Blood online
Home About Blood Authors Subscriptions Permission Advertising Public Access contact us
 

 
Advanced
Current Issue
First Edition
Future Articles
Archives
Submit to Blood
Search
American Society of Hematology
Meeting Abstracts
Email Alerts
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jenkins, B. J.
Right arrow Articles by Gonda, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jenkins, B. J.
Right arrow Articles by Gonda, T. J.
Related Collections
Right arrow Hematopoiesis and Stem Cells
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

arrow to previous article Previous Article  |  Table of Contents  |  Next Article next article arrow

Blood, Vol. 92 No. 6 (September 15), 1998: pp. 1989-2002

Saturation Mutagenesis of the beta  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 beta  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 alpha subunits and a common signal-transducing beta  subunit (hbeta c). 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 hbeta c. We report here a comprehensive screen of the entire hbeta c 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 hbeta c that encompasses the extracellular membrane-proximal domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain. Interestingly, most hbeta c 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 beta  subunits were constitutively tyrosine phosphorylated. Taken together, these results highlight key regions involved in hbeta c activation, dissociate hbeta c tyrosine phosphorylation from MAP kinase and STAT activation, and suggest the involvement of distinct mechanisms by which proliferative signals can be generated by hbeta c.

© 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 alpha subunits (hGMRalpha , hIL-3Ralpha , and hIL-5Ralpha ) associated with a common signal-transducing beta  subunit (hbeta c).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 alpha  and beta  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 beta  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 alpha  and beta  subunit heterodimerization,17,18 although the precise stoichiometry of receptor subunits in the active complexes remains unresolved. Chimeric receptors containing the hbeta c cytoplasmic domain fused to the extracellular domains of hGMRalpha or hIL-5Ralpha have been reported to confer cytokine-dependent growth on hematopoietic cells, suggesting that dimerization of the hbeta c cytoplasmic domain is sufficient for cellular proliferation.17,19,20 Consistent with a role for beta  subunit dimerization in receptor activation, it has been shown that beta  subunit homodimers are found in active hGMR complexes.21 More recently, it was observed that the functional hGMR complex may contain at least two alpha  subunits.22 In the context of receptor stoichiometry, these results suggest that the alpha  and beta  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 hbeta c. This led to the identification of two mutations, one of which is located in the transmembrane domain of hbeta c (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 hbeta c homodimerization.24 The other constitutive point mutation lies in the extracellular region of hbeta c (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 hbeta c 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 hbeta c, Leu356, and Trp358 that constitutively activate hbeta c in FDC-P1 cells.28 Interestingly, the Leu356 and Trp358 residues lie within the same membrane-spanning region of hbeta c 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 hbeta c 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 hbeta c, 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 hbeta c, 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 beta  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 Psi 2 cells producing wild-type hGMRalpha 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 hbeta c 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 hbeta c cDNAs were subcloned between the BamHI and HindIII restriction sites of the pRUFNeo retroviral expression vector.33

PCR Mutagenesis and Construction of Point-Mutated hbeta c cDNA Libraries

PCR mutagenesis was performed on the pRUFNeo-hbeta c plasmid in which a Xho I site was silently introduced into wild-type hbeta c 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 hbeta c 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 hbeta c 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 hbeta c 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-hbeta c from which the BamHI/Xho I or Bgl II/Sal I segment of hbeta c had been excised. After transformation of Escherichia coli (DH10B), the resultant point-mutated hbeta c 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 hbeta c mutants. Schematic illustration of hbeta c 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 hbeta c cDNA. The positions in the hbeta c 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 hbeta c 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 hbeta c cDNA library has been described previously.24

Infection of Hematopoietic Cells With Mutant hbeta c Retroviruses

High-titer retroviruses carrying mutant hbeta c 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 hbeta c 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 hbeta c 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 hGMRalpha retrovirus were selected as described previously.28

Recovery of Mutant hbeta c 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 hbeta c 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 hbeta c mutants on the cell surface of infected FDC-P1 or BAF-B03 cells was detected by standard indirect immunofluorescence with the anti-hbeta c 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). hGMRalpha 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 hbeta c 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 beta -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 beta -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 hbeta c Library

We have previously combined PCR-based random mutagenesis with retroviral expression cloning to identify two distinct constitutive point mutations within a region of hbeta c, delimited by Xho I and Bgl II sites in hbeta c 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 hbeta c may have been missed. Indeed, this possibility has been supported by the recent identification of two other constitutive mutations in hbeta c 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 hbeta c cDNA, we devised a strategy, outlined in Fig 1, that involved transient transfection of the Xho I/Bgl II point-mutated hbeta c expression library24 into BOSC 23 retroviral packaging cells to produce high-titer retroviruses carrying the hbeta c point mutants. After transfection, FDC-P1 cells were infected by cocultivation with the mutant hbeta c and, as a control wild-type hbeta c, 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 hbeta c 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 hbeta c retrovirus.

Factor-independent FDC-P1 cell populations expressing the previously identified I374N and V449E constitutive hbeta c mutants were identified by recovery of mutagenized hbeta c 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 hbeta c Point Mutations in the Factor-Independent FDC-P1 Cell Populations

Novel, potentially constitutive hbeta c point mutations in the 41 factor-independent FDC-P1 cell populations were identified by PCR recovery of the mutated hbeta c 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 hbeta c, 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 hbeta c mutant on the cell surface (data not shown). Of the hbeta c 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 hbeta c 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 hbeta c mutants. (A) Proliferation assay of FDC-P1 cells, infected with the indicated hbeta c 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 hbeta c 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-hbeta c 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 hbeta c 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 hbeta c Libraries

Given the efficiency at which constitutive mutations in the Xho I/Bgl II point-mutated hbeta c library were identified, we decided to use the same retroviral expression cloning strategy to screen for constitutively activating point mutations in the remainder of hbeta c. Two independent screens were used, one covering a 770-bp BamHI/Xho I segment of the hbeta c 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 hbeta c cDNA encoding the C-terminal 337 amino acids of the cytoplasmic domain (Fig 1). Point mutations were introduced into both hbeta c 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 hbeta c 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 hbeta c 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 hbeta c 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 hbeta c retrovirus. After several weeks in liquid culture in the absence of factor, only wells seeded with FDC-P1 cells infected with the mutant hbeta c 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 beta  subunits bearing mutations identified in hbeta c 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 hbeta c integrants, suggesting that these populations may have originated from single infected FDC-P1 cells that acquired factor independence without constitutive mutations in hbeta c.

