Multiple Signals Mediate Proliferation, Differentiation, and
Survival from the Granulocyte Colony-stimulating Factor Receptor in
Myeloid 32D Cells*
Alister C.
Ward
§,
Louise
Smith¶
,
John P.
de
Koning
,
Yvette
van Aesch
, and
Ivo P.
Touw
¶
From the
Institute of Hematology, Erasmus University,
3000 DR Rotterdam and ¶ Department of Hematology, Dr. Daniel
den Hoed Cancer Center, 3008AE Rotterdam, The
Netherlands.
 |
ABSTRACT |
Granulocyte colony-stimulating factor (G-CSF)
regulates neutrophil production through activation of its cognate
receptor, the G-CSF-R. Previous studies with deletion mutants have
shown that the membrane-proximal cytoplasmic domain of the receptor is
sufficient for mitogenic signaling, whereas the membrane-distal domain
is required for differentiation signaling. However, the function of the
four cytoplasmic tyrosines of the G-CSF-R in the control of
proliferation, differentiation, and survival has remained unclear. Here
we investigated the role of these tyrosines by expressing a tyrosine
"null" mutant and single tyrosine "add back" mutants in
maturation-competent myeloid 32D cells. Clones expressing the null
mutant showed only minimal proliferation and differentiation, with
survival also reduced at low G-CSF concentrations. Analysis of clones
expressing the add-back mutants revealed that multiple tyrosines
contribute to proliferation, differentiation, and survival signals from
the G-CSF-R. Analysis of signaling pathways downstream of these
tyrosines suggested a positive role for STAT3 activation in both
differentiation and survival signaling, whereas SHP-2, Grb2 and Shc
appear important for proliferation signaling. In addition, we show that
a tyrosine-independent "differentiation domain" in the
membrane-distal region of the G-CSF-R appears necessary but not
sufficient for mediating neutrophilic differentiation in these cells.
 |
INTRODUCTION |
The production of blood cells is regulated by a range of
extracellular stimuli, including a network of hematopoietic growth factors and cytokines. One of these, granulocyte colony-stimulating factor (G-CSF),1 is a major
regulator of neutrophilic granulocyte production and augments the
proliferation, survival, maturation, and functional activation of cells
of the granulocytic lineage (1-4). The actions of G-CSF are mediated
through its interaction with a specific cell surface receptor, the
G-CSF-R, which forms homo-oligomeric complexes upon ligand binding (5).
Typical of other members of the hematopoietin receptor superfamily, the
G-CSF-R has no intrinsic tyrosine kinase activity but activates
cytoplasmic tyrosine kinases (2, 5, 6). Important signaling molecules
utilized by the G-CSF-R include the Janus tyrosine kinases Jak1, Jak2, and Tyk2 (7-10), the Src kinases p55lyn and
p56/59hck (11-13), the signal transducer and
activator of transcription (STAT) proteins STAT1, STAT3, and STAT5 (9,
14-19), and components of the p21ras/Raf/mitogen-activated
protein kinase pathway (8, 20-24).
The cytoplasmic region of the G-CSF-R can be subdivided into a
membrane-proximal domain, which contains two conserved subdomains known
as box 1 and box 2, and a membrane-distal domain, which contains a
less-conserved box 3 sequence (5). In myeloid cells, the
membrane-proximal domain is essential for mitogenic signaling, whereas
the membrane-distal domain is essential for the transduction of
differentiation signals (19, 25-27). In addition, there are four
tyrosine (Tyr) residues in the cytoplasmic region of the G-CSF-R, at
positions 704, 729, 744, and 764 of the human receptor, three of which
lie in the membrane-distal domain (28). Ligation of the G-CSF-R results
in the rapid phosphorylation of these four tyrosines (7, 29), which
form potential binding sites for signaling molecules that contain Src
homology 2 (SH2) or phosphotyrosine binding domains (30, 31). Some
signaling pathways emanating from the different tyrosines of the
G-CSF-R have been identified. For example, we and others have shown
that Tyr-704 and Tyr-744 of the G-CSF-R are involved in the recruitment
and activation of STAT3 by the G-CSF-R (15, 17, 18, 32). In addition, Tyr-764 is necessary for the formation of Shc/Grb2/p140 complexes as
well as the activation of p21ras (22, 33). However, studies to
determine the role of each of the four cytoplasmic tyrosines in
mediating the effects of G-CSF on proliferation, differentiation, and
survival have yielded ambiguous and somewhat conflicting results (15,
17, 21, 22, 29, 33, 34).
