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Blood, Vol. 93 No. 2 (January 15), 1999:
pp. 447-458
RAPID COMMUNICATION
Defective Internalization and Sustained Activation of Truncated
Granulocyte Colony-Stimulating Factor Receptor Found in Severe
Congenital Neutropenia/Acute Myeloid Leukemia
By
Alister C. Ward,
Yvette M. van Aesch,
Anita M. Schelen, and
Ivo P. Touw
From the Institute of Hematology, Erasmus University, Rotterdam, The
Netherlands; and the Department of Hematology, Dr. Daniel den Hoed
Cancer Center, Rotterdam, The Netherlands.
 |
ABSTRACT |
Acquired mutations truncating the C-terminal domain of the
granulocyte colony-stimulating factor receptor (G-CSF-R)
are found in about 20% of severe congenital neutropenia (SCN)
patients, with this cohort of patients predisposed to acute myeloid
leukemia (AML). In myeloid cells, such mutations act in a
dominant-negative manner leading to hyperproliferation and lack of
differentiation in response to G-CSF. However, why these truncated
receptors are dominant in function over wild-type receptors has
remained unclear. We report that ligand-induced internalization of
truncated G-CSF-R is severely impaired compared with the
wild-type receptor, which results in sustained activation of STAT
proteins. Strikingly, in cells coexpressing both truncated and
wild-type forms, the truncated receptors acted dominantly with regard
to both internalization and sustained activation. Site-directed
mutagenesis of the C-terminus showed that receptor tyrosines in this
region were dispensable for internalization, whereas a
di-leucine-containing motif in Box B3 played some role. However, loss
of the di-leucine motif was not the critical determinant of the
sustained activation status of truncated receptors. These data suggest
that defective internalization, leading to extended receptor
activation, is a major cause of the dominant hyperproliferative effect
of truncated G-CSF receptors, which is only partially due to the loss
of a di-leucine motif present in the Box B3 region of the full-length
receptor.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GRANULOCYTE colony-stimulating factor
(G-CSF) is a major regulator of neutrophil production.1-4
Its effects are mediated by a receptor of the hematopoietin
superfamily, the G-CSF-R, which forms homo-oligomeric complexes upon
ligand binding.5 Like other family members, the G-CSF-R
lacks intrinsic tyrosine kinase activity but activates cytoplasmic
tyrosine kinases.2,5,6 Important signaling pathways
activated by the G-CSF-R include those involving various members of the
Janus tyrosine kinase (Jak) and signal transducer and activator of
transcription (STAT) families of proteins,7-15 the Src
kinases p55Lyn and p56/59Hck,16-18
and components of the p21ras/Raf/MAPK
pathway.11,19-22
Severe congenital neutropenia (SCN) is a heterogeneous disorder
characterized by a severe reduction in circulating neutrophils (<0.2 × 109/L). We have previously identified a subset of
SCN patients with acquired nonsense mutations in the gene encoding the
G-CSF-R. These mutations truncate between 82 and 98 amino acids
from the carboxy-terminus of the receptor, a region implicated in
maturation induction and growth arrest.23-26 In patients
carrying these mutations, a neutrophilic progenitor cell that attains a
GCSFR mutation has gained the ability to clonally expand,
suggesting hyperproliferation of an early compartment. In addition,
such patients have a strong predisposition to acute myeloid leukemia
(AML).26 Truncated receptors show normal affinity for
G-CSF.23 However, when expressed in myeloid cells, these
truncated receptors transduce a strong growth signal but fail to induce
maturation.25 Coexpression of wild-type and truncated
receptors in myeloid cell lines has shown that truncated receptors act
in a dominant-negative manner over wild-type receptors to enhance
proliferation at the expense of maturation.25 Similar
dominant hyperproliferation is seen in mice heterozygous for a targeted
G-CSF-R truncation27 and presumably also in SCN/AML
patients, because GCSFR mutations affect just a single
allele.24-26 However, the molecular mechanisms responsible for the dominant hyperproliferative function of truncated G-CSF receptors have remained unknown.
To investigate these mechanisms, we studied receptor activation and
internalization after exposure to ligand in 32D cells expressing
different G-CSF-R forms. We show that activation of STAT complexes by
truncated receptors is significantly prolonged due to defective
receptor internalization. Importantly, mutant receptors were found to
act in a dominant-negative manner over wild-type receptors in both
processes. Site-directed mutagenesis of the G-CSF-R C-terminal domain
showed that three conserved tyrosines contained within this domain are
not essential for efficient receptor internalization or deactivation.