Differential Tyrosine Phosphorylation of hbeta c Mutants

Previous studies have demonstrated that one of the early biochemical events in response to hGM-CSF stimulation is tyrosine phosphorylation of hbeta c.5,7 To assess the tyrosine phosphorylation state of mutant beta  subunits expressed in the factor-independent FDC-P1 cell populations, cell lysates were subjected to immunoprecipitation with an anti-hbeta c 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-hbeta c antibody (Fig 3B) indicated that all immunoprecipitates contained readily detectable levels of beta  subunits. We also determined whether beta  subunit tyrosine phosphorylation could be detected in all cell lines by coexpressing the hGMRalpha subunit with mutant beta  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 beta  subunits demonstrated an increase in phosphorylation on tyrosine residues upon stimulation with hGM-CSF (Fig 3A), despite the fact that hGMRalpha 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 hbeta c mutants in factor-independent FDC-P1 cells. FDC-P1 cells coexpressing hGMRalpha and the indicated beta  subunits were incubated without (-) or with (+) 50 ng/mL hGM-CSF for 10 minutes. Whole cell lysates were immunoprecipitated with an anti-hbeta c antibody and immunoblotted using (A) an antiphosphotyrosine antibody and (B) an anti-hbeta c antibody. The position of hbeta c in each panel is indicated by an arrow.

Constitutive Activation of the Ras-Raf-MAP Kinase and JAK2-STAT5 Signaling Pathways by hbeta c 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 beta  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 hGMRalpha and wild-type hbeta c (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 hbeta c 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 beta -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 hbeta c mutants. Nuclear extracts prepared from factor-independent FDC-P1 cells expressing the indicated hbeta c mutants were subjected to EMSA using a beta -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 hbeta c plus hGMRalpha . These mutants were chosen as they represent different classes based on their location within hbeta c, tyrosine phosphorylation, and responsiveness to hGM-CSF plus hGMRalpha (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 hbeta c plus alpha , 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 hbeta c mutants. Lysates from FDC-P1 cells expressing wild-type hbeta c plus hGMRalpha 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 hbeta c plus hGMRalpha 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 hbeta c Mutants in BAF-B03 Cells

We have previously shown that extracellular hbeta c 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 hbeta c 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 hbeta c mutants in BAF-B03 cells. (A) Factor-dependent and -independent proliferation of BAF-B03 cells infected with retroviruses encoding the indicated beta  subunits and subsequently superinfected with a retrovirus encoding the hGMRalpha 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 hGMRalpha 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 hGMRalpha virus alone are shown. (B) Flow cytometric analysis of hbeta c and hGMRalpha expression on the BAF-B03 cells used in (A). In each case, the cells infected with the indicated beta  subunits were stained with an irrelevant control antibody (dashed line), an anti-hbeta c antibody (thin solid line), and an anti-hGMRalpha 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 hGMRalpha 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 hGMRalpha in BAF-B03 cells.28 To examine the ability of the novel hbeta c 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 hGMRalpha subunit. After selection for puromycin-resistant hGMRalpha 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 hbeta c

Sequence analysis indicated that the constitutive mutations are located exclusively in a central region of hbeta c 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 hbeta c, with several resulting in amino acid substitutions at residues in the B (L356P) and C (Q375P and Y376N,D,S) beta -strands of domain 4, and two other mutations affecting residues in the D (W383R) and E (L399P) beta -strands (Table 1 and Fig 8). Notably, our previous sequence alignment of domain 4 of hbeta c with the corresponding domain of other cytokine receptors indicates that the residues in hbeta c 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 hbeta c. Determination of a molecular model of domain 4 of hbeta c has been described previously.28 The transmembrane sequence of hbeta c 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 alpha -helical conformation for the transmembrane region, and the model was evaluated for stereochemical parameters. The model of the hbeta c domain 4 and transmembrane domain is presented in cartoon form, using Molscript74 and Raster3D.75 beta -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. alpha -Carbon atoms of residues targetted for constitutive mutations are represented by CPK spheres.

Within the remainder of hbeta c, 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 hbeta c, 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 hbeta c mutants may be dependent on the presence of cell type-specific signaling molecules.24,27 Our recent studies have shown that the murine GMRalpha subunit is one such molecule and, when introduced into BAF-B03 or CTLL-2 cells along with the I374N mutant, allows factor-independent proliferation (B.J.J., F. Le, and T.J.G., manuscript submitted).

Notably, with each of the extracellular mutants, receptor activation has resulted from amino acid substitutions that are predicted to severely disrupt the normal beta -sheet structure of domain 4. Indeed, our earlier studies suggested that disruption of interactions between beta -strands B and C by constitutive mutations at positions 356, 358 (on strand B), and 374 (on strand C) leads to receptor activation.28 The fact that such mutations at various locations within domain 4 all lead to receptor activation may indicate that activation results from disruption or relaxation of a constrained conformation of domain 4. This, in turn, may relieve an inhibitory intramolecular or intermolecular interaction and allow assembly of an active receptor complex. In agreement with such a model, several studies have demonstrated that the extracellular membrane-proximal domains of other cytokine receptors (equivalent to domain 4 of hbeta c) play a regulatory role in mediating interactions between receptor subunits.12,47,48 Thus, it is possible that the conformational change induced in domain 4 of hbeta c by the constitutive extracellular mutations mimicks the effect of ligand binding to the normal receptor. Indeed, crucial regions involved in ligand binding have been identified in domain 4 of hbeta c (reviewed in Bagley et al23).

