expressing either wild-type (wt) or I374N beta -subunits were infected with a retrovirus encoding the FmGMRalpha subunit and stained with an irrelevant control antibody (dashed line), the anti-hbeta c antibody 1C1 (thin solid line), and the anti-FLAG antibody M2 (thick solid line) by high sensitivity immunofluorescence. Cell number and fluorescence are in arbitrary units; the latter is plotted on a logarithmic scale. Also shown are analyses of cells not exposed to the FmGMRalpha virus. B, proliferation of the BAF-B03 cells depicted in A in the presence of mIL-3 (300 units/ml) or mGM-CSF (80 units/ml) or in the absence of either factor, as indicated. Proliferation assays were carried out, as described under "Experimental Procedures," with 5 × 103 cells plated in triplicate. Error bars indicate the S.E. of the mean of each triplicate.

The observation that the mouse GMRalpha subunit was required for the activity of I374N raised the possibility that another component(s) of the mouse GMR or IL-3R (i.e. mIL-3Ralpha mbeta c or mbeta IL-3) present in FDC-P1 and BAF-B03 cells might also be needed. We therefore introduced I374N and, as a control, wild-type hbeta c with FmGMRalpha into mouse IL-2-dependent CTL-EN cells, which do not express any receptor components belonging to the GMR or IL-3R. CTL-EN cells are a derivative of CTLL-2 cells engineered for increased expression of the ecotropic retroviral receptor (41),2 thereby rendering them more susceptible to retroviral infection. We also included the V449E transmembrane hbeta c mutant in this experiment, since it is inactive when expressed in CTLL-2 cells, although, unlike the I374N mutant, it does confer factor independence on BAF-B03 cells (16). The expression of these subunits was confirmed by flow cytometry (data not shown), following which these cells were tested for factor-independent proliferation. As shown in Fig. 2, only CTL-EN cells expressing both FmGMRalpha and I374N were factor-independent, thereby indicating that components of the mouse IL-3R are not required for the constitutive activity of I374N. In view of this result, all subsequent experiments were performed in BAF-B03 cells.


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Fig. 2.   Factor-independent proliferation of CTL-EN cells co-expressing FmGMRalpha and the I374N mutant. Proliferation of CTL-EN cells expressing the indicated subunits in the absence of factor. Cells were maintained in mouse IL-2 (4 ng/ml) and the appropriate drug selection prior to assay for factor-independent growth.

The I374N Mutation Induces Constitutive Association of hbeta c with mGMRalpha in BAF-B03 Cells-- To examine whether the requirement for mGMRalpha by I374N might reflect a physical association between these two subunits, BAF-B03 cells co-expressing FmGMRalpha with I374N or, as a control, wild-type hbeta c were subjected to immunoprecipitation with an anti-hbeta c antibody, followed by immunoblot analysis with an anti-FLAG antibody. As shown in Fig. 3A, a protein of 60-kDa, consistent with the predicted size of mGMRalpha , was detected only in immunoprecipitates from cell lysates expressing FmGMRalpha and the I374N mutant. Importantly, the converse immunoprecipitation (with anti-FLAG antibody) and immunoblot analysis (with anti-hbeta c antibody) confirmed the physical association between mGMRalpha and the I374N mutant (data not shown). Reprobing the immunoblot with an anti-hbeta c antibody indicated that both wild-type and I374N beta -subunits were immunoprecipitated from the appropriate cell lysates (Fig. 3B). Furthermore, immunoblot analysis of whole cell lysates with an anti-FLAG antibody indicated that the total levels of FmGMRalpha protein present in lysates from all cell populations were comparable (Fig. 3C). Together, these observations indicate that the I374N mutation acts, at least in part, by inducing constitutive association of hbeta c with mGMRalpha .


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Fig. 3.   Co-immunoprecipitation of FmGMRalpha and I374N from BAF-B03 cells. A and B, uninfected BAF-B03 cells and cells expressing the indicated subunits were incubated in medium without factor, and lysates were immunoprecipitated (IP) with the anti-hbeta c antibody 8E4. Immunoprecipitated proteins were analyzed by immunoblotting (IB) with the anti-FLAG antibody M2 (A) or with the anti-hbeta c antibody 1C1 (B). C, whole cell lysates from the indicated BAF-B03 cells were subjected to immunoblotting with the anti-FLAG antibody M2.

