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Related Collections Hematopoiesis Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, Vol. 92 No. 6 (September 15), 1998: pp. 1989-2002 Saturation Mutagenesis of the  Subunit of the Human Granulocyte-Macrophage Colony-Stimulating Factor Receptor Shows Clustering of Constitutive Mutations, Activation of ERK MAP Kinase and STAT Pathways, and Differential  Subunit Tyrosine Phosphorylation By Brendan J. Jenkins, Timothy J. Blake, and Thomas J. Gonda From the Hanson Centre for Cancer Research and Division of Human Immunology, Institute of Medical and Veterinary Science, Adelaide, South Australia, Australia.     ABSTRACT Abstract Introduction Methods Results Discussion References The high-affinity receptors for human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and IL-5 are heterodimeric complexes consisting of cytokine-specific subunits and a common signal-transducing  subunit (hc). We have previously demonstrated the oncogenic potential of this group of receptors by identifying constitutively activating point mutations in the extracellular and transmembrane domains of hc. We report here a comprehensive screen of the entire hc molecule that has led to the identification of additional constitutive point mutations by virtue of their ability to confer factor independence on murine FDC-P1 cells. These mutations were clustered exclusively in a central region of hc that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hc mutants exhibited cell type-specific constitutive activity, with only two transmembrane domain mutants able to confer factor independence on both murine FDC-P1 and BAF-B03 cells. Examination of the biochemical properties of these mutants in FDC-P1 cells indicated that MAP kinase (ERK1/2), STAT, and JAK2 signaling molecules were constitutively activated. In contrast, only some of the mutant  subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hc activation, dissociate hc tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hc. © 1998 by The American Society of Hematology.     INTRODUCTION Abstract Introduction Methods Results Discussion References THE HIGH-AFFINITY RECEPTORS for human granulocyte-macrophage colony-stimulating factor (GM-CSF; hGMR), interleukin-3 (IL-3; hIL-3R), and IL-5 (hIL-5R) are composed of cytokine-specific subunits (hGMR, hIL-3R, and hIL-5R) associated with a common signal-transducing  subunit (hc).1-5 Each receptor subunit belongs to the cytokine receptor family, members of which are characterized by an extracellular cytokine receptor module (CRM) containing several conserved sequence elements, including the distinctive WSXWS (Trp-Ser-Xaa-Trp-Ser) motif, and a cytoplasmic domain that lacks any intrinsic enzymatic activity associated with signal transduction (reviewed in Mui and Miyajima6). Despite the absence of intrinsic tyrosine kinase activity in the  and  subunits of the hGMR, hIL-3R, and hIL-5R, cytokine binding to these receptors results in the induction of several cellular responses, such as tyrosine phosphorylation of intracellular substrates, including the  subunit itself, and activation of the Ras-Raf-MAP kinase and JAK2-STAT5 signaling pathways.5,7-11 Although the definitive mechanisms underlying both cytokine receptor activation and the subsequent activation of signaling pathways in response to ligand binding have not been fully elucidated, an essential event in cytokine receptor activation is the ligand-induced multimerization of receptor subunits. Direct evidence for this has been provided by the crystal structure of the ligand-bound human growth hormone receptor complex in which a receptor homodimer is bound by one ligand molecule.12 The isolation of constitutively active cytokine receptor mutants has also provided a useful tool for examining the normal activation process of some receptors, because these mutant receptors most likely mimic the structure of the normal cytokine-activated receptors. For example, constitutive point mutations that replace specific residues with cysteines in the extracellular region of the receptors for erythropoietin and thrombopoietin (c-Mpl) result in constitutive disulphide-linked receptor homodimerization, suggesting that ligand-induced homodimerization is required for signaling by the normal receptors.13-15 Indeed, the recently published crystal structure of an erythropoietin receptor homodimer bound to a peptide agonist also provides strong evidence for the involvement of homodimerization in erythropoietin receptor activation.16 In the case of the normal hGMR, hIL-3R, and hIL-5R, there is increasing evidence that the formation of active hGMR and hIL-3R complexes involves ligand-induced  and  subunit heterodimerization,17,18 although the precise stoichiometry of receptor subunits in the active complexes remains unresolved. Chimeric receptors containing the hc cytoplasmic domain fused to the extracellular domains of hGMR or hIL-5R have been reported to confer cytokine-dependent growth on hematopoietic cells, suggesting that dimerization of the hc cytoplasmic domain is sufficient for cellular proliferation.17,19,20 Consistent with a role for  subunit dimerization in receptor activation, it has been shown that  subunit homodimers are found in active hGMR complexes.21 More recently, it was observed that the functional