To better define the role of receptor tyrosines in signaling from the
G-CSF-R, we examined the ability of a tyrosine null mutant and a series
of single tyrosine add back mutants to transduce biological signals in
response to G-CSF. Furthermore, we performed these studies in
maturation-competent myeloid 32D cells, which are able to closely
recapitulate many of the cellular responses to G-CSF, including
proliferation, survival, and, importantly, terminal differentiation
into mature neutrophils (27). Thus, 32D cells provide an appropriate
cellular context to assess the physiological relevance of signals from
the G-CSF-R. This analysis revealed that multiple tyrosines contribute
to proliferation, differentiation, and survival signaling from the
human G-CSF-R. Analysis of signaling pathways downstream from these
tyrosines suggest a positive role for STAT3 activation in both
differentiation and survival, whereas SHP-2, Grb2, and Shc appear
important for proliferation. In addition, we show that a
tyrosine-independent "differentiation domain" in the
membrane-distal region of the G-CSF-R appears necessary, although not
sufficient, for mediating neutrophilic differentiation in 32D cells.
 |
EXPERIMENTAL PROCEDURES |
Cell Culture and Transfection of G-CSF-R Constructs--
The
pLNCX expression constructs of human G-CSF-R wild-type (WT), a series
of triple Tyr
Phe add back mutants (mA, mB, mC, and mD), a
quadruple Tyr
Phe null mutant (mO), and a truncation mutant derived
from a patient with severe congenital neutropenia (mDA) have been
described previously (18, 27). A subline of the
IL-3-dependent murine myeloid cell line 32D.cl3, called
32D.cl8.6, which lacked endogenous G-CSF-R expression but remained
maturation-competent, was generated as described previously (27). This
was maintained in RPMI 1640 medium supplemented with 10% fetal calf
serum and 10 ng/ml murine IL-3. The expression constructs were
linearized by PvuI digestion and transfected into 32D.cl8.6
cells by electroporation. After 48 h of incubation, cells were
selected with G418 (Life Technologies, Inc.) at a concentration of 0.8 mg/ml. Multiple clones were expanded for further analysis. To check
G-CSF-R expression levels, cells were incubated at room temperature for
60 min with 10 µg/ml biotinylated mouse anti-human G-CSF-R monoclonal
antibody LMM741 (PharMingen, San Diego, CA), then at 4 °C for 60 min sequentially with 5 µg/ml phycoerythrin-conjugated
streptavidin, 5 µg/ml biotinylated anti-streptavidin antibody, and
finally 2 µg/ml phycoerythrin-conjugated streptavidin, with washing
between each antibody step. Samples were analyzed by flow cytometry
using a FACScan (Becton Dickinson, San Jose, CA). Several independently
derived cell lines of each construct were selected on the basis of
equivalent receptor expression.
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 1-2 × 105 cells/ml in RPMI medium supplemented with 10% fetal
calf serum and either 100 ng/ml human G-CSF, 10 ng/ml of murine IL-3,
or without growth factors. The medium was replenished every 1-2 days, and the cell densities were adjusted to 1-2 × 105
cells/ml. Viable cells were counted on the basis of trypan blue exclusion. To analyze the morphological features, cells were spun onto
glass slides and examined by May-Grünwald-Giemsa staining. To
quantify the neutrophilic maturation of 32D.cl8.6 transfectants in
response to G-CSF, the number of terminally differentiated cells was
determined and expressed as a percentage of total living cells (% neutrophils).
Preparation of Cell Lysates and Western Blotting--
Cells were
deprived of serum and factors for 4 h at 37 °C in RPMI 1640 medium at a density of 1 × 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 volumes of ice-cold
phosphate-buffered saline supplemented with 10 µM
Na3VO4 were added. Subsequently, cells were
pelleted and lysed by incubation for 30 min at 4 °C in Tyr(P) lysis
buffer (1% Triton X-100, 100 mM NaCl, 50 mM
Tris-HCl, pH 8.0, 0.1 mM Na3VO4, 1 mM dithiothreitol, 1 mM Pefabloc SC, 50 µg/ml
aprotinin, 50 µg/ml leupeptin, 50 µg/ml bacitracin) followed by
centrifugation at 13,000 × g for 15 min. The soluble
proteins were mixed with sample buffer, separated by SDS-polyacrylamide
gel electrophoresis (SDS-polyacrylamide gel electrophoresis), and
transferred onto nitrocellulose (0.2 µm; Schleicher & Schuell).
