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HEMATOPOIESIS
Distinct domains of the human granulocyte-macrophage colony-stimulating factor
receptor ␣ subunit mediate activation of Jak/Stat signaling and differentiation
Michael B. Lilly, Marina Zemskova, Arthur E. Frankel, Jonathan Salo, and Andrew S. Kraft
The ␣ subunit of the human granulocytemacrophage colony-stimulating factor
(GM-CSF) receptor has several isoforms
that result from alternative splicing
events. Two forms, ␣-1 and ␣-2, have
intracytoplasmic sequences that are identical within a membrane-proximal domain
but differ completely distally. Variant and
mutated GM-CSF receptor ␣ subunits,
along with the ␤ subunit (␤c protein) were
expressed in M1 murine leukemia cells.
and the ability of the receptors to signal
for differentiation events and to activate
Jak/Stat signaling pathways was examined. All cell lines expressing both ␣ and
␤c proteins exhibited high-affinity bind-
ing of radiolabeled human GM-CSF. Receptor ␣ subunits with intact membraneproximal intracellular domains could
induce expression of the macrophage
antigen F4/80 and down-regulate the expression of CD11b. Addition of recombinant human GM-CSF to cells expressing
␣-1 subunits induced the expression of
CD86 and tyrosine phosphorylation of
Jak-2 and its putative substrates SHPTP-2,
Stat-5, and the GM-CSF receptor ␤c subunit. Cells containing ␣ subunits that
lacked a distal domain (term-3) or had the
alternatively spliced ␣-2 distal domain
showed markedly decreased ability to
support tyrosine phosphorylation of Jak-2
and its substrates or to up-regulate CD86.
Ligand binding induced stable association of the ␣-1 subunit and ␤c protein. In
contrast, the ␣-2 subunit did not stably
associate with the ␤c subunit. These data
identify potential molecular mechanisms
for differential signaling of the ␣-1 and
␣-2 proteins. The association of unique
signaling events with the 2 active GMCSF ␣ subunit isoforms offers a model
for variable response phenotypes to the
same ligand. (Blood. 2001;97:1662-1670)
© 2001 by The American Society of Hematology
Introduction
The mechanisms by which hematopoietic cytokines effect multiple
biologic responses have been the subjects of many studies.1
Granulocyte-macrophage colony-stimulating factor (GM-CSF)
stimulates both the growth and the differentiation of hematopoietic
cells. The GM-CSF receptor consists of an ␣ and a ␤ subunit. The ␣
subunit binds GM-CSF, whereas the ␤ subunit complexes with the
␣ protein and reconstitutes the high-affinity receptor. Eight alternatively spliced variants of the GM-CSF ␣ subunit have been
detected. The ␣-1 subunit (400 amino acids) is largely external and
has a short, 54–amino acid internal tail.2 In contrast, the ␣-2 subunit
is 410 amino acids long.3 In the ␣-2 subunit, the C-terminal 25
amino acids of the ␣-1 sequence are replaced by a 35–amino acid
stretch that is rich in serine and proline residues. Six additional
splicing variants of the ␣ subunit lack the transmembrane and
intracellular domains, and some of these are likely secreted.4-7
Receptors for IL-3, IL-5, and GM-CSF share a common ␤
subunit (␤c) but each receptor has a unique ␣ subunit. These
cytokines generally induce similar signal transduction events and
response phenotypes when tested in cell cultures. Nevertheless,
certain differences have been described. In factor-dependent human leukemia cells, IL-3 and GM-CSF induce proliferation by
protein kinase C–dependent mechanisms, whereas IL-5–induced
proliferation is not inhibited by protein kinase C antagonists.8 In a
subline of HL60 cells that expresses receptors for both GM-CSF
and IL-5, the former cytokine was able to stimulate leukotriene
synthesis, but the latter was ineffective.9 In some cell lines
GM-CSF is able to prevent cell death induced by the phosphatidylinositol-3–kinase inhibitor wortmannin, but IL-3 is ineffective.10
Other cell lines can be induced to differentiate by GM-CSF,
whereas IL-3 supports blast-like proliferation.11 Because all 3
cytokine receptors share a common ␤c receptor component,
differences in their response phenotypes likely result from signaling events regulated by the ␣ subunits. Furthermore, because the
membrane-proximal regions of the ␣ subunits are highly homologous, differences in response phenotypes could be particularly
dependent on signaling events regulated by the variable carboxyterminal domains of the various ␣ subunits.
Specific regions of the GM-CSF receptor ␤c subunit have been
shown to be important in the binding of signal transduction
intermediates12,13 and the regulation of response phenotypes.14,15 In
contrast, the ␣ subunit is less well studied. The ␣ subunit protein
does not appear to bind Jak-2 directly.13 However, it does regulate
GM-CSF–induced responses in hematopoietic cells. Deletion of
the entire ␣ intracytoplasmic domain blocked GM-CSF–induced
cell growth and differentiation.16 These effects appear to require a
proline-rich membrane-proximal domain that can also be found in
the ␣ subunits of the IL-3 and IL-5 receptors.