hGMR complex may contain at least two  subunits.22 In the context of receptor stoichiometry, these results suggest that the  and  subunits of these receptors may form higher order complexes.22,23 We have previously combined polymerase chain reaction (PCR)-based random mutagenesis with retroviral expression cloning to screen for constitutive point mutations within a membrane-spanning region of hc. This led to the identification of two mutations, one of which is located in the transmembrane domain of hc (V449E) and is able to confer factor independence on FDC-P1 and BAF-B03 cells.24 This mutation is similar to a constitutive mutation in the neu/erbB-2 oncogene25,26 and, by analogy, has been proposed to act by inducing constitutive hc homodimerization.24 The other constitutive point mutation lies in the extracellular region of hc (I374N) and confers factor independence on FDC-P1 cells, but not BAF-B03 cells, suggesting that there are alternate mechanisms, possibly involving cell type-specific signaling molecules, by which hc can be activated.24,27 In addition to the above-mentioned I374N and V449E mutants, we have recently used a site-directed mutagenesis approach to identify amino acid substitutions at two other residues in the extracellular region of hc, Leu356, and Trp358 that constitutively activate hc in FDC-P1 cells.28 Interestingly, the Leu356 and Trp358 residues lie within the same membrane-spanning region of hc that was screened for constitutive point mutations, raising the possibility that other constitutive point mutations were missed in this screen. To address this issue, we report here a comprehensive screen of the entire hc molecule for constitutive point mutations by combining random mutagenesis with a simplified retroviral expression strategy and a more sensitive screen. The efficiency of this strategy was demonstrated by the identification of several novel constitutive point mutations in hc, most of which exhibit cell type-specific differences in constitutive activity. Interestingly, these mutations are clustered exclusively in the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain of hc, reflecting, we suggest, key sites involved in normal GMR/IL-3R/IL-5R activation. We have also initiated an investigation into the effect these constitutively active mutants have on certain intracellular signaling events in factor-independent FDC-P1 cells. Surprisingly, examination of the tyrosine phosphorylation state of the mutant  subunits indicated that only some mutants were constitutively tyrosine phosphorylated. In contrast, ERK1/2 MAP kinase and STAT signaling molecules involved in the Ras-Raf-MAP kinase and JAK-STAT pathways, respectively, were constitutively activated by all mutants.     MATERIALS AND METHODS Abstract Introduction Methods Results Discussion References Cell Lines BOSC 23 retroviral packaging cells29 and 2 cells producing wild-type hGMR retrovirus24 were maintained as described previously.28 The mouse IL-3/GM-CSF-dependent myeloid cell line, FDC-P1,30 and the BAF-B03 subline31 of the mouse IL-3-dependent pro-B-cell line, Ba/F3, were maintained as described previously.24 Site-Directed Mutagenesis and Construction of Expression Plasmids The hc cDNA used here was that described by Barry et al32; amino acids are numbered from the initiating codon. Site-directed mutagenesis was performed on double-stranded DNA with mutagenic oligonucleotides using the Altered Sites in vitro mutagenesis system (Promega, Madison, WI) in accordance with the manufacturer's instructions. All mutations were confirmed by DNA sequencing, after which mutant hc cDNAs were subcloned between the BamHI and HindIII restriction sites of the pRUFNeo retroviral expression vector.33 PCR Mutagenesis and Construction of Point-Mutated hc cDNA Libraries PCR mutagenesis was performed on the pRUFNeo-hc plasmid in which a Xho I site was silently introduced into wild-type hc to facilitate cloning of PCR products (Fig 1). Random point mutations were introduced into the N-terminal 770-bp BamHI/Xho I segment, bases 1-770, and the C-terminal 1,017-bp Bgl II/Sal I segment, bases 1705-2722, of the hc cDNA2 (sequence accession no. M38275) at a mutation frequency of approximately 0.3% (1/350) under the mutagenic reaction conditions described by Jenkins et al.24 The primers used for amplification of the N-terminal region were the RCF1 primer,33 corresponding to the gag sequence in the pRUFNeo vector, and an internal hc primer (5-AGCTGGCCACCTCCTTCCTCACCT-3, bases 839-816) defining a 937-bp fragment. The primers used for amplification of the C-terminal region were an internal hc primer (5-CCCCAAGCATGTCTGTGATCCACC-3, bases 1651-1674) and the RCR1 primer,33 corresponding to the MC1Neo sequence in the pRUFNeo vector, defining a 1,105-bp fragment. After digestion with the appropriate restriction enzymes, mutant fragments were agarose gel-purified and ligated directionally into pRUFNeo-hc from which the BamHI/Xho I or Bgl II/Sal I segment of hc had been excised. After transformation of Escherichia coli (DH10B), the resultant point-mutated hc cDNA libraries of approximately 3.5 × 104 (BamHI/Xho I) and approximately 9.5 × 104 (Bgl II/Sal I) independent plasmid clones were further amplified as described previously.33 View larger version (25K): [in this window] [in a new window]   Fig 1. Outline