Filters were blocked by incubation in TBST (10 mM Tris-HCl,
pH 7.4, 150 mM NaCl, 0.05% (v/v) Tween 20) containing
0.6% (w/v) bovine serum albumin. Antibodies used for Western blotting
were anti-phospho-STAT3[Y705] (New England Biolabs, Inc. Beverly,
MA), anti-phospho-STAT3[S727] (New England Biolabs), and anti-STAT3
(Santa Cruz Biotechnology Inc., Santa Cruz, CA) and were diluted in
TBST containing 0.6% (w/v) bovine serum albumin. After washing with
TBST, immune complexes were detected with horseradish
peroxidase-conjugated species-specific antiserum (DAKO, Glostrup,
Denmark) followed by enhanced chemiluminescence reaction (DuPont). In
some instances, membranes were stripped in 62.5 mM
Tris-HCl, pH 6.7, 2% SDS, and 100 mM
-mercaptoethanol at 50 °C for 30 min, reblocked, washed, and reprobed.
Far Western Analysis--
The cytoplasmic domain of the human
G-CSF-R was cloned into pET-15b (Novagen, Madison, WI) as described
(18). In addition, the following glutathione S-transferase
fusion constructs were made utilizing standard polymerase chain
reaction protocols to amplify the appropriate coding regions, which
were cloned into pGEX-2T (Amersham Pharmacia Biotech): Grb2-FL
(SH3-SH2-SH3), Grb2-FL(mut) (SH3-SH2mutant-SH3), Shc-SH2, SHP-1-SH2(N),
SHP-1-SH2(C), SHP-2-SH2(N), SHP-2-SH2(C), Syk-SH2(N), and Syk-SH2(C).
The authenticity of all constructs was verified by DNA sequencing. In
addition, constructs were obtained for the production of glutathione
S-transferase fusions with GAP-SH2(N) and GAP-SH2(C) from
Tony Pawson, Vav-SH2 and Fps-SH2 from Lewis Cantley, CrkL-SH2 and
Abl-SH2 from Wallace Langdon, and Grb14-SH2 from Roger Daly. For the
production of tyrosine-phosphorylated G-CSF-R cytoplasmic domain, the
pET clone was introduced into the E. coli strain TKB1
(Stratagene, La Jolla, CA), which contains an inducible tyrosine
kinase. Fusion protein was produced and purified according to the
manufacturer's instructions, then 32P-labeled using heart
muscle kinase. For the production of glutathione S-transferase fusions, plasmids were transformed into XL-1
Blue (Stratagene), with proteins expressed and purified on
glutathione-Sepharose 4B beads as described (35). These proteins were
then electrophoresed on 10% SDS-polyacrylamide gel electrophoresis
gels (36) and electrophoretically transferred to Hybond-C membranes
(Amersham Pharmacia Biotech). The membranes were processed through a
denaturation-renaturation cycle (37) and probed with the
32P-labeled G-CSF-R, as described (38).
 |
RESULTS |
Expression of G-CSF-R Mutants in Maturation-competent 32D
Cells--
The contribution of receptor tyrosines to G-CSF-R function
has remained unclear (15, 17, 21, 22, 29, 33, 34). Therefore, to better
understand the role of these tyrosines, we analyzed a quadruple Tyr
Phe or null mutant with no cytoplasmic tyrosines (mO) and a series of
triple Tyr
Phe or add back mutants, which each retain a single
cytoplasmic tyrosine (mA, mB, mC, and mD). Expression vectors encoding
these mutant receptors along with WT and truncated (mDA) G-CSF-Rs (Fig.
1A) were introduced into a
subline of the IL-3-dependent murine myeloid cell line 32D.cl3 called 32D.cl8.6 that does not express endogenous G-CSF-R. Surface expression of the G-CSF-R was determined using
fluorescence-activated cell sorter analysis, and cell lines expressing
equivalent levels of receptor were selected for further analysis, with
at least three independent clones studied for each construct. Examples of clones expressing WT or mutant G-CSF-R proteins are shown in Fig.
1B.

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Fig. 1.