Transmembrane mutations.   We have previously isolated a point mutation in the transmembrane domain of hbeta c (V449E) that confers factor independence on FDC-P1 and BAF-B03 cells.24 The V449E mutation is similar to the constitutive mutation found in the transmembrane domain of the Neu/c-erbB2 receptor tyrosine kinase that stimulates receptor homodimerization.25,26 By analogy, it is likely that V449E generates ligand-independent proliferative signals by inducing constitutive beta  subunit homodimerization. The exact mechanism by which the mutation in Neu promotes receptor dimerization is still unclear. It has been proposed that introduction of strongly polar or hydrophilic residues within the hydrophobic transmembrane environment induces hydrogen bonding between receptor subunits, thus enhancing receptor dimerization.49 Consistent with this finding, the residues substituted at Leu445 (Gln), Val449 (Glu), and Ala459 (Asp and Ser) contain polar side-chains that could participate in subunit-subunit hydrogen bonding. We might, therefore, expect the constitutive mutations at the Leu445, Val449, and Ala459 residues to act by a similar mechanism. However, the inability of the L445Q mutant to confer factor independence on BAF-B03 cells (Fig 7) suggests that it is activated by a different mechanism, possibly by inducing association with cell type-specific signaling molecules in a manner similar to that proposed for the extracellular mutants.24,27,28 Alternatively, the inactivity of L445Q could be attributed to a quantitative phenomenon whereby dimerization, and therefore the subsequent signal, is weaker than that of the other mutants and below a threshold required for proliferation in BAF-B03 cells, but not FDC-P1 cells.

Cytoplasmic mutations.   To our knowledge, this is the first report of mutations within the cytoplasmic domain of a cytokine receptor that results in constitutive activation. Accordingly, it is difficult at the moment to envisage how the cytoplasmic mutations in hbeta c (R461C,H and H544R) activate the receptor. The Arg461 residue was originally assigned to the transmembrane domain of hbeta c2; however, our own analysis of the hbeta c sequence with a transmembrane domain prediction program (Tmpred50) indicated that this residue lies outside of the transmembrane domain of hbeta c and is predicted to be the first cytoplasmic residue. Interestingly, the Cys and His residues substituted at this position are less hydrophilic than the wild-type Arg and, therefore, may only weakly anchor the transmembrane domain in its normal position. In such a scenario, one might speculate that the increased flexibilty or length of the transmembrane domain may relieve an inhibitory constraint that exposes a structure within hbeta c involved in inter-subunit interactions.

The H544R mutation is located within the box 2 motif that is loosely conserved among cytokine receptors.23,51 The exact role of this motif in mitogenesis is somewhat controversial52-56 and possibly reflects the differential requirement of this motif for proliferative signaling among cytokine receptors. Nonetheless, impaired mitogenesis of several cytokine receptors that either lack or contain mutations in this motif correlates with a loss in JAK association and activation,53,56,57 suggesting that this motif plays a role in JAK activation. Given the general requirement of cytokine receptors for JAK association and activation (reviewed in Ihle40), the H544R mutation may alter the conformation of the membrane-proximal region and enhance JAK2 association and/or activation. Alternatively, this mutation may trigger receptor activation by facilitating dimerization of the cytoplasmic domains of beta  subunits in a manner similar to a mutation (V559G) in the membrane-proximal cytoplasmic domain of the c-kit receptor tyrosine kinase, which stimulates constitutive receptor homodimerization.58

Biochemical Consequence of Constitutive Mutations in FDC-P1 Cells

beta subunit phosphorylation.   One of the early events indicative of hGMR activation in response to GM-CSF binding is tyrosine phosphorylation of hbeta c.5,7 Phosphorylation of receptor tyrosine residues facilitates binding and activation of signaling molecules that link the receptor to signaling pathways involved in various cellular responses, including survival and proliferation. For example, mutation of a key site for hbeta c phosphorylation, Tyr750, severely compromised both tyrosine phosphorylation of hbeta c and the survival of cells expressing this mutant under low serum conditions.59 Our results suggest that tyrosine phosphorylation of major sites in hbeta c is not essential for either constitutive or GM-CSF-induced survival and proliferation, at least in serum-containing cultures. None of the extracellular mutant beta  subunits, or the L445Q transmembrane mutant or the H544R cytoplasmic mutant, were constitutively phosphorylated in the absence of hGM-CSF (Fig 3A). Moreover, only the I374N, Q375P, Y376N, W383R, and L445Q mutants (and, as expected, wt hbeta c) displayed hGM-CSF-induced tyrosine phosphorylation when coexpressed with hGMRalpha . This is somewhat surprising, considering that all of these mutants, with the exception of L356P and W358N, can confer hGM-CSF growth responsiveness on BAF-B03 cells when coexpressed with hGMRalpha (Fig 7 and Jenkins et al28). Both V449E and A459D transmembrane domain mutants, and the R461C cytoplasmic mutant, were constitutively phosphorylated. Interestingly, constitutive phosphorylation of the two transmembrane mutants correlated with their ability to confer factor independence on BAF-B03 cells, thus providing further evidence for a similar mechanism of activation by these two mutants.