of the strategy used to generate and express hc mutants. Schematic illustration of hc showing the signal sequence (shading), the two cytokine receptor modules (CRMs72) containing the conserved cysteine residues (thin vertical lines) and the characteristic WSXWS motifs (thick vertical lines; see Bazan73 for a description of these elements), and the transmembrane and cytoplasmic domains. Also included is a schematic diagram of the pRUFNeo retroviral expression vector containing the hc cDNA. The positions in the hc cDNA of the BamHI, Xho I, Bgl II, and Sal I restriction sites that define the regions subjected to random mutagenesis are shown underneath. The arrows above the cDNA represent the primers used for PCR amplification/mutagenesis of the N-terminal and C-terminal hc fragments; they lie just outside the restriction sites defining the mutagenized regions (see Materials and Methods). The construction of the Xho I/Bgl II point-mutated hc cDNA library has been described previously.24 Infection of Hematopoietic Cells With Mutant hc Retroviruses High-titer retroviruses carrying mutant hc cDNAs were generated by transiently transfecting BOSC 23 retroviral packaging cells with retroviral DNA essentially as described by Jenkins et al.28 For the generation of retroviruses representing the three point-mutated hc libraries, one 60-mm dish (BamHI/Xho I library), five 60-mm dishes (Xho I/Bgl II library), and two 60-mm dishes (Bgl II/Sal I library) were each seeded with 2 × 106 BOSC 23 cells and transfected with 20 µg of the appropriate retroviral DNA. Infections of FDC-P1 cells were performed by cocultivation as described previously,28 with 2.5 × 105 FDC-P1 cells added to each dish. FDC-P1 cells from each dish were harvested, washed, and selected for factor-independent growth in 24-well multidishes (84 wells for BamHI/Xho I library, 204 wells for Xho I/Bgl II library, and 102 wells for Bgl II/Sal I library, each seeded with 2 × 104 cells) in liquid culture medium without mouse (m) GM-CSF. Factor-independent cells were expanded in liquid culture to 25-cm2 flasks for further analysis. FDC-P1 cell plating in soft agar was performed essentially as described by Johnson34; mGM-CSF (80 U/mL) or G418 (1 mg/mL) was added as required. Retroviral infection of FDC-P1 and BAF-B03 cells with hc point mutants generated by site-directed mutagenesis was performed using BOSC 23 cells, and cells were harvested and selected as described previously.28 FDC-P1 and BAF-B03 cells infected with wild-type hGMR retrovirus were selected as described previously.28 Recovery of Mutant hc cDNAs From Factor-Independent Cells Genomic DNA was isolated from cells using a proteinase K/SDS procedure essentially as described by Hughes et al.35 PCR was performed on 100 ng of genomic DNA with Pfu DNA polymerase (Stratagene, La Jolla, CA) under conditions recommended by the manufacturer. The primers and cycling parameters used were those for generating the point-mutated hc cDNA libraries. PCR products were agarose gel-purified and directly sequenced with the PCR primers using a Taq DyeDeoxy Terminator Cycle Sequencing Kit (Perkin Elmer, Norwalk, CT), followed by analysis on an ABI Prism 377 DNA Sequencer (ABI, Foster City, CA). Flow Cytometric Analysis of Receptor Subunit Expression Expression of hc mutants on the cell surface of infected FDC-P1 or BAF-B03 cells was detected by standard indirect immunofluorescence with the anti-hc monoclonal antibody 1C118 and fluorescein isothiocyanate (FITC)-conjugated antimouse IgG (Silenus, Hawthorn, Victoria, Australia) followed by flow cytometry on an Epics-Profile II analyser (Coulter, Hialeah, FL). hGMR expression was analyzed as described above with the 8G6 monoclonal antibody.36 Cell Proliferation Assays Infected FDC-P1 or BAF-B03 cells were washed twice and triplicate samples of equal cell number (103 or 5 × 103) were cultured in 96-well microtiter plates with or without appropriate growth factor for 72 hours. Cell proliferation was measured by the CellTiter 96 Non-Radioactive Cell Proliferation Assay (Promega). Antibodies The monoclonal hc antibodies, 8E4 and 1C1, were a kind gift from Angel Lopez (Hanson Centre for Cancer Research, Adelaide, Australia). An anti-MAP kinase (ERK1/2) antibody was purchased from Zymed Laboratories (San Francisco, CA), and the antibody specific for the active phosphorylated form of MAP kinases ERK1/2 was purchased from Promega. Horseradish peroxidase-conjugated antiphosphotyrosine antibody, RC20, was purchased from Transduction Laboratories (Lexington, KY). The antiphosphotyrosine antibody, 4G10, and an anti-JAK2 polyclonal antibody were purchased from Upstate Biotechnology (Lake Placid, NY). Immunoprecipitation and Western Blot Analysis For stimulation with human GM-CSF (hGM-CSF), cells (2 to 4 × 107) were initially starved for 12 hours in growth media without cytokine. Cells were left unstimulated or were stimulated with hGM-CSF (50 ng/mL) at 37°C for 10 minutes or, in some cases, cells were grown continuously in the presence of 2 ng/mL hGM-CSF. Cells were washed with cold phosphate-buffered saline (PBS) containing 20 mmol/L sodium orthovanadate and lysed on ice in lysis buffer (50 mmol/L HEPES [pH 7.5], 150 mmol/L NaCl, 1% NP-40, 2 mmol/L sodium orthovanadate, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L EDTA, 1 mmol/L