In contrast, mutation of a conserved di-leucine- containing motif in
Box B3, which was previously shown to be important for internalization
of the related gp130 receptor,28 caused delayed
internalization and prolonged receptor activation, although not as
pronounced as the effect of C-terminal truncation. Moreover,
steady-state surface expression and activation status of these
di-leucine mutant receptors was only marginally different from
wild-type G-CSF-R, which was reflected in a minor enhancement of
proliferation, with maturation signaling remaining intact. Together,
these data provide a plausible explanation for the dominant hyperproliferative function of truncated G-CSF-Rs in response to G-CSF,
mediated by defective receptor internalization after ligand binding,
leading to extended growth signals, which is only partially due to the
loss of a di-leucine motif in Box B3 of the receptor.
| |
MATERIALS AND METHODS |
Cell culture.
32D.cl8.6, a subline of the interleukin-3
(IL-3)-dependent murine
myeloid 32D cell line,29 was maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) and 10 ng of murine IL-3
per milliliter at 37°C and 5% CO2.
Site-directed mutagenesis and transfections.
The pLNCX expression clones of human WT (wild-type) G-CSF-R, the
C-terminal deletion mutant mDA ( 715), and the single
tyrosine-to-phenylalanine (Y F) substitution mutants Y704F,
Y729F, Y744F, and Y764F have been described
previously.23,30 Double YF mutants were created from the single mutants using site-directed mutagenesis, as
described,30 and from these a triple YF mutant,
mA (Y729F, Y744F, Y764F), was constructed by recombinant polymerase
chain reaction (PCR), using the following oligonucleotide primers:
GRRV14 (5 -CCTGGGCTTGTGGGGCTGC), GRFR11
(5-TGCTGGGCAGCCCCACAAG), LNCXRV
(5-CCCTTACTTTCTGGGGTGGACATC), and GRFR7
(5-GTCCTCACCCTGATGACC). The 5 segment of each mutant was
amplified using GRFR7 and GRRV14, and the 3 segment was
amplified using GRFR11 and LNCXRV. Products of the primary PCR were
isolated, mixed 1:1, and used as a template for a secondary PCR with
GRFR7 and LNCXRV. To produce the mutant mLALA (L753A, L754A), which lacks a conserved di-leucine motif in Box B3 of the receptor, a similar
strategy was employed, except using GRFR12
(5-CAGCCCGCTGCAGCGGGCCTCACCCCCAG) in place of GRFR11, using
GRRV15 (5-GCCCGCTGCAGCGGGCTGAGTGGAGTCAC) instead of GRRV14, and
with DNA encoding the WT G-CSF-R as a template. In each case, the
resultant product was digested with Hpa I and Bgl II
and cloned into pLNCX containing WT G-CSF-R, which had also been
digested with these enzymes. The authenticity of all mutants was
verified by restriction enzyme analysis and DNA sequencing. For stable
transfections, parental 32D.cl8.6 cells were electroporated with 10 µg Pvu I-digested pLCNX clones, using a Progenetor II apparatus (Hoefer Scientific Instruments, San Francisco,
CA) set at 260 V, 100 µF, and 1 second. After 48 hours of incubation, cells were selected with G418 (GIBCO-BRL, Breda, The Netherlands) at a
concentration of 0.8 mg/mL. Multiple clones were expanded for further
analysis. In addition, clones from a previous study were used that
contain pLNCX expressing WT G-CSF-R in combination with either pBabe
alone (32D[WT/vec]) or with pBabe expressing the mDA (32D[WT/mDA]),
selected with 0.8 mg/mL G418 and 1 µg/mL puromycin.25
Flow cytometric analysis.
To determine G-CSF-R expression levels in of 32D.cl8.6 transfectants,
cells (106) were incubated at 4°C for 1 hour
sequentially with 10 µg/mL of biotinylated mouse antihuman G-CSF-R
monoclonal antibody LMM741 (PharMingen, San Diego, CA), 5 µg/mL of
phytoerythrin-conjugated streptavidin (SA-PE), 5 µg/mL of
biotinylated antistreptavidin antibody, and finally 2 µg/mL of SA-PE,
with washing between each antibody step. Samples were analyzed by flow
cytometry using a FACScan (Becton Dickinson, San Jose, CA). At least
three independently derived clones of each construct were selected on
the basis of homogeneous receptor expression. For internalization
experiments, cells (106) were incubated at 4°C for 1 hour with 0.2 µg/mL biotinylated G-CSF and then for various times at
37°C. Subsequently, cells were incubated for 30 minutes at 4°C
with SA-PE in the presence of 0.02% NaN3 before flow
cytometric analysis.