The constitutive association of mGMRalpha with the I374N mutant was reminiscent of the ability of human GMRalpha to associate with wild-type hbeta c in the absence of GM-CSF (32). We therefore examined the ability of I374N to associate with hGMRalpha in the absence of ligand, since a failure to do so could explain our previous observation that co-expression of hGMRalpha did not allow constitutive activity of I374N in BAF-B03 cells (Refs. 16 and 27; Fig. 7). The experiment illustrated in Fig. 4A shows, however, that both mutant and wild-type hbeta c could associate equally well with hGMRalpha in the absence (or presence) of ligand, as judged by co-immunoprecipitation from BAF-B03 cells expressing both subunits. Equivalent levels of expression of the beta - and alpha -subunits are confirmed by the analyses of Fig. 4, B and C, respectively.


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Fig. 4.   Ligand-independent association of hGMRalpha with I374N and wild-type hbeta c. A and B, uninfected BAF-B03 cells and cells expressing the indicated hGMR subunits were incubated in medium with (+) or without (-) hGM-CSF (10 ng/ml), and lysates were immunoprecipitated (IP) with the anti-hbeta c antibody 8E4. Immunoprecipitated proteins were analyzed by immunoblotting (IB) with the anti-hGMRalpha antibody 8D10 (A) or with the anti-hbeta c antibody 1C1 (B). C, whole cell lysates from the indicated BAF-B03 cells were subjected to immunoblotting with the anti-hGMRalpha antibody 8D10.

Both the N-terminal and C-terminal Regions of mGMRalpha Are Essential for Activation of and Association with the I374N Mutant-- To broadly define the regions of the mGMRalpha extracellular domain required for the constitutive activation of I374N, two FLAG-tagged extracellular truncation mutants were generated. One of these, Fmalpha D1, lacked residues Leu15-Ala96, which comprise the N-terminal domain, whereas the other, Fmalpha D2, lacked residues Leu15-Glu194, which also includes domain 1 of the cytokine receptor module (CRM; Fig. 4A). Although these truncation mutants (and full-length FmGMRalpha ) were efficiently expressed on the surface of G418-resistant cells (Fig. 4B), neither truncation mutant was able to confer factor independence on BAF/I374N cells (Fig. 4C), indicating that the N-terminal domain of mGMRalpha is required for constitutive activation of I374N. Furthermore, the inability of BAF/I374N cells expressing the malpha D1 mutant to proliferate in the presence of mGM-CSF suggests that the N-terminal domain of mGMRalpha is also important in normal mGMR function.

Considering that the cytoplasmic domain of GMRalpha is essential for normal GM-CSF-mediated cell growth (33), we also investigated whether the cytoplasmic domain of mGMRalpha was required for constitutive signaling by I374N. We therefore generated a cytoplasmic truncation mutant, Fmalpha t3, which lacked the C-terminal 14 amino acids of mGMRalpha (Fig. 5A). Although G418-resistant BAF/I374N infectants efficiently expressed Fmalpha t3 (Fig. 5B), these cells failed to grow in the absence of factor (Fig. 5C) or in response to mGM-CSF. This implies that the C-terminal 14 amino acids of mGMRalpha are essential for mediating factor-independent growth conferred by I374N and also for normal mGM-CSF-mediated growth.


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Fig. 5.   Analysis of BAF/I374N cells expressing FmGMRalpha extracellular and cytoplasmic truncation mutants. A, schematic illustration of the truncated FmGMRalpha subunits showing the regions deleted in each truncation mutant. For comparison, the full-length FmGMRalpha is also shown. The asterisks represent the C terminus of the depicted subunits. B, flow cytometric analysis of BAF/I374N cells superinfected with full-length and truncated FmGMRalpha subunits. Procedures, nomenclature, and axes are as in Fig. 1A. Also shown are "parental" BAF/I374N cells. C, proliferation assay of the BAF/I374N cells depicted in B in the presence of mIL-3 (300 units/ml) or mGM-CSF (80 units/ml) or in the absence of either factor, as indicated.