EGTA, 2 mg/mL iodoacetamide, 0.2 mg/mL trypsin inhibitor [Boehringer Mannheim, Indianapolis, IN], and Complete protease inhibitor [Boehringer Mannheim]) for 15 minutes. Insoluble material was removed by centrifugation at 12,000 rpm for 2 minutes and samples were taken for protein estimation (Dc protein assay kit; Bio-Rad Laboratories, Richmond, CA). Cell lysates were incubated with the indicated antibody for 1 hour at 4°C. Immune complexes were precipitated with 75 µL (50% slurry) of protein A-sepharose (Pharmacia) for 1 hour at 4°C, washed three times with lysis buffer, and boiled for 2 minutes in 1× reducing sodium dodecyl sulfate (SDS) sample buffer. In the case of total protein analysis, samples were lysed and insoluble material was removed and boiled for 2 minutes in 1× reducing SDS sample buffer. JAK2 phosphorylation was detected in cells (1 × 108) treated (±hGM-CSF) as described above, except that 500 nmol/L sodium orthovanadate was added to the medium for 10 minutes before lysis. Cells were lysed on ice in lysis buffer with the addition of 0.1% deoxycholic acid and 0.1% SDS and immunoprecipitated as described. Proteins were electrophoresed on SDS-polyacrylamide gels and electrophoretically transferred to 0.2-µm nitrocellulose (Schleicher and Schuell, Keene, NH). After blocking with TBS-T (50 mmol/L Tris [pH 7.4], 135 mmol/L NaCl, 0.1% Tween 20) containing 3% bovine serum albumin (BSA; Fraction V; Boehringer Mannheim), membranes were incubated with the primary antibody and washed three times in TBS-T. After incubation with goat antirabbit or antimouse secondary antibodies coupled with horseradish peroxidase (Pierce, Rockford, IL), filters were washed with TBS-T three times and subjected to enhanced chemiluminescence detection (Pierce). Before reprobing with the indicated antibodies, membranes were stripped in 50 mmol/L Tris (pH 7.4), 2% SDS, 100 mmol/L -mercaptoethanol at 55°C for 20 minutes; washed three times in TBS-T; and blocked in TBS-T containing 3% BSA. Electrophoretic Mobility Shift Assay (EMSA) Nuclei from cells washed and lysed as described above were pelleted by centrifugation at 12,000 rpm for 2 minutes. Nuclei were resuspended in lysis buffer (without NP-40) supplemented with 150 mmol/L NaCl and 10% glycerol. After incubation at 4°C for 15 minutes, insoluble material was removed by centrifugation at 12,000 rpm for 5 minutes and samples were stored at 70°C. EMSAs were performed using a -casein promoter probe that contains a binding site for STAT1, STAT3, and STAT5 essentially as described by Barry et al.37     RESULTS Abstract Introduction Methods Results Discussion References Isolation of Factor-Independent FDC-P1 Cells Infected With the Membrane-Spanning Xho I/Bgl I Point-Mutated hc Library We have previously combined PCR-based random mutagenesis with retroviral expression cloning to identify two distinct constitutive point mutations within a region of hc, delimited by Xho I and Bgl II sites in hc cDNA (see Fig 1), by virtue of their ability to confer factor independence on the murine factor-dependent hematopoietic cell line, FDC-P1.24 The retroviral library used in that study was of moderate size, and the selection for factor independence was biased towards strongly activating mutations present in large pools of infected FDC-P1 cells, suggesting that other less frequent or weakly activating point mutations present in this region of hc may have been missed. Indeed, this possibility has been supported by the recent identification of two other constitutive mutations in hc that lie within this region.28 To investigate whether other sites for oncogenic activation by point mutation are present in the Xho I/Bgl II region of the hc cDNA, we devised a strategy, outlined in Fig 1, that involved transient transfection of the Xho I/Bgl II point-mutated hc expression library24 into BOSC 23 retroviral packaging cells to produce high-titer retroviruses carrying the hc point mutants. After transfection, FDC-P1 cells were infected by cocultivation with the mutant hc and, as a control wild-type hc, virus-producing BOSC 23 cells. FDC-P1 cells were then selected for factor-independent growth in 24-well multidishes, with each well containing 2 × 104 cells in medium without mGM-CSF. After several weeks in culture in the absence of factor, 96/204 wells seeded with FDC-P1 cells infected with the mutant hc retroviral library contained viable, proliferating cells, whereas no such cells were present in control wells seeded with uninfected FDC-P1 cells or FDC-P1 cells infected with wild-type hc retrovirus. Factor-independent FDC-P1 cell populations expressing the previously identified I374N and V449E constitutive hc mutants were identified by recovery of mutagenized hc fragments by PCR from genomic DNA, followed by restriction enzyme digestions diagnostic of the I374N (BstYI) and V449E (Bgl II) mutants.24 Of the 96 factor-independent FDC-P1 cell cultures, 23 contained the extracellular I374N mutant and 32 contained the transmembrane domain V449E mutant (data not shown). Conditioned medium from the 41 factor-independent cell cultures containing unidentified constitutive mutations failed to support the growth of uninfected FDC-P1 cells, thus eliminating the possibility of factor independence being due to autocrine growth factor production (data not shown). Identification