Measurement of 125I-G-CSF internalization.
Cells (2 to 4 × 106) were incubated for 1 hour at
4°C in 100 µL  -minimal essential medium containing 10% FCS
and 1500 pmol/L 125I-G-CSF (Amersham Nederland BV, Den
Bosch, The Netherlands), with or without excess nonlabeled G-CSF, and
then transferred to 37°C for various times. Cells were washed
either in phosphate-buffered saline (PBS; total ligand) or sodium
citrate, pH 4 (internalized ligand), before centrifugation through an
FCS cushion. Specific binding was determined as the difference in total
binding in the absence or presence of unlabeled G-CSF. Internalized
ligand was expressed as a percentage of total specific binding at each
time point.
Cell proliferation and morphological analysis.
To determine the proliferation and differentiation characteristics of
32D.cl8.6 clones, cells were incubated at an initial density of 1 to 2 × 105 cells/mL in RPMI medium supplemented with 10%
FCS with 100 ng/mL of human G-CSF, with 10 ng/mL of murine IL-3, or
without growth factors. The medium was replenished every 2 to 4 days,
and the cell densities were adjusted to 1 to 2 × 105
cells/mL. Viable cells were counted on the basis of trypan blue exclusion. To analyze morphological features, cells were spun onto
glass slides and examined after May-Grünwald-Giemsa staining. Without IL-3 or G-CSF, all transfectants died within 1 to 2 days and
showed no signs of neutrophilic differentiation, whereas parental 32D.cl8.6 cells also died within 1 to 2 days in G-CSF-containing medium. To quantify the neutrophilic maturation of 32D.cl8.6
transfectants in response to G-CSF, the number of cells showing signs
of differentiation (ie, band neutrophils or more mature) was determined
and expressed as a percentage of total living cells (percentage of
neutrophils).
Preparation of nuclear extracts.
Cells were deprived of serum and factors for 4 hours at 37°C in
RPMI 1640 medium at a density of 1 to 2 × 106/mL and then stimulated with either RPMI 1640 medium
alone or in the presence of 100 ng/mL human G-CSF. At different time
points, 10 vol of ice-cold PBS supplemented with 10 µmol/L
Na3VO4 were added to the cells, which were then
pelleted and resuspended in ice-cold hypotonic buffer (20 mmol/L HEPES,
pH 7.8, 20 mmol/L NaF, 1 mmol/L Na3VO4, 1 mmol/L Na4P2O7, 1 mmol/L
dithiothreitol [DTT], 1 mmol/L EDTA, 1 mmol/L EGTA,
0.2% Tween-20, 0.125 µmol/L okadaic acid, 1 mmol/L Pefabloc SC, 50 µg/mL aprotinin, 50 µg/mL leupeptin, 50 µg/mL bacitracin, and 50 µg/mL iodoacetamide).31 Cells were vortexed for 10 seconds and the nuclei were pelleted by centrifugation at
15,000g for 30 seconds. Nuclear extracts were prepared by
resuspension of the nuclei in high-salt buffer (hypotonic buffer with
420 mmol/L NaCl and 20% glycerol) followed by rocking for 30 minutes
at 4°C. Insoluble materials were removed by centrifugation at
4°C for 15 minutes at 15,000g.
Electrophoretic mobility shift assay (EMSA).