Few data exist examining the differential signaling events
From the Departments of Medicine and Surgery and the Center for Molecular
Biology and Gene Therapy, Loma Linda University, Loma Linda, CA; the
Department of Medicine and the Comprehensive Cancer Center, Wake Forest
University, Winston-Salem, NC; and the Department of Medicine, University of
Colorado Health Science Center, Denver, CO.
Submitted March 27, 2000; accepted November 20, 2000.
Supported in part by National Institutes of Health grants CA45672 (M.B.L.) and
DK44741 (A.S.K.).
Reprints: Michael Lilly, Department of Medicine, Loma Linda University, Loma
Linda, CA 92354; e-mail: [email protected].
1662
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2001 by The American Society of Hematology
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
triggered by the GM-CSF receptor ␣-1 and ␣-2 subunits. Both
receptor isoforms appear to be expressed in the primary and
immortalized hematopoietic cell lines examined to date.17 Both ␣
subunits can support the propagation of a proliferation signal in
factor-dependent murine cells.17 Thus, it is unclear whether there
are unique biologic or biochemical events mediated by the
alternative isoforms of the GM-CSF receptor.
To further evaluate the function of the ␣-1 and ␣-2 subunits and
the importance of specific segments of the ␣ subunits in signaling,
we have produced murine leukemia cells containing the human
GM-CSF receptor common ␤c subunit in combination with variant
or mutant ␣ subunits. Our results suggest the existence of at least 2
signaling domains of the ␣ subunit that variably regulate Jak/Stat
signaling and the expression of the differentiation antigens F4/80,
CD11b, and CD86.
Materials and methods
Cell lines and culture methods
The murine leukemia cell line M1 was used for all experiments. Cells were
maintained in Dulbecco modified Eagle medium (DMEM) with 10% fetal
calf serum. For studies of clonogenic growth, the cells were plated at 200
cells/35-mm dish, in DMEM 10% fetal calf serum, and 0.3% agar. Seven
days later colonies were counted with an inverted-stage microscope and
scored for number and morphology.
Plasmids and gene transduction
cDNAs encoding the ␣-1 subunit and the common ␤c subunit of the human
GM-CSF receptor have previously been described,2,14 as have the truncated
mutants term-1 and term-3.18 The cDNA for the human ␣-2 subunit was
kindly provided by Dr Colin Sieff.3 A chimeric ␣-1 subunit protein
(hereafter GM5), in which the C-terminal amino acids have been replaced
by the corresponding amino acids of the human IL-5 receptor ␣ subunit
protein, was constructed by a polymerase chain reaction–based technique,
using an IL-5 receptor ␣ chain cDNA provided by Dr J. Tavernier. All ␣
subunit cDNAs were ligated into the retroviral transduction plasmid
pLXSN19 and sequenced fully to ensure fidelity of the sequence. The ␤c
subunit cDNA was in the mammalian expression plasmid pEF/BOS.20
The human GM-CSF receptor ␤c cDNA was introduced into M1 cells
by electroporation, and a single clone (M1/␤c #7) was used for all
subsequent experiments requiring coexpression of ␣ and ␤c subunits.
␣ subunits were then expressed in M1/␤c cells by retroviral transduction. Plasmids encoding ␣ subunit variants were packaged in the amphotropic packaging cell line PA317, as described previously.21 Briefly,
recombinant retroviruses were produced by transiently transfecting HEK293
cells with plasmids encoding pol and ecotrophic env, along with pLXSN
plasmid containing retroviral backbone, gag sequences, and transgene of
choice. Supernatant from the transfected cells was used to infect PA317
cells, and pools of retrovirus-producing packaging cells were selected by
growth in G418. M1/␤c cells were cocultivated with packaging cells for 48
hours, and nonadherent cells were removed and selected in G418. At least 3
independently derived pools of retrovirally infected cells were produced for
each ␤c–␣ subunit combination. Parental M1 cells were also cocultivated
with packaging cells to produce pools of cells expressing only ␣-1 or
␣-2 subunits.
Reagents
Recombinant human GM-CSF (rhGM-CSF) was from Immunex (Seattle,
WA), and MG132 and tyrosine kinase inhibitors were from Alexis (San
Diego, CA). Sodium iodide I 125–labeled rhGM-CSF was obtained
from DuPont (Boston, MA).
GM-CSF RECEPTOR ␣ CHAINS
1663
Receptor characterization
Transduced M1 cells were characterized initially by flow cytometry, using
an anti-␤c monoclonal antibody (Chemicon, Temecula, CA) and an anti-␣
subunit monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Functional characterization was performed by radioligand-binding studies
using the program Ligand for Scatchard analysis.
Immunochemical studies
Signal transduction events associated with the various receptors were
characterized by combined immunoprecipitation–immunoblotting experiments. Cells were cultured with rhGM-CSF (100 ng/mL) for various
periods and then were washed once in ice-cold phosphate-buffered saline
with 1 mM sodium orthovanadate. Pelleted cells were lysed in detergent
buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 5 mM EDTA, 1% Triton X-100,
1 mM sodium orthovanadate, 50 mM NaF, plus protease inhibitors),
clarified by centrifugation, and used for immunochemical studies. Lysate
derived from approximately 20 million cells was used for each immunoprecipitation. The following antibodies were used: anti–Jak-2, anti–SHPTP-2,
anti-Stat-5 (Santa Cruz); anti-␤c (Chemicon [monoclonal], Santa Cruz
[polyclonal]), and antiphosphotyrosine (UBI, Lake Placid, NY). Detection
was with either protein G-peroxidase or anti-(species) IgG-peroxidase
conjugates, followed by enhanced chemiluminescence reagents (Amersham, Piscataway, NJ).