MAP kinase, STAT, and JAK activation.   In contrast to the differential tyrosine phosphorylation of the mutant beta  subunits, all factor-independent FDC-P1 cell lines exhibited a similar level of constitutive ERK1/2 MAP kinase activation that resembled that induced by the wild-type hGMR in response to hGM-CSF (Fig 4A). Moreover, we have shown that, with all of the mutants tested, JAK2 is constitutively tyrosine phosphorylated at a level similar to that seen in cells grown continuously in GM-CSF. These results imply that beta  subunit phosphorylation at the major site(s) detected is not essential for the activation of the Ras-Raf-MAP kinase pathway, although by analogy with other cytokine receptors, JAK2 activation may be.60 Other mechanisms may therefore exist that link hbeta c to this pathway in the absence of receptor tyrosine phosphorylation. These could be mediated by JAK2, which can link the receptor to this pathway indirectly through its asssociation with the SHP-2 phosphatase61 or directly through its association with Raf-1,62 thus bypassing the requirement for receptor phosphotyrosines. Alternatively, it is conceivable that mutants which do not exhibit hbeta c tyrosine phosphorylation instead phosphorylate tyrosine residues on other molecules and that the resultant phosphotyrosines act as binding sites for components of the Ras-Raf-MAP kinase pathway such as Shc or Grb2. It is tempting to speculate that the endogenous murine GMR/IL-3R beta  subunits might fulfil such a role. However, the ability of the I374N mutant to confer factor independence on CTLL-2 cells in the presence of the murine GMRalpha subunit, as mentioned above (B.J.J., F. Le, and T.J.G., manuscript submitted), renders this less likely, because CTLL-2 cells do not express any components of the murine GMR/IL3R complex. Furthermore, our results also suggest that activation of MAP kinase may be important for these mutants to stimulate proliferation. Indeed, preliminary data suggest that ERK1/2 MAP kinases are constitutively activated in BAF-B03 cells only by hbeta c mutants able to confer factor independence on these cells (T.J.B., B.J.J., T.J.G., unpublished data). This is consistent with previous studies that have observed constitutive activation of the Ras-Raf-MAP kinase pathway in spontaneously derived factor-independent variants of human TF-1 cells and factor-independent BAF-B03 cells expressing a constitutively active c-Mpl mutant.63,64

We also demonstrated that all factor-independent FDC-P1 cell populations exhibited constitutive STAT DNA-binding activity (Fig 5), which is both indicative of and consistent with constitutive JAK2 activation. Whereas activation of STAT5 has predominantly been associated with the GMR/IL3R (eg, Mui et al8), only a proportion of each DNA-protein complex was supershifted with an anti-STAT5 antibody, and the extent of these supershifts varied among the cell populations (data not shown). Therefore, it is possible that other STAT proteins may be present in these constitutive DNA-binding complexes. Indeed, activation of STAT1, STAT3, and STAT5 in response to GM-CSF stimulation has been reported in various hematopoietic cells.8,41,65

Recently, conflicting reports have emerged as to the role of STAT5 in cytokine-induced mitogenic signaling.66-71 However, constitutive activation of STAT5 has been observed in factor-independent hematopoietic cell lines.63 Together with our data, this is consistent with a role for STAT5 activation in cell proliferation; on the other hand, constitutive STAT5 activation may simply reflect constitutive JAK2 activation.

Induction of other signals, eg, c-myc, is also likely to be required for a proliferative response. In this respect, we are currently investigating the activation state of other signaling molecules implicated in cytokine-induced proliferation by the hbeta c mutants.

Conclusions

In this report we have coupled saturation mutagenesis with a simple retroviral expression cloning strategy to comprehensively screen the entire hbeta c molecule for constitutive mutations. The identification of mutations clustered exclusively within the membrane-proximal extracellular domain, transmembrane domain, and membrane-proximal region of the cytoplasmic domain of hbeta c provides new insights into the role these regions in hbeta c and, by analogy, other members of the cytokine receptor family play in receptor activation. We observed that most of these mutants exhibited cell type-specific differences in their constitutive activity, which suggests the involvement of different mechanisms in the activation of hbeta c. Consistent with this, only some mutants were constitutively tyrosine phosphorylated. Intriguingly, this latter observation also dissociates receptor tyrosine phosphorylation from proliferative signaling, JAK2 activation, ERK MAP kinase, and STAT activation and most likely reflects the emerging theme of redundancy in mechanisms underlying activation of downstream signaling molecules.

    FOOTNOTES

   Submitted December 1, 1997; accepted May 11, 1998.
   Supported by a research grant from the National Health and Medical Research Council (NHMRC) of Australia. T.J.G. is a Senior Research Fellow of the NHMRC. B.J.J. is the recipient of a Dawes postgraduate scholarship from the Royal Adelaide Hospital.
   Address reprint requests to Thomas J. Gonda, PhD, Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Frome Road, Adelaide, South Australia 5000, Australia; e-mail: Tom.Gonda{at}imvs.sa.gov.au.
   The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.

    ACKNOWLEDGMENT

The authors are grateful to Sun Qiyu and Angel Lopez for supplying their antireceptor antibodies, Chris Bagley for advice and assistance with molecular modelling, and our various colleagues who generously supplied us with growth factors. We thank Angel Lopez, Mathew Vadas, Richard D'Andrea, and Leonie Ashman for critical reading of this manuscript; Alan Bishop and Judy Haywood for assistance with flow cytometry; and Arthur Mangos for automated sequencing analyses.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

1. Gearing DP, King JA, Gough NM, Nicola NA: Expression cloning of a receptor for human granulocyte-macrophage colony-stimulating factor. EMBO J 8:3667, 1989[Medline] [Order article via Infotrieve]

2. Hayashida K, Kitamura T, Gorman DM, Arai K, Yokota T, Miyajima A: Molecular cloning of a second subunit of the receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF): Reconstitution of a high-affinity GM-CSF receptor. Proc Natl Acad Sci USA 87:9655, 1990[Abstract/Free Full Text]

3. Kitamura T, Sato N, Arai K, Miyajima A: Expression cloning of the human IL-3 receptor cDNA reveals a shared beta  subunit for the human IL-3 and GM-CSF receptors. Cell 66:1165, 1991[Medline] [Order article via Infotrieve]