Nuclear extracts were incubated for 20 minutes at room temperature with
0.2 ng of 32P-labeled double-stranded oligonucleotide and 2 µg of poly(dI-dC) in 20 µL of binding buffer (13 mmol/L HEPES, pH
7.8, 80 mmol/L NaCl, 3 mmol/L NaF, 3 mmol/L NaMoO4, 1 mmol/L DTT, 0.15 mmol/L EDTA, 0.15 mmol/L EGTA, and 8%
glycerol).32 The oligonucleotide probes used in this study
were m67 (5-CATTTCCCGTAAATC), a high-affinity mutant of the
sis-inducible element (SIE) of the human c-fos
gene,33 which binds STAT1 and STAT3, and  -cas
(5-AGATTTCTAGGAATTCAATCC), derived from the 5 region of
the -casein gene,34 which binds STAT5 and STAT1. The
DNA-protein complexes were separated by electrophoresis on 5%
polyacrylamide gels containing 5% glycerol in 0.25× TBE. The
gels were dried and subsequently analyzed by autoradiography.
| |
RESULTS |
STAT activation from truncated G-CSF receptors is prolonged.
Activation of STAT proteins has been implicated in the control of
G-CSF-mediated proliferation and differentiation.35,36 In
addition, STAT activation represents a sensitive measure of receptor
activation.37 To investigate how stimulation of truncated G-CSF receptors might lead to hyperproliferation at the expense of
maturation, we examined the effect of receptor truncation on STAT
activation in 32D cells, in which the dominant hyperproliferative function of truncated receptors has been clearly
documented.25 32D.cl8.6 clones expressing either wild-type
(WT) G-CSF-R, or a truncated form derived from an SCN patient (mDA;
Figs 1 and 2A) were stimulated with
G-CSF, and the kinetics of STAT activation were examined (Fig 2B). At
early time points (up to 15 minutes), G-CSF-induced activation of
STAT5 from WT and mDA receptors was similar, whereas STAT3 activation
from mDA was reduced. However, at later time points of stimulation,
when STAT5 activation from the WT G-CSF-R decreased, activation from
mDA persisted. A similar result was seen with STAT3, although not as
marked. In addition, activation of STAT1-containing complexes from mDA
was greater and more sustained compared with the WT G-CSF-R.
IL-3-induced activation of STAT5 complexes was equivalent in cells
expressing either receptor type (data not shown).
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| Fig 1.
Schematic representation of G-CSF-R proteins
studied. Cytoplasmic domains of wild-type and mutant
receptors are shown. Boxes B1 and B2 denote subdomains conserved in
members of the hematopoietin receptor superfamily, whereas Box B3
is conserved only with a limited number of family members,
including gp130.62,63 Y, tyrosine; F, phenylalanine; L,
leucine; A, alanine.
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| Fig 2.
Activation of STAT complexes by wild-type and truncated
receptors. (A) Flow cytometric analysis of G-CSF-R
expression on representative 32D.cl8.6 clones expressing
wild-type (WT) or truncated (mDA) G-CSF-Rs. Cells were either stained
with biotinylated mouse antihuman G-CSF-R antibodies, followed by
PE-conjugated streptavidin, biotinylated antistreptavidin,
and finally PE-conjugated streptavidin (unshaded), or without
the anti-G-CSF-R step (shaded). (B) EMSA of nuclear
extracts prepared from 32D[WT] and 32D[mDA] cells stimulated
with G-CSF for the times indicated, analyzed using m67 and -cas
probes. Supershift analysis with antibodies against various STAT
proteins was used to identify which STATs were present in each
complex: S1, STAT1; S3, STAT3; S5, STAT5. This is representative
of four independent experiments. (C) 32D[WT] and
32D[mDA] cells were stimulated with G-CSF for 10 minutes,
washed extensively, and incubated in media alone for the times
indicated (G-off). Nuclear extracts were prepared at the indicated
times and assayed by EMSA using the m67 and -cas probes. This is
representative of three independent experiments.
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To further examine the activation kinetics of truncated receptors,
cells expressing WT or mDA receptors were stimulated with G-CSF for 10 minutes, extensively washed to remove the cytokine, and then analyzed
for STAT activation. Under these conditions, extended activation of mDA
receptors, as shown by prolonged activation of STATs 1, 3, and 5, was
again observed (Fig 2C). This result shows that the truncated receptors
that bound ligand during the first 10 minutes of stimulation were
responsible for the prolonged STAT activation, and not new receptors
recruited to the cell surface, implying that truncated receptors have
extended off-rates after ligand binding. Because WT and mDA receptors
show equivalent Kd,23 this suggests that
receptor deactivation is altered in truncated receptors.
Impaired ligand-mediated G-CSF-R internalization by truncated
receptors.