For coimmunoprecipitation studies, cell pellets (typically derived from
30 ⫻ 106 cells) were lysed in coimmunoprecipitation buffer (50 mM Tris,
pH 7.5, 150 mM NaCl, NP-40 1%, 1 mM EDTA, 1% glycerol, 1 mM
dithiothreitol, and protease inhibitors). The precipitates were washed twice
in high-salt buffer (coimmunoprecipitation buffer with 500 mM NaCl, 0.1%
NP-40) and twice in low-salt buffer (coimmunoprecipitation buffer with
0.1% NP-40, but no NaCl). Precipitates were then solubilized and analyzed
by immunoblotting with the appropriate antibodies.
Differentiation studies
Pools of M1 cells were treated with rhGM-CSF (100 ng/mL) in culture for 3
days. Differentiation of M1 cells in response to cytokine treatment was
assessed through flow cytometry and morphology. In each flow cytometry
experiment, fluorescence with specific antibodies was compared with that
from an isotype-matched control antibody. Specific fluorescence was
quantitated by comparing the 2 curves through Kolmogorov–Smirnoff
analysis. The parameter D/s(n) was used to represent the overall difference
between the curves and, hence, specific fluorescence. The antibodies:
anti-F4/80 and anti-CD11b (Biosource, Camarillo, CA) and anti-CD86
(Caltag, Burlingame, CA) were used for this analysis. Morphology was
assessed on Giemsa-stained cytocentrifuge preparations.
Results
Expression of human GM-CSF receptors in M1 leukemia cells
The human GM-CSF receptor ␤c cDNA was introduced into M1
cells by electroporation, along with a puromycin resistance plasmid. A single clone of puromycin-resistant cells, in which expression of the ␤c protein was detected by flow cytometry, was used for
all subsequent studies in which ␤c and ␣ subunits were coexpressed. These M1/␤c cells were cocultured with retroviral packaging lines that produced amphotropic viruses encoding 5 variant
human GM-CSF receptor ␣ subunits (Figure 1). The intracytoplasmic domain of the ␣-1 subunit of the receptor is 54 amino acids
long. In comparison, in the ␣-2 splicing variant, the C-terminal 25
amino acids of the ␣-1 cytoplasmic domain are replaced by 35
amino acids rich in serine and proline. Three additional ␣ subunit
mutants were created by polymerase chain reaction–based mutagenesis. In the term-3 ␣ subunit, a stop codon was inserted after 34
intracytoplasmic amino acids, and in the term-1 ␣ subunit, a stop
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1664
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
LILLY et al
Figure 1. Sequence of variant and mutant GM-CSF receptor ␣ subunits. The ␣-1
and ␣-2 isoforms are natural splicing variants of the ␣ subunit. They differ completely
from their C-terminal amino acids (italics). The GM5 chimeric ␣ subunit consists of
sequences from the proximal proline-rich domain of the GM-CSF receptor ␣ subunit
(bold) fused to distal sequences from the human IL-5 ␣ subunit (underlined). The
term-3 mutant ␣ subunit lacks the C-terminal amino acids because of placement of a
stop codon after the codons for 34 intracellular amino acids. The term-1 mutant ␣
subunit has only 5 intracellular amino acids.
codon was placed after only 5 amino acids of the intracytoplasmic
portion of the receptor. Finally, a GM5 ␣ subunit was constructed
containing the first 18 amino acids of the GM-CSF receptor ␣
subunit intracytoplasmic domain (including the critical proline
residues) and 36 amino acids derived from the carboxy terminal
amino acids of the IL-5 ␣ subunit.22 Additional pools of M1 cells,
expressing only the ␣-1 or ␣-2 subunits, were also prepared.
Three pools of G418-resistant cells were isolated and
characterized for each ␣ subunit variant. Expression of the ␣
subunit proteins, as detected by flow cytometry, was variable
(data not shown). All pools of transduced cells contained
specific binding sites for 125I–rhGM-CSF (Table 1; Figure 2).
Scatchard analysis demonstrated both high-affinity and lowaffinity binding sites for cells coexpressing ␣ and ␤c subunits. The
high-affinity binding of each of these receptor complexes was
similar to that reported for natural GM-CSF receptors (kd ⫽ 10-66
pM). In contrast, M1/␣-1 and M1/␣-2 cells did not express
high-affinity binding sites but showed a single class of low-affinity
binding sites (kd ⫽ 1.2-2.2 nM).