Expression of G-CSF-R mutants in myeloid 32D
cells. A, schematic diagram of the cytoplasmic domains
of mutant G-CSF-R proteins. Boxes 1,
2, and 3 denote subdomains conserved in several
members of the hematopoietin receptor superfamily (5). B,
flow cytometric analysis of G-CSF-R expression on parental 32D.cl8.6
cells (par) or transfectants. Cells were either stained with
biotinylated mouse anti-human G-CSF-R antibodies followed by
phycoerythrin-conjugated streptavidin, biotinylated anti-streptavidin,
and finally phycoerythrin-conjugated streptavidin
(unfilled) or without the anti-G-CSF-R step
(filled) and analyzed for fluorescence
(FL2-Height).
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Receptor Tyrosines Are Required for Proliferation and
Differentiation but Are Dispensable for Survival at High Ligand
Concentration--
To ascertain whether receptor tyrosines were
required at all to mediate proliferation, survival, and differentiation
responses in response to G-CSF in 32D cells, we first compared the WT
and tyrosine null mutant (mO) G-CSF-Rs. To this end, 32D[WT] and
32D[mO] transfectants were switched from IL-3- to G-CSF-containing
medium following extensive washing to remove any traces of IL-3. In the absence of IL-3 or G-CSF, all transfectants died within 1 to 2 days
without showing any signs of differentiation. However, in response to
100 ng/ml G-CSF, 32D[WT] cells showed transient proliferation for 5 to 7 days (Fig. 2). After 6 to 10 days,
32D[WT] cells developed into terminally differentiated neutrophils,
as shown by an increased cytoplasm-to-nucleus ratio, a neutrophilic
granule-containing cytoplasm, and lobulated nuclei (Fig.
3, A and B). In
contrast, 32D[mO] cells showed only minimal proliferation and
differentiation in response to G-CSF, although cell viability was
largely maintained at this saturating ligand concentration (Figs. 2 and
3). This result establishes that receptor tyrosines are required to
facilitate proliferation and differentiation responses from the
full-length G-CSF-R.

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Fig. 2.
Proliferation of 32D clones in response to
G-CSF. Cell-proliferation of 32D.cl8.6 clones expressing WT or
mutant G-CSF-Rs, as indicated, in response to 100 ng/ml G-CSF. Data
represent the mean growth of three independent clones for each receptor
construct. , WT; , mA; , mB; ; mC; , mD; , mO; ,
mDA.
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Fig. 3.
Neutrophilic differentiation of 32D.cl8.6
transfectants in response to G-CSF. A, 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 to G-CSF. B, maturation of 32D.cl8.6 cells
expressing wild-type or mutant G-CSF-Rs, as indicated, expressed as
the percentage of living cells showing terminal differentiation (% neutrophils) at each time point. Data represent the mean of three
independent clones of each. , WT; , mA; , mB; ; mC; ,
mD; , mO; , mDA.
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Multiple Tyrosines Contribute to Proliferation and Differentiation
Signaling--
The almost complete abrogation of proliferation and
differentiation signaling from the tyrosine null mutant receptor
provided the opportunity to determine the contribution of individual
tyrosines to these pathways through the analysis of clones expressing
the tyrosine add back mutants (Figs. 2 and 3). This revealed that Tyr-704 alone (mA) could contribute to both G-CSF-mediated
proliferation and differentiation signaling. This mutant elicited
initial proliferation responses equivalent to those from the wild-type
receptor, although the proliferative phase terminated slightly earlier.
Concurrently, mA produced an earlier and more complete differentiation
compared with the WT receptor. In contrast, Tyr-729 (mB) gave only a
very weak signal for differentiation, with no significant increase in
proliferation compared with the null mutant. The Tyr-744-containing mutant (mC) produced a phenotype similar to the Tyr-704-containing mA,
although with slightly less proliferation and differentiation. Finally,
analysis of 32D[mD] cells revealed that Tyr-764 projects strong
proliferative signals, such that these cells continued to proliferate
indefinitely on G-CSF, with no significant differentiation observed.