of Novel Constitutive hc Point Mutations in the Factor-Independent FDC-P1 Cell Populations Novel, potentially constitutive hc point mutations in the 41 factor-independent FDC-P1 cell populations were identified by PCR recovery of the mutated hc region and sequencing (see Materials and Methods). Mutations of potential interest were selected for further analysis on the basis of (1) being the only mutation in a factor-independent cell population, (2) presence in more than one factor-independent cell population, or (3) proximity to sequences implicated in receptor activation or signaling. To test whether selected mutations could induce constitutive activity, they were recreated independently by site-directed mutagenesis. After insertion into the pRUFNeo retroviral vector and transient transfection into the BOSC 23 retroviral packaging cells, these mutants, as well as wild-type hc, were introduced into FDC-P1 cells and then selected for either G418-resistance or for growth in factor-free medium. Flow cytometric analysis of G418-resistant cell populations indicated that a substantial proportion (8.5% to 42%) of infected cells expressed each hc mutant on the cell surface (data not shown). Of the hc mutants tested, 13 (Table 1 and Fig 8) were able to confer factor-independent growth on FDC-P1 cells (Fig 2), with several mutants containing different amino acid substitutions for the same wild-type residue. Of these mutants, Tyr376 was replaced with Ser, Asp, and Asn; Ala459 with Asp and Ser; and Arg461 with Cys and His. Importantly, the factor independence exhibited by each of the 41 FDC-P1 cell populations could be attributed to at least one of these constitutive mutations.   View this table: [in this window] [in a new window]   Table 1. Summary of Biological and Biochemical Activities Conferred by Constitutively Active hc Mutants View larger version (30K): [in this window] [in a new window]   Fig 2. Proliferation of factor-independent FDC-P1 cells infected with novel constitutive hc mutants. (A) Proliferation assay of FDC-P1 cells, infected with the indicated hc mutants, which had been selected before assay for growth in the absence of factor. Also shown are uninfected cells (uninf) that were washed and assayed in medium with (+) and without () mGM-CSF. Cells (103) were plated in triplicate and cell proliferation was measured at 72 hours as described in Materials and Methods. The mean and standard error of each triplicate is shown. (B) Flow cytometric analysis of constitutive hc mutant expression on the factor-independent FDC-P1 cells depicted in (A). Cells were stained by standard indirect immunofluorescence; dashed lines represent cells stained with an irrelevant control antibody and solid lines indicate staining with an anti-hc antibody. Cell number and fluorescence are in arbitrary units; the latter is plotted on a logarithmic scale. Also shown are analyses of uninfected FDC-P1 cells. Note that data for only the novel mutants isolated in this study are shown. Over several independent experiments, the proliferation rates of some infected factor-independent cell populations were consistently different to each other and to that of uninfected FDC-P1 cells grown in the presence of mGM-CSF (Fig 2A). Interestingly, these differences in factor-independent growth rates could not be attributed to corresponding differences in cell-surface expression (Fig 2B). Furthermore, the proliferation rates of factor-independent cell populations grown in the presence of mGM-CSF were similar to those of uninfected FDC-P1 cells, indicating that all factor-independent cell populations retained a similar responsiveness to mGM-CSF-generated mitogenic signals (data not shown). Taken together, these data suggest that some constitutive hc mutants may be more strongly activating than others, as also seen in a previous study.28 Absence of Constitutive Mutations in the N-Terminal BamHI/Xho I and C-Terminal Bgl II/Sal I Point-Mutated hc Libraries Given the efficiency at which constitutive mutations in the Xho I/Bgl II point-mutated hc library were identified, we decided to use the same retroviral expression cloning strategy to screen for constitutively activating point mutations in the remainder of hc. Two independent screens were used, one covering a 770-bp BamHI/Xho I segment of the hc cDNA encoding the first 239 amino acids that contain the N-terminal extracellular cytokine receptor module (CRM) and the other a 1,017-bp Bgl II/Sal I segment of the hc cDNA encoding the C-terminal 337 amino acids of the cytoplasmic domain (Fig 1). Point mutations were introduced into both hc regions at a rate of approximately 0.3% (1 in 350 bp), and libraries of approximately 3.5 × 104 (BamHI/Xho I) and approximately 9.5 × 104 (Bgl II/Sal I) plasmid clones representing hc cDNAs bearing point mutations in the targeted regions were generated. Considering that there was no overwhelming mutational bias in these procedures (data not shown), the libraries should adequately represent all of the possible point mutations in both hc segments. In separate experiments, retroviruses generated by transient transfection of BOSC 23 cells with the BamHI/Xho I and Bgl II/Sal I point-mutated hc libraries were used to infect FDC-P1 cells by cocultivation with infection frequencies of 38% and 25%, respectively. Again, parallel