GM-CSF regulates expression of differentiation antigens in M1
leukemia cells
The murine leukemia cell line M1 has commonly been used to
identify signaling events associated with macrophage-like differentiation. A wide variety of cytokines can induce cell adhesion,
macrophage-like morphology, and terminal differentiation with
clonal extinction in this line. Treatment of M1/␤c/␣-1 cells and
M1/␤c/GM5 cells with rhGM-CSF produced an increase in size, a
decrease in nuclear/cytoplasmic ratio, and a moderate degree of
vacuolization. However there was no decrease or increase in
growth rate in suspension culture. Furthermore, no decrease in
plating efficiency or clonogenic growth in semisolid medium,
Table 1. Binding affinity of human GM-CSF receptors in M1 cells expressing
human ␤c and various ␣ subunit constructs
High affinity
Cell line
kd (pM)*
Low affinity
Sites†
kd (nM)
Sites
M1/␣-1
0
0
2.2
22 415
M1/␣-2
0
0
1.2
17 200
M1/␤c /␣-1
10
3130
1.1
12 320
M1 /␤c /␣-2
50
3160
3.0
14 300
M1 /␤c /GM5
10
4190
0.9
20 105
M1 /␤c /term-3
66
2560
8.8
24 600
M1/␤c /term-1
56
3520
7.2
32 800
*Values are the means of duplicate determinations from a pool of 3 independently
derived pools of retrovirally transduced cells.
†Sites per cell.
Figure 2. Scatchard analysis of 125I-labeled rhGM-CSF binding to M1 cells
expressing ␣ and ␤c subunits of GM-CSF receptor. Cells were incubated with
decreasing concentrations of labeled rhGM-CSF in the presence or absence of
100-fold excess of unlabeled rhGM-CSF, on ice for 3 hours. Bound and free
rhGM-CSF fractions were separated by centrifugation through fetal calf serum. The
bound and free fractions were quantitated in a gamma-counter. Scatchard transformation of the data was performed assuming 100% binding ability of labeled ligand.
Graphs show representative curves from a single pool of transduced cells. At least 3
pools were produced for each receptor combination.
dispersed colony morphology in semisolid medium, or other
evidence of terminal differentiation or clonal extinction was seen.
Treatment of M1/␤c/␣-2, M1/␤c/term-1, M1/␤c/term-3, M1/␣-1, or
M1/␣-2 cells with rhGM-CSF had no obvious effect on cell
morphology (data not shown).
We further examined the expression of several monocyte–
dendritic cell differentiation markers in cytokine-treated M1 cells.
Neither untreated nor rhGM-CSF–treated cells expressed major
histocompatibility complex class II, CD80, or CD40 proteins on
their membranes. All cell pools expressed the antigen recognized
by the antidendritic cell antibody NLDC145.23 Cytokine treatment
neither increased nor decreased expression of this marker.
The monocytic–dendritic protein F4/80 was minimally expressed under basal conditions by M1 cells. GM-CSF treatment
induced strong expression of F4/80 in all M1/␤c/␣ cell lines
containing at least the membrane-proximal 18 amino acids of
the GM-CSF receptor ␣ subunits (M1/␤c/␣-1, M1/␤c/␣-2,
M1/␤c/GM5, and M1/␤c/term-3; Figure 3A-B). The M1/␤c/term-1
cells, which lack almost the entire intracytoplasmic portion of the
␣ subunit, showed no up-regulation of F4/80 in response to
cytokine treatment. Similarly, cells expressing only ␣-1 or ␣-2
subunit proteins did not show cytokine-dependent expression of
F4/80 antigen.
CD11b was expressed under basal conditions by the M1 cells.
As with F4/80 antigen, all M1/␤c/␣ cells containing at least the
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
Figure 3. Expression of F4/80 antigen in rhGM-CSF–treated M1 cells expressing ␣ or ␤c subunits, or both, of GM-CSF receptor. Cells were incubated with
rhGM-CSF 100 ng/mL for 3 days, then analyzed by flow cytometry using anti-F4/80 or
an irrelevant isotype-matched control antibody. (A) Bars show mean ⫾ SD of D/s(n)
(derived from Kolmogorov-Smirnoff analysis) of 3 independently derived pools. (B)
Histograms of F4/80 fluorescence with or without rhGM-CSF treatment in M1/␤c/␣-1
(left) and M1/␤c/␣-2 (right) cells.
proximal intracytoplasmic portion of the ␣ subunit were able to
regulate CD11b expression in response to cytokine treatment.
(Figure 4). Interestingly, rhGM-CSF suppressed expression of the
protein. As before, the M1/␤c/term-1 cells did not respond to
cytokine treatment.
GM-CSF RECEPTOR ␣ CHAINS
1665
Figure 5. Expression of CD86 antigen in rhGM-CSF–treated M1 cells expressing ␣ or ␤c subunits, or both, of GM-CSF receptor. Cells were incubated with
rhGM-CSF 100 ng/mL for 3 days, then analyzed by flow cytometry using anti-CD86 or
an irrelevant isotype-matched control antibody. (A) Bars show mean ⫾ SD of D/s(n)
(derived from Kolmogorov-Smirnoff analysis) of 3 independent derived pools. (B)
Histograms of CD86 fluorescence with and without rhGM-CSF treatment in M1/␤c /
␣-1 (left) and M1/␤c /␣-2 (right) cells.