The Membrane-distal Region of the Human G-CSF-R Contains a
Tyrosine-independent Differentiation Domain Essential for Neutrophilic
Differentiation in 32D Cells--
We had previously identified the
membrane-distal domain of the G-CSF-R as essential for the transduction
of differentiation signals based on the observation that a truncation
mutant isolated from a patient with severe congenital neutropenia, mDA,
which lacks this domain, was unable to elicit differentiation in
myeloid cells (19, 25-27). The construction of mutant mA, which lacks all three tyrosines in this domain, allowed an assessment of whether tyrosines were required for these effects. Comparison of the 32D[mA] and 32D[mDA] clones revealed that the membrane-distal domain lacking all tyrosines still augments differentiation (Figs. 2 and 3).
Signals for Survival at Low Ligand Concentration Parallel Those for
Differentiation--
We have shown above that 32D[mO] cells are
largely able to survive at 100 ng/ml G-CSF, without undergoing
significant proliferation. In contrast, survival at lower G-CSF
concentrations was impaired relative to 32D[WT] cells, such that
32D[WT] cells can survive on 0.1 ng/ml G-CSF, whereas 32D[mO] cells
cannot (Fig. 4). We could then determine
the contribution of individual tyrosines to this low dose
G-CSF-mediated survival signaling by exposing clones to different
concentrations of ligand. Results with the add-back mutants showed a
strong role for Tyr-704 and Tyr-744 and a weak role for Tyr-729 in
mediating survival, with Tyr-764 apparently having no role. This
clearly parallels the effects of these tyrosines in mediating
differentiation signals.

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Fig. 4.
Survival signaling from G-CSF-R tyrosine
mutants. Cell proliferation/survival analysis of 32D clones
expressing the indicated G-CSF-Rs performed as described in Fig. 2,
except at 100 ( ), 10 ( ), 1 ( ), and 0.1 ( ) ng/ml
G-CSF.
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STAT3 Activation Correlates with Both Survival at Low Ligand
Concentration and Differentiation--
The concordant pattern of
survival at low doses of G-CSF and differentiation resembled that for
STAT3 activation from the G-CSF-R, which we have recently reported in
Ba/F3 cells (18). In the various 32D cells clones, G-CSF-mediated STAT3
activation, as measured by both tyrosine and serine phosphorylation,
was greatest with mA (Tyr-704) and mC (Tyr-744), with a lesser
contribution from mB (Tyr-729) (Fig. 5).
In contrast, mO was only weakly able to activate STAT3, whereas mD
(Tyr-764) showed no more activation than mO. Thus, the ability of
mutants to activate STAT3 showed a striking correlation with the
strength of their respective differentiation and survival signals,
suggesting a positive role for STAT3 in the control of both
processes.

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Fig. 5.
STAT3 activation by G-CSF-R tyrosine
mutants. Western blot analysis of total lysates from 32D cells
expressing wild-type or mutant G-CSF-Rs, either starved ( ) or
stimulated with G-CSF for 10 min (+), probed with the antibodies
indicated. pY, Tyr(P); pS, Ser(P);
C-term, C terminus.
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Identification of Pathways Important for Proliferative Responses
from the G-CSF-R--
Finally, we also sought to identify which
signaling molecules might play a role in the tyrosine-mediated
proliferation signaling from the G-CSF-R, i.e. via Tyr-704,
Tyr-744, and Tyr-764. To achieve this we employed a novel strategy to
identify SH2 domains that could interact directly with the
phosphorylated tyrosines of the G-CSF-R. As an initial screen, we used
a 32P-labeled, tyrosine-phosphorylated WT G-CSF-R
cytoplasmic domain simulating an activated receptor to probe a range of
SH2 domains immobilized on nitrocellulose (Fig.
6A). This showed that the isolated SH2 domains of SHP-2 and Shc could bind to the receptor. In
addition, full-length Grb2 could bind to the receptor, whereas a
specific SH2 domain mutant of Grb2 could not, implying that the Grb2
SH2 domain also interacts directly with the G-CSF-R. We were next able
to map the specific tyrosine binding site(s) of these SH2 domains by
probing them with tyrosine-phosphorylated receptor proteins
generated from the single tyrosine add-back mutants (Fig.
6B). This revealed direct binding of SHP-2, preferentially via its N-terminal SH2 domain, to both Tyr-704 and Tyr-764, and Grb2
and Shc via their respective SH2 domains to Tyr-764. Because these
tyrosines also elicit strong proliferation signals, we can posit a
direct role for SHP-2, Grb2, and Shc in mediating these responses. No
interactions of these molecules with Tyr-744 were identified.