cocultivations were performed in both experiments with untransfected BOSC 23 cells and BOSC 23 cells producing wild-type hc retrovirus. After several weeks in liquid culture in the absence of factor, only wells seeded with FDC-P1 cells infected with the mutant hc retroviruses (2/84 for BamHI/Xho I library and 3/102 for Bgl II/Sal I library) contained viable, proliferating cells. Conditioned medium from these cultures again demonstrated that factor independence was not due to autocrine growth factor production (data not shown). However, retesting of  subunits bearing mutations identified in hc PCR fragments recovered from these factor-independent cell populations in FDC-P1 cells failed to generate factor-independent cells (data not shown). Sequencing of these PCR fragments showed that the factor-independent cell populations isolated from each screen contained identical, full-length hc integrants, suggesting that these populations may have originated from single infected FDC-P1 cells that acquired factor independence without constitutive mutations in hc. Differential Tyrosine Phosphorylation of hc Mutants Previous studies have demonstrated that one of the early biochemical events in response to hGM-CSF stimulation is tyrosine phosphorylation of hc.5,7 To assess the tyrosine phosphorylation state of mutant  subunits expressed in the factor-independent FDC-P1 cell populations, cell lysates were subjected to immunoprecipitation with an anti-hc antibody and immunoblotting with an antiphosphotyrosine antibody. As shown in Fig 3A, only V449E, A459D, and R461C mutants were constitutively tyrosine phosphorylated in the absence of factor, despite the fact that immunoblotting with an anti-hc antibody (Fig 3B) indicated that all immunoprecipitates contained readily detectable levels of  subunits. We also determined whether  subunit tyrosine phosphorylation could be detected in all cell lines by coexpressing the hGMR subunit with mutant  subunits (and wild-type, as a control) in each FDC-P1 cell population. Only I374N, Q375P, Y376N, W383R (weakly), L445Q, and, as expected, wild-type  subunits demonstrated an increase in phosphorylation on tyrosine residues upon stimulation with hGM-CSF (Fig 3A), despite the fact that hGMR expression could be detected on the surface of all cell lines (data not shown). View larger version (39K): [in this window] [in a new window]   Fig 3. Tyrosine phosphorylation of constitutive hc mutants in factor-independent FDC-P1 cells. FDC-P1 cells coexpressing hGMR and the indicated  subunits were incubated without () or with (+) 50 ng/mL hGM-CSF for 10 minutes. Whole cell lysates were immunoprecipitated with an anti-hc antibody and immunoblotted using (A) an antiphosphotyrosine antibody and (B) an anti-hc antibody. The position of hc in each panel is indicated by an arrow. Constitutive Activation of the Ras-Raf-MAP Kinase and JAK2-STAT5 Signaling Pathways by hc Mutants One of the major signaling pathways activated in response to cytokines, including GM-CSF, is the Ras-Raf-MAP kinase pathway.10,38 Tyrosine phosphorylation and activation of MAP kinase is dependent on the sequential activation of upstream Ras, Raf-1, and MEK-1 effector molecules (reviewed in Marshall39). We therefore examined the levels of tyrosine phosphorylation on ERK1 and ERK2 MAP kinases in factor-independent FDC-P1 cells expressing the mutant  subunits. Western blot analysis of cell lysates with an antibody specific for activated, ie, phosphorylated, ERK1/2 MAP kinases showed that both proteins were similarly tyrosine phosphorylated in all cells in the absence of factor (Fig 4A). Furthermore, the extent of phosphorylation was similar to that seen in FDC-P1 cells expressing hGMR and wild-type hc (hGMR) in the presence of hGM-CSF (lanes 1 and 3). In contrast, no MAP kinase phosphorylation was detected in cells expressing hGMR in the absence of hGM-CSF (lane 2). As shown in Fig 4B, the levels of total ERK1/2 proteins present in lysates from all cell populations were comparable. View larger version (43K): [in this window] [in a new window]   Fig 4. Constitutive tyrosine phosphorylation of ERK1/2 MAP kinases in factor-independent FDC-P1 cells expressing constitutive hc mutants. Whole cell lysates from factor-independent FDC-P1 cells were analyzed by immunoblotting using (A) an anti-phospho-ERK1/2 antibody and (B) an anti-ERK1/2 antibody. Also shown are analyses of FDC-P1 cells expressing wild-type hGMR that were either starved of growth factor overnight and incubated with (lane 1) or without (lane 2) 50 ng/mL hGM-CSF for 10 minutes or continuously cultured in 1 ng/mL hGM-CSF (lane 3). Another major pathway that has been implicated in cytokine receptor signaling is the recently identified JAK-STAT pathway (reviewed in Ihle40). Indeed, several studies have shown that GM-CSF induces tyrosine phosphorylation and activation of the JAK2 protein tyrosine kinase.11,41-43 Activation of this kinase results in the subsequent phosphorylation and activation of STAT5 and, in some cases, other members of the STAT family of transcription factors.41,44,45 Activation of STAT proteins is reflected in their ability to dimerize and translocate to the nucleus, where they stimulate gene transcription by binding to specific DNA sequences. We therefore examined nuclear extracts from the factor-independent