All cell lines expressed low levels of CD86 antigen, an immune
costimulatory molecule highly expressed by activated dendritic
cells. Treatment of M1/␤c /␣-1 and M1/␤c /GM5 cells with rhGMCSF for 3 days led to a noticeable increase in CD86 expression
(Figure 5A-B). In contrast, M1/␤c /term-1 and M1/␤c /term-3 cells
showed no significant change in CD86 expression after cytokine
treatment. M1/␤c /␣-2 cells actually showed a slight, but statistically significant, decline in expression of the costimulatory molecule after treatment with rhGM-CSF. Again, M1 cells expressing
only ␣-1 or ␣-2 did not show cytokine-dependent expression of
CD86 in response to rhGM-CSF treatment. Thus CD86 expression
paralleled morphologic differentiation in response to cytokine
treatment.
Regulation of Jak phosphorylation by the GM-CSF ␣ subunit
Figure 4. Expression of CD11b antigen in rhGM-CSF–treated M1/␤c cells with
various ␣ subunits. Cells were incubated with rhGM-CSF 100 ng/mL for 3 days,
then analyzed by flow cytometry using anti-CD11b or an irrelevant isotype-matched
control antibody. Bars show mean ⫾ SD of D/s(n) (derived from Kolmogorov-Smirnoff
analysis) of 3 independently derived pools.
Our initial results suggested that the variable C-terminal domain of
the GM-CSF receptor ␣ subunit could regulate morphologic
differentiation and expression of CD86. We next sought to determine whether different signal transduction events could be associated with signaling through the various ␣/␤c receptor complexes
and their response phenotypes.
Treatment of the transduced M1 leukemia cells with rhGM-CSF
resulted in prompt tyrosine phosphorylation of the Jak-2 protein
(Figure 6A). Interestingly, the M1/␤c/␣-1 and M1/␤c/GM5 cells
demonstrated more than 10 times as much tyrosine phosphorylation of the Jak-2 protein as did the M1/␤c/␣-2 and M1/␤c/term-3
cells, even though the amount of total Jak-2 protein in these cells
was similar (Figure 6A-B). M1/␤c/term-1 cells again failed to
demonstrate any response to cytokine treatment, suggesting that the
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LILLY et al
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
thereafter) was similar to that seen with M1/␤c/␣-1 cells. Thus it is
unlikely that the differences in phosphorylation result from untimely sampling.
Effect of variant and mutant ␣ subunits on GM-CSF–induced
tyrosine phosphorylation of putative Jak-2 substrates
To examine whether the observed differences in activation of Jak-2
were reflected in differential phosphorylation of potential Jak-2
substrates, we studied phosphorylation of the human ␤c subunit
after the addition of rhGM-CSF to the transduced cells. Tyrosine
phosphorylation of the ␤c subunit in response to cytokine was
markedly different between cells containing the ␣-1 protein and
those with the ␣-2 protein. Among all the ␣ subunit variant pools,
the level of phosphorylation of the ␤c subunit paralleled the level of
activation of Jak-2 (Figure 8A-B). Thus, even though Jak-2 is
constitutively associated with the ␤c subunit in these M1 clones
(see below), the carboxy terminal portion of the ␣ receptor is
important in regulating rhGM-CSF–mediated tyrosine phosphorylation of the ␤c subunit.
Because Stat-5 proteins play an essential role in mediating
signal transduction by the Jak kinases, the ability of each of these ␣
subunits to regulate Stat phosphorylation after rhGM-CSF treatment was examined (Figure 9A-B). When compared to the ␣-1 and
GM5 clones, incubation of the ␣-2 or term-3 subunit containing
M1 clones with rhGM-CSF stimulated 50% lower levels of
tyrosine phosphorylation of Stat-5. This decrease in phosphorylation was less than the decrease in Jak-2 phosphorylation. In the
Figure 6. Tyrosine phosphorylation of Jak-2 in M1/␤c cells with various ␣
subunits. (A) Immunoprecipitation–immunoblotting analysis. Cells were treated with
rhGM-CSF 100 ng/mL for 10 minutes. Lysates were then subjected to immunoprecipitation with anti–Jak-2, followed by blotting with antiphosphotyrosine (top panel). The
blot was then stripped and reprobed with anti–Jak-2 (bottom panel). (B) Quantitation
by densitometry of tyrosine-phosphorylated Jak-2 band after rhGM-CSF treatment.
intracytoplasmic portion of the receptor is necessary for Jak/Stat
activation. A time-course analysis after cytokine treatment showed
that the difference in phosphotyrosine content of Jak-2 isolated
from M1/␤c/␣-1 and M1/␤c/␣-2 cells persisted at least 1 hour
(Figure 7). In this experiment, the level of Jak-2 protein declined by
40% to 50% in the M1/␤c/␣-2 cells after cytokine treatment. This
finding was inconsistently seen (compare with Figure 6) and could
not account for the near-complete absence of Jak-2 tyrosine
phosphorylation in these cells. The time course of the faint Jak-2
phosphorylation in M1/␤c/␣-2 cells (peak at 10 minutes, decline
Figure 7. Time course of Jak-2 tyrosine phosphorylation in M1/␤c/␣-1 and
M1/␤c/␣-2 cells, after rhGM-CSF treatment. Cells were treated with rhGM-CSF 100
ng/mL for the indicated periods, then immunoprecipitated with anti–Jak-2, followed by
blotting with antiphosphotyrosine (top panel). The blot was then stripped and
reprobed with anti–Jak-2 (bottom panel).