4. Tavernier J, Devos R, Cornelis S, Tuypens T, Van der Heyden J, Fiers W, Plaetinck G: A human high affinity interleukin-5 receptor (IL5R) is composed of an IL5-specific alpha  chain and a beta  chain shared with the receptor for GM-CSF. Cell 66:1175, 1991[Medline] [Order article via Infotrieve]

5. Sakamaki K, Miyajima I, Kitamura T, Miyajima A: Critical cytoplasmic domains of the common beta  subunit of the human GM-CSF, IL-3 and IL-5 receptors for growth signal transduction and tyrosine phosphorylation. EMBO J 11:3541, 1992[Medline] [Order article via Infotrieve]

6. Mui AL, Miyajima A: Cytokine receptors and signal transduction. Prog Growth Factor Res 5:15, 1994[Medline] [Order article via Infotrieve]

7. Duronio V, Clark Lewis I, Federsppiel B, Wieler JS, Schrader JW: Tyrosine phosphorylation of receptor beta  subunits and common substrates in response to interleukin-3 and granulocyte-macrophage colony-stimulating factor. J Biol Chem 267:21856, 1992[Abstract/Free Full Text]

8. Mui AL, Wakao H, O'Farrell AM, Harada N, Miyajima A: Interleukin-3, granulocyte-macrophage colony stimulating factor and interleukin-5 transduce signals through two STAT5 homologs. EMBO J 14:1166, 1995[Medline] [Order article via Infotrieve]

9. Silvennoinen O, Witthuhn BA, Quelle FW, Cleveland JL, Yi T, Ihle JN: Structure of the murine Jak2 protein-tyrosine kinase and its role in interleukin 3 signal transduction. Proc Natl Acad Sci USA 90:8429, 1993[Abstract/Free Full Text]

10. Sato N, Sakamaki K, Terada N, Arai K, Miyajima A: Signal transduction by the high-affinity GM-CSF receptor: Two distinct cytoplasmic regions of the common beta  subunit responsible for different signaling. EMBO J 12:4181, 1993[Medline] [Order article via Infotrieve]

11. Quelle FW, Sato N, Witthuhn BA, Inhorn RC, Eder M, Miyajima A, Griffin JD, Ihle JN: JAK2 associates with the beta c chain of the receptor for granulocyte-macrophage colony-stimulating factor, and its activation requires the membrane-proximal region. Mol Cell Biol 14:4335, 1994[Abstract/Free Full Text]

12. De Vos P, Ultsch M, Kossiakoff AA: Human growth hormone and extracellular domain of its receptor: Crystal structure of the complex. Science 255:306, 1992[Abstract/Free Full Text]

13. Alexander WS, Metcalf D, Dunn AR: Point mutations within a dimer interface homology domain of c-Mpl induce constitutive receptor activity and tumorigenicity. EMBO J 14:5569, 1995[Medline] [Order article via Infotrieve]

14. Watowich SS, Yoshimura A, Longmore GD, Hilton DJ, Yoshimura Y, Lodish HF: Homodimerization and constitutive activation of the erythropoietin receptor. Proc Natl Acad Sci USA 89:2140, 1992[Abstract/Free Full Text]

15. Watowich SS, Hilton DJ, Lodish HF: Activation and inhibition of erythropoietin receptor function: Role of receptor dimerization. Mol Cell Biol 14:3535, 1994[Abstract/Free Full Text]

16. Livnah O, Stura EA, Johnson DL, Middleton SA, Mulcahy LS, Wrighton NC, Dower WJ, Jolliffe LK, Wilson IA: Functional mimicry of a protein hormone by a peptide agonist: The Epo receptor complex at 2.8 A. Science 273:464, 1996[Abstract]

17. Eder M, Ernst TJ, Ganser A, Jubinsky PT, Inhorn R, Hoelzer D, Griffin JD: A low affinity chimeric human alpha /beta -granulocyte-macrophage colony-stimulating factor receptor induces ligand-dependent proliferation in a murine cell line. J Biol Chem 269:30173, 1994[Abstract/Free Full Text]

18. Stomski F, Sun Q, Bagley C, Woodcock J, Goodall G, Andrews R, Berndt M, Lopez A: Human interleukin-3 (IL-3) induces disulphide-linked IL-3 receptor alpha  and beta  chain heterodimerization which is required for receptor activation but not high affinity binding. Mol Cell Biol 16:3035, 1996[Abstract]

19. Takaki S, Kanazawa H, Shiiba M, Takatsu K: A critical cytoplasmic domain of the interleukin-5 (IL-5) receptor alpha  chain and its function in IL-5-mediated growth signal transduction. Mol Cell Biol 14:7404, 1994[Abstract/Free Full Text]

20. Muto A, Watanabe S, Miyajima A, Yokota T, Arai K: High affinity chimeric human granulocyte-macrophage colony-stimulating factor receptor carrying the cytoplasmic domain of the beta  subunit but not the alpha  subunit transduces growth promoting signals in Ba/F3 cells. Biochem Biophys Res Commun 208:368, 1995[Medline] [Order article via Infotrieve]

21. Muto A, Watanabe S, Miyajima A, Yokota T, Arai K: The beta  subunit of human granulocyte-macrophage colony-stimulating factor receptor forms a homodimer and is activated via association with the alpha  subunit. J Exp Med 183:1911, 1996[Abstract/Free Full Text]

22. Lia F, Rajotte D, Clark SC, Hoang T: A dominant negative granulocytye-macrophage colony-stimulating factor receptor alpha  chain reveals the multimeric structure of the receptor complex. J Biol Chem 271:28287, 1996[Abstract/Free Full Text]