We next examined whether the inability of the extracellular and cytoplasmic truncation mGMRalpha mutants to confer factor independence on BAF/I374N cells was due to a failure to associate with I374N. Lysates from BAF/I374N cells expressing the Fmalpha D1 extracellular truncation and the Fmalpha t3 cytoplasmic truncation were therefore subjected to immunoprecipitation with an anti-FLAG antibody, followed by immunoblot analysis with an anti-hbeta c antibody. As shown in Fig. 6A, the I374N mutant was precipitated when co-expressed with the full-length FmGMRalpha subunit but not with the truncated FmGMRalpha subunits. Reprobing with an anti-FLAG antibody demonstrated that both full-length and truncated FmGMRalpha subunits were themselves immunoprecipitated (Fig. 6B), and immunoblot analysis of whole cell lysates with an anti-hbeta c antibody confirmed that comparable levels of the I374N mutant were expressed in the cells (Fig. 6C). Thus, these data demonstrate that both the N-terminal and C-terminal regions of mGMRalpha are essential for the association with I374N and, together with the data presented in Fig. 5, that the constitutive activity of I374N is dependent upon this association.


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Fig. 6.   Extracellular and cytoplasmic truncations of FmGMRalpha abolish the co-immunoprecipitation of FmGMRalpha and I374N from BAF-B03 cells. A and B, the BAF-B03 cells expressing the indicated subunits were incubated in medium without factor and lysates were immunoprecipitated (IP) with the anti-FLAG antibody M2. Immunoprecipitated proteins were analyzed by immunoblotting (IB) with the anti-hbeta c antibody 1C1 (A) or with the anti-FLAG antibody M2 (B). The asterisk by Fmalpha in B indicates that the small C-terminal deletion mutant Fmalpha t3 ran at the same size as the full-length FmGMRalpha under the gel conditions employed. C, whole cell lysates from the indicated BAF-B03 cells were subjected to immunoblotting with the anti-hbeta c antibody 1C1.

Species Specificity of GMRalpha for the Constitutive Activation of I374N Lies in Its Extracellular and/or Transmembrane Domains-- In view of our previous observations that co-expression of the human GMRalpha subunit with I374N in BAF-B03 and CTLL-2 cells did not lead to factor-independent growth (16, 27), the ability of the mouse GMRalpha subunit to facilitate constitutive activity of I374N in BAF-B03 and CTL-EN cells was somewhat surprising. To define which region(s) of the GMRalpha subunit govern this apparent species specificity, we constructed a series of chimeric GMRalpha subunits containing regions from both species (Fig. 7A). These chimeras, along with the normal FmGMRalpha and hGMRalpha subunits, were then introduced into BAF/I374N cells and tested for their ability to confer factor independence. Flow cytometric analyses confirmed that while the chimeric GMRalpha subunits were co-expressed with the I374N mutant (Fig. 7B), only cells co-expressing the Fmalpha halpha 1 chimera or, as expected, the normal FmGMRalpha subunit with the I374N mutant exhibited factor-independent proliferation (Fig. 7C). Thus, the species specificity lies in the extracellular and/or transmembrane domains of mGMRalpha . Furthermore, since chimeras containing only the mouse N-terminal domain (Fmalpha halpha 2) or the mouse extracellular CRM and transmembrane domain (halpha malpha 2) were unable to confer factor independence on BAF-B03 cells, it is likely that both of the mGMRalpha regions present in these chimeras contribute to the species-specific requirement for mGMRalpha for I374N activity.


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Fig. 7.   Analysis of BAF/I374N cells infected with retroviruses encoding chimeric mouse and human GMRalpha subunits. A, schematic illustration of chimeric GMRalpha subunits. Regions from the mouse GMRalpha are shown in white, whereas regions from the human GMRalpha are shown in black. For comparison, the normal FmGMRalpha and hGMRalpha subunits are also shown. B, flow cytometric analysis of BAF/I374N cells that were superinfected with retroviruses encoding normal and chimeric GMRalpha subunits and stained with the anti-FLAG antibody M2 (dotted line), the anti-hGMRalpha antibody 8G6 (thick solid line), and the anti-hbeta c antibody 1C1 (thin solid line). Axes are as in Fig. 1A. C, proliferation assay of the BAF/I374N cells depicted in B in the presence of mIL-3 (300 units/ml), mGM-CSF (80 units/ml), or hGM-CSF (1 ng/ml) or in the absence of any factor, as indicated.