Figure 8. Tyrosine phosphorylation of ␤c subunit protein in M1/␤c cells with
various ␣ subunits. (A) Immunoprecipitation–immunoblotting analysis. Cells were
treated with rhGM-CSF 100 ng/mL for 10 minutes. Lysates were then immunoprecipitated with anti-␤c (monoclonal), followed by blotting with anti-phosphotyrosine (top
panel). The blot was then stripped and reprobed with anti-␤c (polyclonal; bottom
panel). (B) Quantitation by densitometry of tyrosine-phosphorylated ␤c subunit band
after rhGM-CSF treatment.
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
GM-CSF RECEPTOR ␣ CHAINS
1667
latter case ␣-2 and term-3 receptor complexes induced no more
than 10% of the level of Jak-2 phosphorylation induced in the ␣-1
and GM5 subunits containing clones. These results could suggest
that only small amounts of Jak-2 phosphorylation are sufficient to
regulate the phosphorylation of larger amounts of Stat-5 or that the
level of Stat-5 phosphorylation reflects the activities of other
tyrosine kinases.24
Results similar to those obtained with other Jak-2 substrates
were seen when the dual SH2-containing phosphatase SHPTP-2
was immunoprecipitated from the rhGM-CSF–treated cells (Figure
10A-B). Those M1 cell lines containing the ␣-1 and GM-5
receptors had markedly increased phosphorylation of SHPTP-2,
whereas the term-3 and ␣-2 cell lines had approximately half as
much tyrosine phosphorylation. Again, the term-1–containing M1
cells showed no cytokine-directed phosphorylation of SHPTP-2.
␣ subunits differ in their ability to form stable complexes
with ␤c protein
The human GM-CSF receptor exists as a preformed complex,
consisting of variable combinations of the ␣ subunit, the ␤c
receptor protein, and the tyrosine kinase Jak-2.25,26 We examined
receptor complexes in the presence and absence of ligand binding.
Complex formation between the ␣ subunits and the ␤c subunit
differed markedly among the various samples (Figure 11A).
Neither ␣ subunit form appeared to be constitutively associated
with the ␤c receptor protein (under our assay conditions). After
Figure 9. Tyrosine phosphorylation of Stat-5 protein in M1/␤c cells with various
␣ subunits. (A) Immunoprecipitation–immunoblotting analysis. Cells were treated
with rhGM-CSF 100 ng/mL for 10 minutes. Lysates were then immunoprecipitated
with anti–Stat-5. Precipitated proteins were then solubilized, divided, and subjected
to immunoblotting with either antiphosphotyrosine (top panel) or anti–Stat-5 (bottom
panel). (B) Quantitation by densitometry of tyrosine-phosphorylated Stat-5 band after
rhGM-CSF treatment.
ligand binding, the ␣-1 subunit formed a complex with the larger
receptor component, which was stable in the face of stringent
washing conditions. In contrast, no stable complex between the ␣-2
protein and the ␤c subunit was detected after ligand binding.
Differences in ␣/␤c complex formation did not appear to result
from variation in the amount of ␣ chain expressed by the cells
(Figure 11C). Jak-2 was constitutively associated with the receptor
␤c subunit in both cell lines and was not modified by ligand binding
(Figure 11B).
Discussion
Figure 10. Tyrosine phosphorylation of SHPTP-2 protein in M1/␤c cells with
various ␣ subunits. (A) Immunoprecipitation–immunoblotting analysis. Cells were
treated with rhGM-CSF 100 ng/mL for 10 minutes. Lysates were then immunoprecipitated with anti–SHPTP-2. Precipitated proteins were then solubilized, divided, and
subjected to immunoblotting with either antiphosphotyrosine (top panel) or anti–
SHPTP-2 (bottom panel). (B) Quantitation by densitometry of tyrosine-phosphorylated SHPTP-2 band after rhGM-CSF treatment.
Hematopoietic cytokine receptors often exhibit multiple isoforms.
The human GM-CSF receptor ␣ subunit exists in at least 8
isoforms, which result from alternative splicing. Six of these
appear to have no intracytoplasmic sequences,4-7 and 5 lack the
transmembrane domain as well. Two forms, ␣-12 and ␣-2,3 have
intracytoplasmic sequences that are able to transduce signals.
Similarly, several membrane-associated and soluble isoforms of
the IL-5 receptor ␣ subunit have been described.22 Recently, a
novel isoform of the common ␤c receptor protein has been
characterized.27 As a result of alternative splicing, this receptor
protein has a deletion in the intracytoplasmic domain, resulting in
impaired proliferative signaling. However, no previous reports
have identified differences in signaling events or response phenotypes for ␣ subunit variants.
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1668
LILLY et al
Figure 11. Complex formation among components of human GM-CSF receptor.