Ligand-induced receptor internalization represents a major mechanism of
downmodulating receptor activation.28,38 Therefore, to
investigate the extended receptor off-rates, the kinetics of G-CSF-R
internalization after exposure to G-CSF were examined by measuring the
amount of surface bound biotinylated G-CSF over time
(Fig 3A). Significant internalization of the WT G-CSF-R
was already seen within 15 minutes, and by 90 minutes most
ligand/receptor complexes had been internalized. In contrast,
ligand-induced receptor internalization of mDA was delayed, and even
after 90 minutes a significant proportion of ligand/receptor complexes
were still present on the cell surface. To confirm and quantify this
result, we also studied internalization using 125I-labeled
G-CSF (Fig 3B). This indicated that, after 2 hours, 88% of ligand was
internalized via the WT G-CSF-R, compared with only 33% via mDA.
Similarly, the receptor C-terminus of the IL-8 receptor has been shown
to greatly influence the cellular responses to IL-8.39
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| Fig 3.
Internalization of ligated G-CSF-R complexes. (A)
32D[WT] and 32D[mDA] cells were allowed to bind biotinylated-G-CSF
at 4°C and were subsequently incubated at 37°C for various
times before staining with SA-PE to determine surface-bound G-CSF.
(Bold line) 0 minutes; (dotted line) 30 minutes; (thin line) 90 minutes; (shaded histogram) 0 minutes, with initial binding in the
presence of excess nonbiotinylated-G-CSF. This is representative of
four independent determinations. (B) Rate of internalization of
125I-G-CSF by 32D[WT] ( ) and 32D[mDA] ( ) cells,
expressed as the percentage of total specific binding that was
resistant to acid washing at each time point. Similar results were
obtained in a duplicate experiment.
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Cytochalasin D can mimic the effect of receptor truncation.
Cytochalasin D specifically inhibits actin polymerization, without
affecting other pathways,40-44 and has been used previously to interfere with a range of internalization
processes.45-49 32D[WT] cells pretreated with
cytochalasin D for 20 minutes before G-CSF stimulation showed both
impaired receptor internalization (Fig 4A)
and sustained receptor activation (Fig 4B) compared with cells with no
pretreatment. Thus, blocking the internalization even of WT G-CSF-R is
sufficient to enhance receptor activation, which suggests a causal
relationship between these two processes. In addition, this result
implicates actin microfilaments as effectors of the ligand-induced
internalization of the G-CSF-R.
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| Fig 4.
Effect of cytochalasin D on receptor activation and
internalization. (A) Receptor internalization of 32D[WT] cells in the
presence or absence of cytochalasin D, as indicated. (Bold line) 0 minutes; (dotted line) 30 minutes; (thin line) 90 minutes; (shaded
histogram) 0 minutes, with initial binding in the presence of excess
nonbiotinylated-G-CSF. This is representative of three independent
experiments. (B) 32D[WT] cells, in the presence or absence of
cytochalasin D, were stimulated with G-CSF for 10 minutes, washed
extensively, and incubated in media alone for the times indicated
(G-off). Nuclear extracts were prepared at the indicated times and
assayed by EMSA using the m67 and -cas probes. This is
representative of three independent experiments.
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Truncated receptors act in a dominant-negative manner with regard to
internalization and STAT activation.
Defective internalization and prolonged receptor activation provides a
possible explanation for the enhanced proliferation mediated by
truncated receptors. However, we have previously shown that truncated
receptors exert a dominant hyperproliferative function over wild-type
maturation signals in 32D cells coexpressing WT and mDA
receptors.25 Therefore, we analyzed these cells
(Fig 5A) with regard to receptor
internalization and STAT activation after exposure to ligand. Impaired
receptor downregulation was observed in coexpressing cells
(32D[WT/mDA]) compared with cells expressing only wild-type receptors
(32D[WT/vector]) (Fig 5B). Furthermore, 32D[WT/mDA] cells showed
more sustained STAT activation in response to G-CSF than
32D[WT/vector] cells (Fig 5C). This clearly shows that mutant
receptors exert a dominant effect over wild-type receptors with regard
to internalization and activation, as well as proliferation.
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| Fig 5.