23. Bagley CJ, Woodcock JM, Stomski FC, Lopez AF: The structural and functional basis of cytokine receptor activation: Lessons from the common beta  subunit of the granulocyte-macrophage colony-stimulating factor, interleukin-3 (IL-3) and IL-5 receptors. Blood 89:1471, 1997[Free Full Text]

24. Jenkins BJ, D'Andrea RJ, Gonda TJ: Activating point mutations in the common beta  subunit of the human GM-CSF, IL-3 and IL-5 receptors suggest the involvement of beta  subunit dimerization and cell type-specific molecules in signalling. EMBO J 14:4276, 1995[Medline] [Order article via Infotrieve]

25. Bargmann CI, Hung MC, Weinberg RA: Multiple independent activations of the neu oncogene by a point mutation altering the transmembrane domain of p185. Cell 45:649, 1986[Medline] [Order article via Infotrieve]

26. Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: A point mutation in the neu oncogene mimics ligand induction of receptor aggregation. Nature 339:230, 1989[Medline] [Order article via Infotrieve]

27. Gonda TJ, D'Andrea RJ: Activating mutations in cytokine receptors: Implications for receptor function and role in disease. Blood 89:355, 1997[Free Full Text]

28. Jenkins BJ, Bagley CJ, Woodcock J, Lopez AF, Gonda TJ: Interacting residues in the extracellular region of the common beta  subunit of the human GM-CSF, IL-3 and IL-5 receptors involved in constitutive activation. J Biol Chem 271:29707, 1996[Abstract/Free Full Text]

29. Pear WS, Nolan GP, Scott ML, Baltimore D: Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci USA 90:8392, 1993[Abstract/Free Full Text]

30. Dexter TM, Garland J, Scott D, Scolnick E, Metcalf D: Growth of factor-dependent haemopoietic precursor cell lines. J Exp Med 152:1036, 1980[Abstract/Free Full Text]

31. Hatakeyama M, Mori H, Doi T, Taniguchi T: A restricted cytoplasmic region of IL-2 receptor beta  chain is essential for growth signal transduction but not for ligand binding and internalization. Cell 59:837, 1989[Medline] [Order article via Infotrieve]

32. Barry SC, Bagley CJ, Phillips J, Dottore M, Cambareri B, Moretti P, D'Andrea R, Goodall GJ, Shannon MF, Vadas MA, Lopez AF: Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha - and beta -chains of its receptor and participate in function. J Biol Chem 269:8488, 1994[Abstract/Free Full Text]

33. Rayner JR, Gonda TJ: A simple and efficient procedure for generating stable expression libraries by cDNA cloning in a retroviral vector. Mol Cell Biol 14:880, 1994[Abstract/Free Full Text]

34. Johnson GR: Colony formation in agar by adult bone marrow multipotential hemopoietic cells. J Cell Physiol 103:371, 1980[Medline] [Order article via Infotrieve]

35. Hughes SH, Payvar F, Spector D, Schimke RT, Robinson HL, Payne GS, Bishop JM, Varmus HE: Heterogeneity of genetic loci in chickens: Analysis of endogenous viral and normal genes by cleavage of DNA with restriction endonucleases. Cell 18:347, 1979[Medline] [Order article via Infotrieve]

36. Woodcock JM, Zacharakis B, Plaetinck G, Bagley CJ, Qiyu S, Hercus TR, Tavernier J, Lopez AF: Three residues in the common beta  chain of the human GM-CSF, IL-3 and IL-5 receptors are essential for GM-CSF and IL-5 but not IL-3 high affinity binding and interact with Glu21 of GM-CSF. EMBO J 13:5176, 1994[Medline] [Order article via Infotrieve]

37. Barry SC, Moretti PAB, Korpeleinen E, D'Andrea R, Lopez AF, Vadas MA, Goodall GJ: Roles of the N-terminal and C-terminal domains of the IL-3 receptor alpha  chain in receptor function. Blood 89:842, 1997[Abstract/Free Full Text]

38. Durstin M, Inhorn RC, Griffin JD: Tyrosine phosphorylation of Shc is not required for proliferation or viability signalling by granulocyte-macrophage colony-stimulating factor in hematopoietic cell lines. J Immunol 157:534, 1996[Abstract]

39. Marshall CJ: Specificity of receptor tyrosine kinase signalling: Transient versus sustained extracellular signal-regulated kinase activation. Cell 80:179, 1995[Medline] [Order article via Infotrieve]

40. Ihle JN: The Janus protein tyrosine kinase family and its role in cytokine signaling. Adv Immunol 60:1, 1995[Medline] [Order article via Infotrieve]

41. Brizzi MF, Aronica MG, Rosso A, Bagnara GP, Yarden Y, Pegoraro L: Granulocyte-macrophage colony-stimulating factor stimulates JAK2 signalling pathway and rapidly activates p93fes, STAT1 p91 and STAT3 p92 in polymorphonuclear leukocytes. J Biol Chem 271:3562, 1996[Abstract/Free Full Text]

42. Watanabe S, Itoh T, Arai K: JAK2 is essential for activation of c-fos and c-myc promoters and cell proliferation through the human granulocyte-macrophage colony-stimulating factor receptor in BA/F3 cells. J Biol Chem 271:12681, 1996[Abstract/Free Full Text]

43. Gouilleux F, Wakao H, Mundt M, Groner B: Prolactin induces phosphorylation of Tyr694 of Stat5 (MGF), a prerequisite for DNA binding and induction of transcription. EMBO J 13:4361, 1994[Medline] [Order article via Infotrieve]

44. Sakai I, Kraft AS: The kinase domain of Jak2 mediates induction of Bcl-2 and delays cell death in hematopoietic cells. J Biol Chem 272:12350, 1997[Abstract/Free Full Text]