(A) Association of GM-CSF receptor ␣ subunits with ␤c protein. Cells were treated
with rhGM-CSF 100 ng/mL for 10 minutes. Lysates were then immunoprecipitated
with anti-␤c polyclonal antibody. Precipitated proteins were solubilized and subjected
to immunoblotting with anti-␣ subunit antibody (top panel). The blot was stripped and
reprobed with anti-␤c (polyclonal; bottom panel). (B) Association of Jak-2 with ␤c
protein. Cells were treated with rhGM-CSF as above. Lysates were immunoprecipitated with anti-␤c polyclonal antibody. Precipitated proteins were then solubilized,
divided, and subjected to immunoblotting with either anti–Jak-2 (top panel) or anti-␤c
(polyclonal; bottom panel). (C) Expression of ␣ subunit proteins in transduced M1
cells. Lysates from either M1/␤c/␣-1 or M1/␤c/␣-2 cells were subjected to immunoprecipitation with an anti-␣ subunit antibody (␣ GM-CSF receptor) or an irrelevant
isotype-matched antibody (NC). Precipitated proteins were then subjected to immunoblotting with anti-␣ subunit antibody. The heavy band at approximately 55 kd
represents IgG heavy chain.
Previous studies comparing the ␣-1 and the ␣-2 GM-CSF
receptor proteins demonstrated that both subunits were able to
support the proliferation of factor-dependent murine hematopoietic
cells treated with human GM-CSF.17 To determine whether the ␣
subunit variants could differentially regulate maturation, we introduced the receptor cDNAs into M1 murine leukemia cells, which
demonstrate cytokine-dependent differentiation. Human GM-CSF
treatment of cells with receptor complexes incorporating ␣-1
proteins induced morphologic changes and increases in F4/80
BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
antigen and CD86 protein expression but a decrease in CD11b
expression. There was no evidence of terminal macrophage-like
differentiation (clonal extinction, dispersed colony morphology)
when cells were cultured with rhGM-CSF in semisolid medium.
The cells did, however, retain the ability to differentiate because
treatment of every pool with leukemia inhibitory factor dramatically up-regulated CD11b expression and induced clonal extinction with dispersed colony morphology in semisolid medium (data
not shown).
Although the ␣-1 and ␣-2 receptor complexes did support some
similar response phenotypes, important differences were seen.
M1/␤c/␣-1 cells increased their expression of CD86 in response to
rhGM-CSF, whereas M1/␤c/␣-2 cells slightly (but significantly)
decreased display of this costimulatory molecule. These data
demonstrate that the distal C-terminal domain of the GM-CSF
receptor ␣ subunit can regulate phenotypic responses after ligand
binding. They also suggest that the C-terminus of the ␣-2 subunit is
actively involved in propagating transmembrane signals because
the term-3 mutant ␣ subunit, which lacks a distal domain, had no
statistically significant effect on CD86 expression. We have also
noted in M1 cells that both ␣-1 and ␣-2 receptor proteins can
support cytokine-induced expression of the pim-1 kinase, whereas
term-3 ␣ subunits cannot (data not shown). At present we cannot
determine whether the ␣-2 receptor complex transmits signals for
unique differentiation or proliferation phenotypes. The lack of a
clear morphologic response in M1/␤c/␣-2 cells may be a reflection
of the limited spectrum of potential differentiation response
phenotypes in M1 cells. Expression of the variant GM-CSF
receptor proteins in other cell lines could result in wider differences
of response phenotypes than we have seen in the M1 cells.
The basis for the different response phenotypes supported by
␣-1– and ␣-2–GM-CSF receptors appears to involve Jak/Stat
signaling. Both the ␣-2– and the term-3–containing complexes
were markedly less efficient at inducing tyrosine phosphorylation
of Jak-2 than were receptors with ␣-1 and GM5 ␣ subunits. This
effect was amplified by additional differences in tyrosine phosphorylation of putative Jak-2 substrates. It is unknown what minimal
level of Jak-2 activation–tyrosine phosphorylation is required to
propagate an effective signal. Therefore, the ␣-2 and term-3
receptors could be supporting some degree of Jak-2–dependent
response. However, the correlation between phenotypic responses
(CD86 expression, morphologic differentiation) and biochemical
responses (tyrosine phosphorylation of Jak-2 and substrates) was
exact for the 5 cell lines examined, suggesting that phenotype and
biochemical events were linked.
The biochemical events underlying the differences in Jak/Stat
signaling by the various receptor complexes are unclear. Certainly
receptor number alone is not an adequate explanation. Although the
term-3 ␣ subunit was expressed in lower numbers than the other
functional receptors, this is unlikely to account for its relatively
poor Jak-2 activation. The numbers of high-affinity binding sites on
M1/␤c/term-3 cells were still higher than typically found on normal
hematopoietic cells, and the receptors were equally capable of
inducing F4/80 antigen expression as the other variants. Furthermore, M1/␤c/␣-2 cells also showed impaired Jak-2 activation, even
though they had higher receptor density than did the M1/␤c/␣-1 or
M1/␤c/GM5 cells. More rapid degradation of phosphorylated Jak-2
in M1/␤c/␣-2 cells appears an unlikely explanation. Treatment with
the proteasome inhibitor MG132, reported to increase levels of
tyrosine phosphorylated proteins,28 had no effect on Jak-2 phosphorylation in M1/␤c/␣-2 cells (data not shown), and Jak-2 appeared
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BLOOD, 15 MARCH 2001 䡠 VOLUME 97, NUMBER 6
GM-CSF RECEPTOR ␣ CHAINS
Figure 12. Schematic diagram representing GM-CSF receptor ␣ subunit domains. Receptor regions are identified at left (TM, transmembrane domain).