Dominant-negative effect of truncated receptors. (A) Flow
cytometric analysis of G-CSF-R expression on 32D[WT/vector] and
32D[WT/mDA] cells. Cells were either stained with
biotinylated mouse antihuman G-CSF-R antibodies, followed by
PE-conjugated streptavidin, biotinylated antistreptavidin, and finally
PE-conjugated streptavidin (unshaded), or without the anti-G-CSF-R
step (shaded). (B) Receptor internalization of 32D[WT/vector] and
32D[WT/mDA] cells, as indicated. (Bold line) 0 minutes; (dotted line)
30 minutes; (thin line) 90 minutes; (shaded histogram) 0 minutes, with
initial binding in the presence of excess nonbiotinylated-G-CSF.
This is representative of three independent determinations. (C)
32D[WT/vector] and 32D[WT/mDA] cells were stimulated with G-CSF for
10 minutes, washed extensively, and incubated in media alone for the
times indicated (G-off). Nuclear extracts were prepared at the
indicated times and assayed by EMSA using the m67 and -cas probes.
This is representative of three independent experiments.
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Mutational analysis of the G-CSF-R C-terminus.
The data presented above suggest that sequences in the C-terminus
mediate ligand-induced G-CSF-R internalization that subsequently affects receptor activation and cell proliferation. Because
internalization motifs based on di-leucine and tyrosine-containing
sequences have been described,50,51 we examined whether
such motifs in the G-CSF-R C-terminus were involved in receptor
internalization. Two specific mutants were constructed: mLALA, which
has a conserved di-leucine motif in Box B3 replaced with a di-alanine,
and mA, a triple YF mutant that lacks the three tyrosines in
the C-terminal region (Fig 1). These mutant receptors were introduced
into 32D.cl8.6 cells and clones expressing equivalent levels of each
receptor were selected for further analysis
(Fig 6A). Mutation of the di-leucine motif
resulted in a delay in receptor internalization (Fig 6B), leading to
extended STAT activation (Fig 6C), although neither of these effects
was as prominent as observed with mDA. In addition, clones expressing
the mLALA mutant showed some variability in growth but with a tendency
for slightly enhanced proliferation compared with the 32D[WT] cells,
although clearly not the sustained exponential growth of 32D[mDA]
(Fig 7A). Furthermore, 32D[mLALA] clones
showed effective, but delayed, neutrophilic maturation in response to
G-CSF (Fig 7B and C), suggesting that the di-leucine motif plays some
role in internalization and subsequent growth control. In contrast,
receptor internalization kinetics for mA were equivalent to those of
the WT G-CSF-R (Fig 6B), whereas STAT activation was similar, albeit
marginally extended (Fig 6C). Moreover, all 32D[mA] clones showed
comparable growth and differentiation to that of 32D[WT] clones (Fig
7). This shows that the tyrosines of the C-terminal region are not
required for receptor internalization, growth inhibition, or
differentiation.
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| Fig 6.
Receptor internalization and activation in cells
expressing mLALA and mA mutants. (A) Flow cytometric
analysis of G-CSF-R expression on parental 32D.cl8.6 cells and
representative 32D.cl8.6 transfectants. Cells were either stained with
biotinylated mouse antihuman G-CSF-R antibodies, followed by
PE-conjugated streptavidin, biotinylated antistreptavidin, and finally
PE-conjugated streptavidin (unshaded), or without the
anti-G-CSF-R step (shaded). (B) Receptor internalization
of 32D.cl8.6 cells expressing wild-type or mutant G-CSF receptors, as
indicated. (Bold line) 0 minutes; (dotted line) 30 minutes; (thin line)
90 minutes; (shaded histogram) 0 minutes, with initial binding in the
presence of excess nonbiotinylated-G-CSF. This is representative of
three independent determinations. (C) 32D.cl8.6 cells, expressing
wild-type or mutant G-CSF receptors, were stimulated with G-CSF for 10 minutes, washed extensively, and incubated in media alone for the times
indicated (G-off). Nuclear extracts were prepared at the indicated
times and assayed by EMSA using the m67 and -cas probes. This is
representative of three independent experiments.
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| Fig 7.
Growth and differentiation of cells expressing mLALA and
mA mutants. (A) Cell-proliferation of 32D.cl8.6 clones expressing WT
( ; thick line), mDA (; thick dashed line), mLALA (; thin
line), and mA ( ; thin dashed line) receptors. Data represent
growth of individual mLALA and mA clones, whereas for WT and mDA
the mean growth of three clones is shown as a reference. (B) Maturation
of 32D.cl8.6 cells expressing wild-type or mutant G-CSF receptors, as
indicated in (A), expressed as the percentage of living cells showing
signs of maturation at each time point. Data represent the mean of
three clones. (C) Morphological features of 32D[WT] cells in the
presence of IL-3 or representative clones expressing wild-type or
mutant G-CSF-R after 7 days of exposure on G-CSF.