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Fig. 6.
Far Western analysis of SH2-domains
with recombinant tyrosine-phosphorylated G-CSF-R. A,
glutathione S-transferase (GST) fusions of
various SH2 domain-containing signaling molecules were separated on
replicate SDS-polyacrylamide gel electrophoresis gels and either
stained with Coomassie or transferred to nitrocellulose for Far Western
analysis with 32P-labeled, tyrosine-phosphorylated
(Tyr(P)) WT G-CSF-R cytoplasmic domain as a probe.
B, far Western as described in A, except using as
probes 32P-labeled, tyrosine-phosphorylated cytoplasmic
domains from WT or mutant G-CSF-Rs, as indicated.
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DISCUSSION |
Previous studies to determine the role(s) of the cytoplasmic
tyrosines of the G-CSF-R in eliciting cellular responses have been
performed with deletion and single tyrosine substitution mutants. These
studies have yielded ambiguous results. This is likely due, at least in
part, to the use of inappropriate cell models, which are unable to
recapitulate the complete gamut of G-CSF responses (15, 17, 21, 22, 29,
34). Furthermore, use of truncation mutants to make inferences about
tyrosine-specific effects (17, 21, 34) is also problematic because of
altered receptor trafficking of such mutants (19, 39). Analysis of single Tyr
Phe substitution mutants has provided some insight into
the function of the receptor tyrosines, although even here the results
are somewhat conflicting, implicating Tyr-44 and, to a lesser extent,
Tyr-704 and Tyr-729 in the macrophage differentiation of M1 cells (15),
Tyr-703 or Tyr-728 of the murine G-CSF-R (equivalent to Tyr-704 and
Tyr-729 of the human receptor) in the neutrophilic differentiation of
L-GM-1 cells (29), and Tyr-764 in the proliferative responses of 32D
cells (33). However, a major problem with these studies is that they
may fail to reveal redundant pathways emanating from these tyrosines.
Analysis of tyrosine null and add-back mutants has provided a useful
approach to investigate the complex roles of individual cytoplasmic
tyrosine residues in cytokine receptor signaling (40, 41). Therefore,
we used this approach to delineate the function of tyrosine-mediated
pathways from the G-CSF-R. In addition, we chose a cell system, myeloid
32D cells, which most closely mimics in vivo
differentiation, because these cells are able to differentiate from
blast-like cells into mature neutrophils. The results of this analysis
revealed multiple signals emanating from the receptor tyrosines to
control proliferation, differentiation, and survival (Fig.
7). In addition, these studies show that
signals independent of receptor tyrosines also contribute to
differentiation and survival.

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Fig. 7.
Model for signaling from the G-CSF-R leading
to specific cellular responses. Potential
tyrosine-dependent pathways leading to proliferation,
differentiation, and survival are indicated. In addition, it is obvious
that tyrosine-independent pathways also contribute to these processes,
although the mechanism(s) remain unclear.
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We found that signals from the G-CSF-R for both survival at low G-CSF
concentration and differentiation largely overlapped, being mediated
most strongly by Tyr-704 and Tyr-744, with a lesser contribution from
Tyr-729. This showed a close correlation with the ability of these
tyrosines to activate STAT3 in these cells. The involvement of STAT3 in
G-CSF-mediated granulocytic differentiation is consistent with a recent
study showing that expression of dominant-negative STAT3 could totally
block this process (42). However, our data also suggest that STAT3
activation may represent the key pathway in maturation signaling from
the full-length G-CSF-R. Furthermore, we provide evidence suggestive of
a role for STAT3 in G-CSF-dependent survival signaling, as
has also been postulated for the related gp130 receptor component (43).
However, it is clear that, at high G-CSF concentrations,
tyrosine-independent survival mechanisms also operate, potentially
involving phosphatidylinositol 3-kinase (34) or mitogen-activated
protein kinase (21). Finally, there appears to be subtle differences in
G-CSF-mediated STAT3 activation in 32D cells compared with that we have
reported in Ba/F3 cells (18), such that the contribution of
tyrosine-independent STAT3 activation is less important, whereas the
Tyr-729-mediated route represents a novel pathway of STAT3 activation
in 32D cells. The reason for these cell-specific differences remains unclear.