45. Quelle FW, Thierfelder WE, Witthuhn BA, Tang B, Cohen SL, Ihle JN: Phosphorylation and activation of the DNA binding activity of purified Stat1 by the Janus protein-tyrosine kinases and the epidermal growth factor receptor. J Biol Chem 270:20775, 1995[Abstract/Free Full Text]

46. Watanabe S, Itoh T, Arai K: Roles of JAK kinases in human GM-CSF receptor signal transduction. J Allergy Clin Immunol 98:S183, 1996[Medline] [Order article via Infotrieve]

47. Grube BJ, Cochrane CG: Identification of a regulatory domain of the interleukin-6 receptor. J Biol Chem 269:20791, 1994[Abstract/Free Full Text]

48. Yawata H, Yasukawa K, Natsuka S, Murakami M, Yamasaki K, Hibi M, Taga T, Kishimoto T: Structure-function analysis of human IL-6 receptor: Dissociation of amino acid residues required for IL-6-binding and for IL-6 signal transduction through gp130. EMBO J 12:1705, 1993[Medline] [Order article via Infotrieve]

49. Sternberg MJ, Gullick WJ: Neu receptor dimerization. Nature 339:587, 1989[Medline] [Order article via Infotrieve]

50. Hofmann K, Stoffel W: TMbase---A database of membrane-spanning protein segments. Biol Chem Hoppe-Seyler 347:166, 1993

51. Murakami M, Narazaki M, Hibi M, Yawata H, Yasukawa K, Hamaguchi M, Taga T, Kishimoto T: Critical cytoplasmic region of the interleukin 6 signal transducer gp130 is conserved in the cytokine receptor family. Proc Natl Acad Sci USA 88:11349, 1991[Abstract/Free Full Text]

52. Miura O, Cleveland JL, Ihle JN: Inactivation of erythropoietin receptor function by point mutations in a region having homology with other cytokine receptors. Mol Cell Biol 13:1788, 1993[Abstract/Free Full Text]

53. DaSilva L, Howard OMZ, Rui H, Kirken RA, Farrar WL: Growth signalling and JAK2 association mediated by membrane-proximal cytoplasmic regions of prolactin receptors. J Biol Chem 269:18267, 1994[Abstract/Free Full Text]

54. Goldsmith MA, Xu W, Amaral MC, Kuczek ES, Greene WC: The cytoplasmic domain of the interleukin-2 receptor beta  chain contains both unique and functionally redundant signal transduction elements. J Biol Chem 269:14698, 1994[Abstract/Free Full Text]

55. Joneja B, Wojchowski DM: Mitogenic signalling and inhibition of apoptosis via the erythropoietin receptor box-1 domain. J Biol Chem 272:11176, 1997[Abstract/Free Full Text]

56. Drachman JG, Kaushansky K: Dissecting the thrombopoietin receptor: Functional elements of the Mpl cytoplasmic domain. Proc Natl Acad Sci USA 94:2350, 1997[Abstract/Free Full Text]

57. Nicholson SE, Novak U, Ziegler SF, Layton JE: Distinct regions of the granulocyte colony-stimulating factor receptor are required for tyrosine phosphorylation of the signalling molecules JAK2, Stat3, and p42, p44MAPK. Blood 86:3698, 1995[Abstract/Free Full Text]

58. Kitayama H, Kanakura Y, Furitsu T, Tsujimura T, Oritani K, Ikeda H, Sugahara H, Mitsui H, Kanayama Y, Kitamura Y, Matsuzawa Y: Constitutively activating mutations of c-kit receptor tyrosine kinase confer factor-independent growth and tumorigenicity of factor-dependent hematopoietic cell lines. Blood 85:790, 1995[Abstract/Free Full Text]

59. Inhorn RC, Carlesso N, Durstin M, Frank DA, Griffin JD: Identification of a viability domain in the granulocyte/macrophage colony-stimulating factor receptor beta -chain involving tyrosine-750. Proc Natl Acad Sci USA 92:8665, 1995[Abstract/Free Full Text]

60. Winston LA, Hunter T: JAK2, Ras, and Raf are required for activation of extracellular signal-regulated kinase/mitogen-activated protein kinase by growth hormone. J Biol Chem 270:30837, 1995[Abstract/Free Full Text]

61. Yin T, Shen R, Feng GS, Yang YC: Molecular characterization of specific interactions between SHP-2 phosphatase and JAK tyrosine kinases. J Biol Chem 272:1032, 1997[Abstract/Free Full Text]

62. Xia K, Mukhopadhyay NK, Inhorn RC, Barber DL, Rose PE, Lee RS, Narsimhan RP, D'Andrea AD, Griffin JD, Roberts TM: The cytokine-activated tyrosine kinase JAK2 activates Raf-1 in a p21ras-dependent manner. Proc Natl Acad Sci USA 93:11681, 1996[Abstract/Free Full Text]

63. Onishi M, Mui AL, Morikawa Y, Cho LC, Kinoshita S, Nolan GP, Gorman DM, Miyajima A, Kitamura T: Identification of an oncogenic form of the thrombopoietin receptor MPL using retrovirus-mediated gene transfer. Blood 88:1399, 1996[Abstract/Free Full Text]

64. Chao JR, Chen CS, Wang TF, Tseng LH, Tsai JJ, Kuo ML, Yen JJY, Yang Yen HF: Characterization of factor-independent variants derived from TF-1 hematopoietic progenitor cells: The role of the Raf/MAP kinase pathway in the anti-apoptotic effect of GM-CSF. Oncogene 14:721, 1997[Medline] [Order article via Infotrieve]

65. van der Bruggen T, Caldenhoven E, Kanters D, Coffer P, Raaijmakers JAM, Lammers JWJ, Koenderman L: Interleukin-5 signalling in human eosinophils involves JAK2 tyrosine kinase and Stat1alpha . Blood 85:1442, 1995[Abstract/Free Full Text]