The I374N Mutant Confers Factor Independence on Human Hemopoietic Cells: A Possible Role for hGMRalpha in the Constitutive Activity of I374N in Human Cells-- Although the human GMRalpha subunit was unable to facilitate the constitutive activity of I374N in mouse BAF-B03 and CTLL-2 cells (16, 27) (see also Fig. 7), it was conceivable that the I374N mutant might be constitutively active in human cells expressing hGMRalpha . We therefore introduced this mutant and, as a control, wild-type hbeta c into human GM-CSF/IL-3/erythropoietin-dependent UT7 cells and tested these cells for factor-independent proliferation. To distinguish between the introduced beta -subunits and the endogenous beta -subunits expressed by UT7 cells, we inserted an 11-amino acid HSV-derived epitope at the N terminus of both wild-type and I374N beta -subunits. Cells infected with these modified beta -subunits were then selected for G418 resistance or growth in medium without factor. The surface expression of the introduced subunits was confirmed by flow cytometric analysis of infected cells stained with both anti-hbeta c and anti-HSV antibodies (Fig. 8A). In two independent experiments, one of which is shown in Fig. 8B, the I374N mutant allowed factor-independent proliferation of UT7 cells. Factor independence was not the result of low level autocrine growth factor production, since conditioned medium from factor-independent cell pools did not support the growth of uninfected UT7 cells (data not shown).


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Fig. 8.   Analysis of human UT7 cells infected with the I374N mutant. A, flow cytometric analysis of G418-resistant UT7 cells infected with retroviruses encoding the HSV-tagged (as shown by asterisks) wild-type and I374N beta -subunits, except for the panel labeled FI *I374N, which shows staining of cells infected with the HSV-tagged I374N mutant and selected for factor-independent growth. Also shown are analyses of uninfected UT7 cells. Cells were stained with an irrelevant control antibody (dashed line), the anti-hbeta c antibody 1C1 (thin solid line), and the anti-HSV antibody (dotted line) by high sensitivity immunofluorescence. The axes are as in Fig. 1A. B, proliferation assay of the UT7 cells depicted in A in the presence and absence of human GM-CSF (2 ng/ml). C, flow cytometric analysis of hGMRalpha expression on the surface of the UT7 cells depicted in A. Cells were stained with the anti-hGMRalpha antibody 8G6 by high sensitivity immunofluorescence. The axes are as in Fig. 1A.

Unfortunately, to the best of our knowledge, no human factor-dependent hemopoietic cell lines "equivalent" to BAF-B03 cells, i.e. that lack human GMRalpha , are available; thus, we could not directly test the requirement for human GMRalpha by I374N in human hemopoietic cells. Notably, however, flow cytometric analysis with an anti-hGMRalpha antibody revealed that the expression of hGMRalpha was significantly up-regulated on the surface of factor-independent cells expressing I374N (FI *I374N cells) compared with uninfected cells or G418-resistant cells (expressing wild-type hbeta c or I374N) that were not selected for factor independence (Fig. 8C). Importantly, the increase in hGMRalpha expression specifically correlated with the factor independence of I374N-expressing cells. This increase in hGMRalpha expression was not simply a function of high level beta -subunit expression (see FI *I374N histogram in Fig. 8A), since infected UT7 cells that were sorted for comparably high levels of HSV-tagged wild-type hbeta c exhibited a similar low level of hGMRalpha expression to the unsorted cells (*wt) shown in Fig. 8C (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Constitutive Activation of I374N in Mouse Cells Requires mGMRalpha -- The I374N mutation in the extracellular domain of hbeta c confers factor independence on mouse FDC-P1 cells but not BAF-B03 or CTLL-2 cells (16), raising the possibility that cell type-specific signaling molecules are involved in its activation. In this study, we have employed retroviral expression cloning to identify the mGMRalpha subunit as one such molecule, since its introduction into BAF-B03 and CTL-EN (a derivative of CTLL-2) cells expressing the I374N mutant conferred factor independence. Importantly, the absence of the mouse GMR and IL-3R in CTL-EN cells indicates that the mechanism of activation of I374N does not require any subunits, apart from mGMRalpha , of these receptors. In contrast, another hbeta c mutant, V449E, that confers factor independence on both FDC-P1 and BAF-B03 cells (16) is not constitutively active when co-expressed with mGMRalpha in CTL-EN cells. This suggests that the I374N and V449E mutants are activated by fundamentally different mechanisms.