Proliferation signaling from the G-CSF-R was predominantly mediated via
Tyr-704 and Tyr-764, with a minor contribution from Tyr-744. We have
previously reported formation of Grb2/SHP-2, Grb2/p90, and
Grb2/Shc/p140 complexes in response to G-CSF, the latter two being
dependent on Tyr-764 (22). Using in vitro binding studies,
we show that Tyr-704 is a direct docking site for SHP-2, whereas
Tyr-764 is a direct docking site for SHP-2, Grb2, and Shc, largely
consistent with the published consensus binding sites for these
molecules (44, 45). In addition, we and others have shown that Grb2
interacts with SHP-2 and Shc via binding of its SH2 domain with
phosphorylated tyrosines in the SHP-2 C terminus (24, 46) and at
position 317 of Shc (31), respectively. Thus, there appears to be at
least four mechanisms by which Grb2 can interact with the activated
G-CSF-R: directly via Tyr-764 (complexed with p90?) or indirectly via
SHP-2 at Tyr-704 and Tyr-764 or Shc at Tyr-764. The relative role of
these alternate complexes in mediating G-CSF responses remains an
important consideration for future investigations. However, because
SHP-2, Grb2, and Shc have all been implicated in proliferation (31,
47-50), it seems likely that these are the molecules responsible for
the proliferative signals emanating from Tyr-704 and Tyr-764. The
molecule(s) docking to Tyr-744, which mediate its weak proliferation
signal remain to be elucidated.
We have previously identified the membrane-distal domain of the G-CSF-R
as being essential for the transduction of differentiation signals (19,
25-27). Analysis of the 32D[mA] clones in this study has revealed
that the membrane-distal domain is required for differentiation independent of receptor tyrosines (Figs. 2 and 3). However, because the
32D[mO] clones differentiated poorly, we can presume that this domain
alone is insufficient to induce complete maturation but rather
co-operates with additional tyrosine-dependent signals (such as from Tyr-704 or Tyr-744). We have recently shown that this
region is important for receptor internalization and concomitant deactivation (19). Therefore, it is likely that a major function of
this tyrosine-independent differentiation domain is to negatively regulate proliferation. Consistent with this, mD, which contains this
domain, elicits sustained proliferation but at a reduced rate compared
with mDA, which does not. In addition, the minor activation of STAT3 by
this domain could also contribute to its differentiation-inducing function.
The data presented here has unequivocally assigned specific roles to
the tyrosines of the G-CSF-R in mediating differentiation, proliferation, and survival in a myeloid system capable of full neutrophilic differentiation. We have shown that the four tyrosines of
the G-CSF-R possess distinct, yet overlapping, functions. This is a
similar conclusion to that obtained from recent studies with other
cytokine receptors, such as the erythropoietin receptor (41) and the
common
-chain of the granulocyte-macrophage colony-stimulating factor receptor (40). Having now identified the key intracellular mediators downstream of the receptor tyrosines, future studies will aim
to identify the targets of these molecules in eliciting the various
G-CSF-mediated biological responses.
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ACKNOWLEDGEMENTS |
We thank Tony Pawson (Mount Sinai Hospital,
Toronto, Canada), Lewis Cantley (Harvard Medical School, Boston),
Wallace Langdon (Queen Elizabeth II Medical Center, Nedlands,
Australia), and Roger Daly (Garvan Institute for Medical Research,
Sydney, Australia) for plasmids and Karola van Rooijen for exquisite
graphical work.
 |
FOOTNOTES |
*
This work was supported by an EMBO long term fellowship (to
A. C. W.) and grants from the Dutch Cancer Society and the
Netherlands Organization for Scientific Research (N.W.O.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Institute of
Hematology, Erasmus University (Room H Ee 1314), P. O. Box 1738, 3000 DR Rotterdam, The Netherlands. Tel.: 31-10-4087768; Fax: 31-10-4089470;
E-mail: ward{at}hema.fgg.eur.nl.
Current address: Centre for Immunology and Cancer Research,
University of Queensland, Dept. of Medicine, Princess Alexandra Hospital, Woolloongabba 4102 Qld., Australia.
 |
ABBREVIATIONS |
The abbreviations used are:
G-CSF-R, granulocyte
colony-stimulating factor receptor;
STAT, signal transducer and
activator of transcription;
SH2, Src homology 2;
WT, wild type;
IL-3, interleukin 3;
TBST, Tris-buffered saline-Tween;
GAP, GTPase-activating
protein.
 |
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