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The proliferation data for cells expressing the various receptor
mutants correlated well with their ability to inhibit short-term receptor internalization and deactivation. To further corroborate these
findings, we also examined steady-state G-CSF-R expression (Fig 8A) and STAT activation (Fig 8B) upon
continuous exposure to ligand. After 1 day of G-CSF treatment,
32D[mDA] cells showed an increase in G-CSF-R on the cell surface
compared with cells cultured in IL-3, whereas both 32D[WT] and
32D[mLALA] cells showed decreased receptor levels, albeit not as
severe for mLALA. Upon extended exposure to ligand, the STAT complexes
observed changed somewhat in all clones, most notable being the
appearance of a STAT3/STAT5 heteromeric complex, similar to what we
have previously described in NFS-60 cells.13 However,
importantly, total STAT activation remained high in 32D[mDA] cells,
whereas it was considerably less for both 32D[WT] and 32D[mLALA]
cells, although slightly higher for mLALA than for the WT (Fig 8B).
Once again, activation of STAT5 was most affected by the receptor
truncation. 32D[mA] cells gave almost identical results to 32D[WT]
cells (data not shown). Thus, the proliferation properties of the
different clones parallel the steady-state receptor levels and STAT
activation, consolidating the correlation between receptor activation
status and proliferation.
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| Fig 8.
Steady-state receptor levels and STAT activation in the
presence of ligand. (A) Flow cytometric analysis of G-CSF-R levels on
32D.cl8.6 cells expressing wild-type or mutant G-CSF-R either growing
on IL-3 (line indicates median FACS signal) or switched to G-CSF for 1 day (dots). Percentages indicate the proportion of G-CSF-stimulated
cells with G-CSF-R expression either above or below the median level on
IL-3. Similar results were obtained with three independent clones. (B)
STAT activation in 32D.cl8.6 cells expressing wild-type or mutant
G-CSF-R, either growing on IL-3, washed, or switched to G-CSF for the
times indicated, using m67 and -cas probes. This is a representative
of three independent clones.
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| |
DISCUSSION |
Expression of truncated G-CSF receptors leads to hyperproliferation in
response to G-CSF in both mice and myeloid cell lines, with truncated
receptors acting dominantly over wild-type receptors.25,27 Furthermore, SCN patients with mutation of a single GCSFR
allele show clonal expansion of the mutant population and are
predisposed to AML,26 suggesting an equivalent effect in
these patients. We set out to identify a molecular mechanism(s) that
might explain this dominant hyperproliferative function of truncated
receptors. We showed that, compared with wild-type receptors, truncated
receptors showed prolonged activation, due to a much slower off-rate,
which correlated with defective internalization of these receptors. Cytochalasin D could inhibit both internalization and deactivation of
wild-type receptors, supporting the notion that the two processes are
linked mechanistically. These data suggest that a motif(s) in the
C-terminus, absent in truncated receptors, is required for receptor
internalization and concomitant deactivation. We have recently
confirmed these results using bone marrow cells from mice with a
targeted truncation of the G-CSF-R (data not shown).
Both di-leucine and tyrosine-based internalization motifs have been
reported.50,51 Indeed, the C-terminal region of G-CSF-R contains a di-leucine motif in Box B3 that is involved in
ligand-mediated receptor endocytosis of the closely related gp130
receptor.28 In addition, the C-terminus contains three
tyrosines, each of which are candidates for the YXX motif (in which
X is any amino acid, and has a bulky hydrophobic side-chain)
implicated in the control of internalization for a number of other
receptors.51 Mutational analysis showed that these three
tyrosines are not required for receptor internalization or the
growth-inhibitory/differentiation function mediated via the C-terminus.
On the other hand, mutation of the di-leucine motif delayed receptor
internalization, resulting in prolonged STAT activation in the short
term. However, the effect of this mutation was relatively minor in the
long term, reflected in a modest enhancement of G-CSF-mediated
proliferation and a delay in maturation. These findings clearly
indicate that another as yet unknown motif(s) in the C-terminus also
plays a role in the control of these processes. Such sequences may
simply represent other motifs required for efficient internalization.