Physical Association of I374N and mGMRalpha -- Co-immunoprecipitation experiments demonstrated that one effect of the I374N mutation in hbeta c is to induce constitutive association with mGMRalpha . The constitutive association between these subunits is reminiscent of a recent report in which hGMRalpha and wild-type hbeta c were co-immunoprecipitated from cell lines in the absence of GM-CSF (32). Factor-independent association with hbeta c appears to be a unique property of GMRalpha , since similar preformed complexes could not be detected with hIL-3Ralpha or hIL-5Ralpha (32). This may in part explain the specific requirement for mGMRalpha , as opposed to mIL-3Ralpha , for constitutive activity of I374N.

We observed that deletions in the extracellular N-terminal domain of mGMRalpha abolished both the constitutive activity of I374N and the association between I374N and mGMRalpha , as well as mGM-CSF-induced proliferative signaling. While the corresponding domains of the hIL-3Ralpha and hIL-5Ralpha subunits have been reported to play a critical role in ligand binding (34-36), our demonstration that the N-terminal domain of mGMRalpha is required for association with the hbeta c mutant suggests that this domain may also play a role in receptor subunit assembly.

Our observation that the cytoplasmic domain of GMRalpha is needed for the activity of I374N was not unexpected, since deletion of the cytoplasmic domains of GMRalpha , IL-3Ralpha , and IL-5Ralpha renders these receptors inactive in proliferative signaling (33, 34, 37). Normally, however, alpha -subunit cytoplasmic truncations do not detectably affect the association of alpha - and beta -subunits, since truncated alpha -subunits still form high affinity ligand-binding receptors (33, 34, 37), and a cytoplasmic truncation of hGMRalpha could still associate with hbeta c in the preformed hGMR complex described by Woodcock et al. (32). Thus, it is surprising that deletion of the C-terminal 14 amino acids of mGMRalpha also abolished the association between mGMRalpha and I374N. Nevertheless, this observation suggests that there may be a degree of interaction between the intracellular domains of alpha - and beta -subunits and that the effect of such an interaction may only be detectable in the context of weaker extracellular interactions between mGMRalpha and I374N as compared with those between wild-type hbeta c and hGMRalpha .

Most importantly, however, the fact that (i) mGMRalpha associates with the I374N mutant but not with wild-type hbeta c and (ii) association of mGMRalpha mutants with I374N correlates with their ability to allow constitutive receptor activity suggests that induction of this association is essential for hbeta c activation. However, constitutive association of hGMRalpha with hbeta c per se is not sufficient for receptor activation (32); thus, it is likely that the I374N mutation has additional effects such as mimicking a ligand-induced conformational change in hbeta c, as we have suggested previously (27, 38).

Determinants of the Species-specific Requirement for GMRalpha for the Constitutive Activity of I374N-- In view of the ability of mouse GMRalpha to allow constitutive activity of I374N in mouse cells, it is somewhat surprising that co-expression of the human GMRalpha subunit with I374N in mouse BAF-B03 and CTLL-2 cells does not lead to factor-independent proliferation (Refs. 16 and 27; see also Fig. 7C). This is not due to the inability of I374N to interact with hGMRalpha because their co-expression in BAF-B03 and CTLL-2 cells results in the formation of a high affinity receptor and generation of a proliferative signal in response to human GM-CSF (16, 27). Moreover, I374N, like wild-type hbeta c (32), also efficiently co-immunoprecipitates with hGMRalpha in the absence of hGM-CSF (Fig. 4).