66. Mui AL, Wakao H, Kinoshita T, Kitamura T, Miyajima A: Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: Role of Stat5 in proliferation. EMBO J 15:2425, 1996[Medline] [Order article via Infotrieve]

67. Damen JE, Wakao H, Miyajima A, Krosl J, Humphries RK, Cutler RL, Krystal G: Tyrosine 343 in the erythropoietin receptor positively regulates erythropoietin-induced cell proliferation and Stat5 activation. EMBO J 14:5557, 1995[Medline] [Order article via Infotrieve]

68. Klingmuller U, Bergelson S, Hsiao JG, Lodish HF: Multiple tyrosine residues in the cytosolic domain of the erythropoietin receptor promote activation of STAT5. Proc Natl Acad Sci USA 93:8324, 1996[Abstract/Free Full Text]

69. Iwatsuki K, Endo T, Misawa H, Yokouchi M, Matsumoto A, Ohtsubo M, Mori KJ, Yoshimura A: STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line. J Biol Chem 272:8149, 1997[Abstract/Free Full Text]

70. Chin H, Nakamura N, Kamiyama R, Miyasaka N, Ihle JN, Miura O: Physical and functional interactions between Stat5 and the tyrosine-phosphorylated receptors for erythropoietin and interleukin-3. Blood 88:4415, 1996[Abstract/Free Full Text]

71. Fujii H, Nakagawa Y, Schindler U, Kawahara A, Mori K, Gouilleux F, Groner B, Ihle JN, Minami Y, Miyazaki T, Taniguchi T: Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta  chain but is not essential for the proliferative signal transmission. Proc Natl Acad Sci USA 92:5482, 1995[Abstract/Free Full Text]

72. Goodall GJ, Bagley CJ, Vadas MA, Lopez AF: A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands. Growth Factors 8:87, 1993[Medline] [Order article via Infotrieve]

73. Bazan JF: Structural design and molecular evolution of a cytokine receptor superfamily. Proc Natl Acad Sci USA 87:6934, 1990[Abstract/Free Full Text]

74. Kraulis P: MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J Appl Cryst 24:946, 1991

75. Merrit EA, Murphy MEP: Raster3D Version 2.0, a program for photorealistic molecular graphics. Acta Crsyt D50:869, 1994

© 1998 by the American Society of Hematology.
 
0006-4971/98/92-0008$3.00/0

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Leukoc. Biol.Home page
A. L. Brown, C. R. Wilkinson, S. R. Waterman, C. H. Kok, D. G. Salerno, S. M. Diakiw, B. Reynolds, H. S. Scott, A. Tsykin, G. F. Glonek, et al.
Genetic regulators of myelopoiesis and leukemic signaling identified by gene profiling and linear modeling
J. Leukoc. Biol., August 1, 2006; 80(2): 433 - 447.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
A. L. Brown, M. Peters, R. J. D'Andrea, and T. J. Gonda
Constitutive mutants of the GM-CSF receptor reveal multiple pathways leading to myeloid cell survival, proliferation, and granulocyte-macrophage differentiation
Blood, January 15, 2004; 103(2): 507 - 516.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
V. T. Phan, D. B. Shultz, B.-T. H. Truong, T. J. Blake, A. L. Brown, T. J. Gonda, M. M. Le Beau, and S. C. Kogan
Cooperation of Cytokine Signaling with Chimeric Transcription Factors in Leukemogenesis: PML-Retinoic Acid Receptor Alpha Blocks Growth Factor-Mediated Differentiation
Mol. Cell. Biol., July 1, 2003; 23(13): 4573 - 4585.
[Abstract] [Full Text] [PDF]

Home page
 J. Leukoc. Biol.Home page
T. J. Blake, B. J. Jenkins, R. J. D'Andrea, and T. J. Gonda
Functional cross-talk between cytokine receptors revealed by activating mutations in the extracellular domain of the {beta}-subunit of the GM-CSF receptor
J. Leukoc. Biol., December 1, 2002; 72(6): 1246 - 1255.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
F. Le, F. Stomski, J. M. Woodcock, A. F. Lopez, and T. J. Gonda
The Role of Disulfide-linked Dimerization in Interleukin-3 Receptor Signaling and Biological Activity
J. Biol. Chem., February 18, 2000; 275(7): 5124 - 5130.
[Abstract] [Full Text] [PDF]

Home page
 BloodHome page
M. P. McCormack and T. J. Gonda
Novel murine myeloid cell lines that exhibit a differentiation switch in response to IL-3 or GM-CSF, or to different constitutively active mutants of the GM-CSF receptor beta subunit
Blood, January 1, 2000; 95(1): 120 - 127.
[Abstract] [Full Text] [PDF]

Home page
 EndocrinologyHome page
K. E. Borg, M. Zhang, D. Hegge, R. L. Stephen, D. J. Buckley, N. S. Magnuson, and A. R. Buckley
Prolactin Regulation of pim-1 Expression: Positive and Negative Promoter Elements
Endocrinology, December 1, 1999; 140(12): 5659 - 5668.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
B. J. Jenkins, F. Le, and T. J. Gonda
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
J. Biol. Chem., March 26, 1999; 274(13): 8669 - 8677.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
H. Sakamoto, T. Kitamura, and A. Yoshimura
Mitogen-activated Protein Kinase Plays an Essential Role in the Erythropoietin-dependent Proliferation of CTLL-2 Cells
J. Biol. Chem., November 10, 2000; 275(46): 35857 - 35862.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Right arrow Rights and Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jenkins, B. J.
Right arrow Articles by Gonda, T. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jenkins, B. J.
Right arrow Articles by Gonda, T. J.
Related Collections
Right arrow Hematopoiesis and Stem Cells
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

click for free articles
home about blood authors subscriptions permissions advertising public access contact us
  Copyright © 1998 by American Society of Hematology         Online ISSN: 1528-0020