FDC-P1 cells for the presence of STAT DNA-binding activity by performing EMSAs. All of these extracts contained a protein complex that specifically bound to a -casein oligonucleotide probe containing a DNA-binding motif for STAT1, STAT3 and STAT5 (Fig 5). As expected, this DNA-binding activity was also induced in cells expressing wild-type hGMR that had been stimulated with hGM-CSF (lanes 1 and 3), but was absent in unstimulated cells (lane 2). Furthermore, the level of induction in hGM-CSF-stimulated cells expressing the hGMR was similar to that seen in factor-independent cells expressing the constitutively active mutants. View larger version (82K): [in this window] [in a new window]   Fig 5. Constitutive STAT activation in factor-independent FDC-P1 cells expressing constitutive hc mutants. Nuclear extracts prepared from factor-independent FDC-P1 cells expressing the indicated hc mutants were subjected to EMSA using a -casein promoter oligonucleotide probe. Also shown are FDC-P1 cells expressing wild-type hGMR that were either continuously cultured in 1 ng/mL hGM-CSF (lane 1) or starved of growth factor overnight and incubated without (lane 2) or with (lane 3) 50 ng/mL hGM-CSF for 10 minutes. The DNA-binding complexes are marked by an arrow. Constitutive activation of STAT DNA-binding activity by cytokine receptors is believed to be a direct consequence of phosphorylation by activated JAKs.46 Thus, the constitutive STAT activation seen in FDC-P1 cells expressing each of the mutants (Fig 5) implies that JAK2 is also constitutively activated. To confirm this, we analyzed JAK2 tyrosine phosphorylation in cells expressing each of five different mutants (I374N, V449E, L356P, L399P, and H544R) and, as a control, wild-type hc plus hGMR. These mutants were chosen as they represent different classes based on their location within hc, tyrosine phosphorylation, and responsiveness to hGM-CSF plus hGMR (see also Table 1). Detection of JAK2 tyrosine phosphorylation proved technically difficult in these cells and required the use of large numbers of cells, inhibition of cellular phosphatase activity by vanadate pretreatment, and a very sensitive two-layer immunodetection protocol. As shown in Fig 6, JAK2 phosphorylation was undetectable in factor-deprived cells expressing wild-type hc plus , but was readily detected in response to acute or continuous stimulation by hGM-CSF. Figure 6 also shows that, in cells expressing each of the five mutants tested, JAK2 was constitutively tyrosine phosphorylated in the absence of GM-CSF. View larger version (60K): [in this window] [in a new window]   Fig 6. Activation of JAK2 by wild-type and constitutively active hc mutants. Lysates from FDC-P1 cells expressing wild-type hc plus hGMR or the indicated constitutive mutants were subject to immunoprecipitation with anti-JAK2 antibodies followed by immununoblotting with (A) antiphosphotyrosine or (B) anti-JAK2 antibodies. Cells expressing wild-type hc plus hGMR were either factor-deprived (GM), restimulated for 10 minutes (+GM), or grown continuously in hGM-CSF (cont. GM). Because the loading of JAK2 for the I374N and V449E mutants was higher than that for the other samples in the upper panels, a second experiment showing these two mutants (plus wild-type controls) is shown in the lower panels. Biological Activity of hc Mutants in BAF-B03 Cells We have previously shown that extracellular hc mutants, while constitutively active in FDC-P1 cells, are unable to confer factor independence on murine IL-3-dependent BAF-B03 cells,24,28 whereas the V449E transmembrane domain mutant confers factor independence on both cell types.24 To examine whether constitutive activation of the extracellular, transmembrane, and cytoplasmic domain mutants identified in this report was also cell type-specific, retroviruses encoding the wild-type and mutant forms of hc were used to infect BAF-B03 cells. For mutants containing multiple amino acid substitutions at a given residue, the more strongly activating mutant, as determined by the growth rates of factor-independent FDC-P1 cells (see Fig 2A), was used. Figure 7A shows that only the transmembrane domain A459D mutant could confer factor independence on BAF-B03 cells, as indicated by proliferation assays and prolonged monitoring of liquid cultures without growth factor (data not shown), even though each mutant was expressed on the surface of infected cells (Fig 7B). Conditioned medium from these cells again contained no detectable autocrine growth factor (data not shown). View larger version (35K): [in this window] [in a new window]   Fig 7. Biological activity of hc mutants in BAF-B03 cells. (A) Factor-dependent and -independent proliferation of BAF-B03 cells infected with retroviruses encoding the indicated  subunits and subsequently superinfected with a retrovirus encoding the hGMR subunit. Proliferation assays were performed using triplicates of 5 × 103 cells in the presence of mIL-3 or hGM-CSF or in the absence of either factor, as indicated. The asterisk by A459D indicates that, in this case, the cells were not superinfected with the hGMR retrovirus. A459D(FI) represents a population of A459D-infected cells that were selected for factor-independent growth before analysis. For comparison, assays of uninfected BAF-B03 cells and cells infected with the hGMR