Our studies with mouse/human chimeric GMRalpha subunits showed that only the chimera containing the entire extracellular and transmembrane domains of mGMRalpha conferred factor independence on BAF/I374N cells, while in contrast, the human and mouse cytoplasmic domains were interchangeable. This suggests two possible explanations for species specificity: (i) that the extracellular domain of mGMRalpha interacts with I374N in a different manner from that of its human homologue, to allow formation of an active complex in the absence of ligand or (ii) that mGMRalpha interacts with a membrane-spanning accessory signaling molecule in a species-specific manner. The latter explanation would also suggest that species specificity might also be a function of the host cell species, i.e. that the accessory molecule might interact preferentially with the GMRalpha subunit of the same species. However, the fact that the I374N mutant was able to confer factor-independent proliferation on human GM-CSF/IL-3/erythropoietin-dependent UT7 cells argues against an exclusive requirement for murine GMRalpha . Moreover, the expression of hGMRalpha on the surface of the factor-independent cells was significantly up-regulated, suggesting that selection for factor independence also selected for hGMRalpha subunit expression. This is consistent with the notion that GMRalpha is involved in the constitutive activation of I374N in these cells also and that the species-specific requirement of GMRalpha for the constitutive activity of I374N may reflect the species of cell in which the mutant is expressed.

The relevance of the requirement for GMRalpha by I374N, and indeed other extracellular hbeta c mutants, to the activity of these mutants in primary hemopoietic cells should be noted. Retroviral infection of mouse fetal liver progenitors with the I374N mutant and another extracellular hbeta c mutant (FIDelta ; Ref. 39) results in the formation of factor-independent cells of only granulocytic and monocytic lineages (40), the two major lineages controlled by GM-CSF (reviewed in Ref. 1). This observation is consistent with the notion that the constitutive activity of I374N and other extracellular hbeta c mutants is restricted to cells expressing GMRalpha .

Finally, the prediction that the extracellular (I374N) and transmembrane (V449E) constitutive mutants of hbeta c appear to act (at least in part) by inducing alpha -beta or beta -beta dimerization, respectively, is interesting in light of evidence that the wild-type GMR/IL3R/IL5R may contain both alpha -beta and beta -beta dimers (see the Introduction). It is tempting to speculate that each class of mutant might activate a subset of the multiple, overlapping signaling pathways activated by the wild-type receptor complex. Thus, they may constitute useful tools for dissecting receptor signaling.

    ACKNOWLEDGEMENTS

We are grateful to Dr. John Rayner for assistance with construction of the FDC-P1 cDNA retroviral expression library, Sun Qiyu and Prof. Angel Lopez for supplying anti-receptor antibodies, and various colleagues who generously supplied growth factors. We thank Alan Bishop and Sandy McIntyre for assistance with flow cytometry and Arthur Mangos for automated sequencing analyses. We also thank Dr. John Norton (Paterson Institute for Cancer Research, Manchester) for the CTL-EN cell line and Dr. Richard D'Andrea for discussions and critical reading of the manuscript.

    FOOTNOTES

* This work was supported by a research grant from the National Health and Medical Research Council (NHMRC) of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a Dawes postgraduate scholarship from the Royal Adelaide Hospital. Present address: Fred Hutchinson Cancer Research Center, 1100 Fairview Ave. N., Seattle, WA 98109-1024.

§ A Senior Research Fellow of the NHMRC. To whom correspondence should be addressed: Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Rd., Adelaide, South Australia 5000, Australia. Tel.: 61-8-8222-3305; Fax: 61-8-8232-4092; E-mail: Tom.Gonda{at}imvs.sa.gov.au.

2 J. Norton, personal communication.

    ABBREVIATIONS

The abbreviations used are: GM-CSF granulocyte-macrophage colony-stimulating factor, mGM-CSF, mouse GM-CSF; GMR, GM-CSF receptor; hGMR, human GMR; mGMR, mouse GMR; GMRalpha , GMR alpha -subunit; IL, interleukin; hIL, human IL; IL-3R and IL-5R, interleukin-3 and -5 receptors, respectively; beta c, common beta -subunit of the GM-CSF, IL-3 and IL-5 receptors; hbeta c, human beta c; CRM, cytokine receptor module; PCR, polymerase chain reaction; kb, kilobase pair(s); wt, wild type; HSV, herpes simplex virus.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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