However, the regulation may be more complex than this. For example, in the experiments involving cytochalasin D (Fig 4), the 32D[WT/mDA] cells (Fig 5), or cells expressing the mLALA mutant (Fig 6), in which
the receptor C-terminus is present in some form, it was apparent that
the effects on internalization were more severe than those on STAT
deactivation, if compared with 32D[mDA] cells. Conversely, 32D[mA]
cells showed a slightly longer STAT activation than 32D[WT] cells
(Fig 6C). Therefore, it is likely that the C-terminus activates
alternate inhibitory mechanisms of receptor activation, which may
involve receptor tyrosines.
Although multiple mechanisms apparently de-activate the WT G-CSF-R, we
showed that truncated receptors acted in a dominant manner over
wild-type receptors with regard to both internalization and activation,
thereby paralleling their dominant hyperproliferative function and
suggesting a causal relationship. This was further supported with
studies of different receptor forms in the continuous presence of
ligand, in which a correlation between steady-state receptor levels,
STAT activation, and proliferation was observed. Thus, truncated
receptors may exert their dominant function by interfering with the
ligand-induced internalization of wild-type receptors, leading to
enhanced proliferative signaling, which suggests that the motifs in the
C-terminus required for internalization must be present in more than
one copy on a given ligated receptor complex to do so efficiently. One
explanation for this is that the molecules that mediate the
internalization process may need to be multimeric to function
effectively. Alternatively, we cannot rule out the possibility that the
presence of active complexes consisting solely of truncated receptors
in these cells is sufficient to produce the hyperproliferative effect.
Whatever the precise details, it is clear that truncated receptors,
either homo-oligomeric or complexed with wild-type receptors, are
capable of generating extended signals in response to G-CSF stimulation, which ultimately leads to hyperproliferation. It is
possible that the hyperproliferation may be due to the sustained activation of STAT proteins per se, because STAT proteins are known to
be constitutively activated in a number of leukemias,52-55 as well as cell lines transformed by viruses56 or
oncogenes.57 Indeed, it has been shown that introduction of
v-Src into 32D cells leads to both constitutive STAT activation and
sustained proliferation with a corresponding block in G-CSF-mediated
differentiation, similar to what is observed with mutant
mDA.58 In addition, it has recently been shown that STAT3
activation is required for cell transformation by Src,59,60
whereas a constitutively active STAT5 mutant has been identified that
functions to promote cell proliferation.61 In the case of
extended G-CSF-R signaling, the most important contribution for
mediating hyperproliferation would presumably be from STAT5, which is
known to play a role in G-CSF-mediated growth signaling36
and whose activation status is most affected by the receptor
truncation. However, because STAT3 has been implicated in
G-CSF-mediated differentiation,35 it may be that it is the
altered balance between STAT5 and STAT3 that is important.
Nevertheless, it is obvious that the sustained activation of
other G-CSF-R signaling pathways may also contribute to the
observed hyperproliferation.
This report provides a plausible explanation for the dominant
hyperproliferative function of truncated G-CSF-Rs in response to G-CSF
mediated by extended growth signals after ligand binding due to
defective receptor internalization. These data further implicate
G-CSF-R truncation as a possible preleukemic event, because cells
carrying a GCSFR mutation could be expected to have a growth
advantage due to prolonged receptor activation in the presence of
ligand. In this light, therefore, because SCN patients are routinely
treated with G-CSF, our data would suggest that further examination
should be afforded to the effects of chronic G-CSF administration on
the incidence of AML in SCN patients with a GCSFR mutation.
| |
ACKNOWLEDGMENT |
The authors thank Drs Mirjam Hermans, Marieke von Lindern, and Tania de
Koning-Ward for helpful discussions and critical reading of the
manuscript and thank Karola van Rooyen for exquisite graphical work.
| |
FOOTNOTES |
Submitted August 17, 1998;
accepted October 26, 1998.
Supported by an EMBO Long Term Fellowship (A.C.W.) and grants from the
N.W.O. and Dutch Cancer Society (I.P.T.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Alister C. Ward, PhD, Institute of
Hematology, Erasmus University (Room H Ee 1314), PO Box 1738, 3000 DR
Rotterdam, The Netherlands; e-mail: ward{at}hema.fgg.eur.nl.
| |
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Top
Abstract
Introduction