Biochemical events (within minutes) or response phenotypes (within hours to days)
dependent on the various domains are identified. Data are from Matsuguchi et al,16
Polotskaya et al,18 and the present report (*).
equally able to bind to the ␤c protein in the presence of either ␣-1 or
␣-2 subunits.
Our data suggest that the molecular mechanisms underlying
differential signaling may involve the stability of ␣/␤c complexes.
After ligand binding, the ␣-1 subunit clearly was more stably
associated with ␤c protein than was the ␣-2 subunit. These
differences may result from the secondary structure of the Cterminal domain of the ␣ subunits. Both the ␣-1 and the GM5 ␣
chains were able to support intense tyrosine phosphorylation of
Jak-2 and its substrates and up-regulation of CD86. There are no
areas of high homology in the membrane-distal region, though both
proteins show alternating charged and hydrophobic amino acids in
this area. Secondary structure analysis does show, though, that both
receptor molecules are likely to have a ␤ strand configuration near
the C-terminus. In contrast, the ␣-2 subunit is predicted to lack this
secondary structure. Possibly, the carboxy terminal domain of the
␣-2 protein destabilizes receptor complexes, leading to impaired
mutual phosphorylation of the associated Jak-2 molecules after
ligand binding.
The current studies extend our previous work in mapping
significant domains of the GM-CSF receptor ␣ subunit.16,18 Our
data suggest the existence of 2 domains (Figure 12). A membraneproximal domain (amino acids 346-370) contains important, conserved proline residues. The distal domain extends from amino acid
370 to the C-terminus and differs entirely between the ␣-1 and ␣-2
subunits. The current report demonstrates that the primary (and
1669
likely, secondary) structure of the distal domain, as much as its
presence or absence, can modulate signal transduction events and
ligand-dependent responses.
Although distinct biochemical events can be associated with
these domains, it is more difficult to generalize about response
phenotypes. The participation of the distal domain in supporting
proliferation is clearly cell-type dependent. Thus the term-3
mutant, containing only the proximal domain, is able to fully signal
for cell growth in BaF3 cells18 but not in WT19 cells.16 Differentiation is a complex process involving many molecular events. We
here find that a particular form of the distal domain is required for
morphologic changes and expression of CD86. As we have seen
before,16 however, the proximal domain is adequate to support
expression of the differentiation antigen F4/80. Likely, the degree
of differentiation reflects the sum of many signaling events, some
of which may show marked quantitative differences based on the
stability of the involved receptor complexes.
Although distal regions of the GM-CSF receptor ␣ subunit
appear to modulate Jak-2 activation, tyrosine kinases activated by
the proximal domain of the ␣ subunit are unknown. We have used
kinase inhibitors to explore the signaling events behind expression
of F4/80, which requires the membrane-proximal domain of the ␣
subunit. The tyrosine kinase inhibitor tyrphostin AG490 was able
to completely inhibit F4/80 expression at doses between 10 and 40
␮M, whereas tyrphostin A10 had no effect (data not shown).
Tyrphostin AG490 has been claimed to be a specific inhibitor of the
Jak-229 and Jak330 kinases, though it can also inhibit the epidermal
growth factor receptor kinase.31 Under our test conditions, tyrphostin AG490 had no effect on rhGM-CSF–induced Jak-2 tyrosine
phosphorylation in M1/␤c/␣-1 cells, even at doses that completely
blocked F4/80 up-regulation. These results imply that the proximal
domain of the ␣ subunit activates signaling events that include a
tyrosine kinase distinct from Jak-2. We did not find any evidence of
activation of the Jak-family kinases Jak-1 or Tyk-2 after rhGMCSF treatment of our cells (data not shown). Possibilities might
include a member of the src family of tyrosine kinases.
Our data demonstrate the existence of 2 distinct domains on the
human GM-CSF receptor ␣ subunit and provide evidence for
distinct biologic and biochemical activities of the 2 receptor ␣
subunit isoforms. It is possible that differential signaling through
the variant receptors will provide a mechanism for a wider range of
response phenotypes after exposure of the cells to GM-CSF.
Acknowledgments
We thank Drs G. Begley, D. Metcalf, and N. Nicola for their
encouragement in these studies and for graciously supplying
important reagents. We also thank Drs C. Sieff and J. Tavernier for
generously providing cDNA clones, and John J. Cooper for superb
technical assistance.
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2001 97: 1662-1670
doi:10.1182/blood.V97.6.1662
Distinct domains of the human granulocyte-macrophage colony-stimulating
factor receptor α subunit mediate activation of Jak/Stat signaling and
differentiation
Michael B. Lilly, Marina Zemskova, Arthur E. Frankel, Jonathan Salo and Andrew S. Kraft
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