virus alone are shown. (B) Flow cytometric analysis of hc and hGMR expression on the BAF-B03 cells used in (A). In each case, the cells infected with the indicated  subunits were stained with an irrelevant control antibody (dashed line), an anti-hc antibody (thin solid line), and an anti-hGMR antibody (thick solid line) by standard indirect immunofluorescence. Axes are as for Fig 2B. The I374N mutant, although not constitutively active in BAF-B03 cells, is still able to form a high-affinity receptor and deliver a proliferative signal, in the presence of hGM-CSF, when coexpressed with the hGMR subunit in BAF-B03 cells.28 In contrast, we previously described two other extracellular mutants, L356N and W358N, which, although also constitutively active in FDC-P1 cells, failed to form a high-affinity receptor complex with hGMR in BAF-B03 cells.28 To examine the ability of the novel hc mutants to generate a proliferative signal as part of the hGMR complex, BAF-B03 cells expressing these mutants were superinfected with a retrovirus carrying the wild-type hGMR subunit. After selection for puromycin-resistant hGMR infectants, flow cytometric analysis indicated that infected cells efficiently coexpressed both subunits (Fig 7B). All mutants, except for the L356P mutant, were able to deliver a proliferative signal in response to 1 ng/mL hGM-CSF, as shown by proliferation assays (Fig 7A), or prolonged monitoring of liquid cultures in 0.1 or 1 ng/mL hGM-CSF (data not shown). We believe the failure of L356P to allow hGM-CSF-dependent growth is probably due to an inability to interact productively with hGM-CSF, because we have previously shown that another mutant with a substitution at the same site (L356N) neither responds to hGM-CSF nor forms a high-affinity receptor complex.28     DISCUSSION Abstract Introduction Methods Results Discussion References Location of Constitutive Mutations in hc Sequence analysis indicated that the constitutive mutations are located exclusively in a central region of hc that spans the extracellular, transmembrane, and cytoplasmic domains (Table 1 and Fig 8). The extracellular mutations reported here and in our previous studies24,28 are all clustered in the membrane-proximal domain (domain 4) of hc, with several resulting in amino acid substitutions at residues in the B (L356P) and C (Q375P and Y376N,D,S) -strands of domain 4, and two other mutations affecting residues in the D (W383R) and E (L399P) -strands (Table 1 and Fig 8). Notably, our previous sequence alignment of domain 4 of hc with the corresponding domain of other cytokine receptors indicates that the residues in hc targetted for oncogenic activation are highly conserved (see Fig 1B in Jenkins et al28), suggesting that the homologous residues in other cytokine receptors may be targets for constitutively activating mutations. View larger version (24K): [in this window] [in a new window]   Fig 8. Location of constitutive mutations in the extracellular domain 4, transmembrane domain, and cytoplasmic domain of hc. Determination of a molecular model of domain 4 of hc has been described previously.28 The transmembrane sequence of hc was joined manually and an Indigo2 computer (Silicon Graphics, Mountain View, CA) was used to run the molecular modelling programs Insight II and Discover (Molecular Simulations Inc, San Diego, CA). Manual and automated methods were used to select an appropriate -helical conformation for the transmembrane region, and the model was evaluated for stereochemical parameters. The model of the hc domain 4 and transmembrane domain is presented in cartoon form, using Molscript74 and Raster3D.75 -strands are indicated by arrowed ribbons and italicized letters. The cytoplasmic domain is depicted in an arbitrary conformation and is shown only to illustrate the location of cytoplasmic mutations. -Carbon atoms of residues targetted for constitutive mutations are represented by CPK spheres. Within the remainder of hc, two transmembrane domain residues, in addition to the previously identified Val449 residue (V449E24), were identified as targets for constitutive mutations. Leu445 was replaced by Gln (L445Q), and Ala459 was replaced by Asp or Ser (A459D,S). Finally, mutations at two positions within the cytoplasmic domain of hc, Arg461, replaced by Cys and His (R461C,H), and His544, replaced by Arg (H544R), also resulted in constitutive activity. The locations of the cytoplasmic mutations are likely to indicate key roles for the respective regions in receptor signaling, whereas the transmembrane mutants may be somewhat more adventitious and reflect a role for receptor dimerization (see below). Mechanisms of Activation Extracellular mutations.   The observation that all extracellular mutants identified in this study, as well as previous studies,24,28 are unable to confer factor independence on BAF-B03 cells (Fig 7) is consistent with a common mechanism of activation by these extracellular mutations. Although this mechanism is not clearly understood, we have previously suggested that the constitutive activity of extracellular hc mutants may be dependent on the presence of cell type-specific signaling molecules.24,27 Our recent studies have shown that the murine GMR subunit is one such molecule and, when introduced into BAF-B03 or CTLL-2 cells along with the I374N mutant, allows<