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Blood, 15 March 2001, Vol. 97, No. 6, pp. 1662-1670
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
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.
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Abstract |
The  subunit of the human granulocyte-macrophage
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 binding of radiolabeled human GM-CSF. Receptor subunits with intact membrane-proximal 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 GM-CSF 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.
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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 carboxy-terminal 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 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.
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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).
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.
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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
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.
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| 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.
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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 low-affinity 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).
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Table 1.
Binding affinity of human GM-CSF receptors in M1 cells
expressing human c and various subunit constructs
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| 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.
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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, 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.
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| 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.
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CD11b was expressed under basal conditions by the M1 cells. As with
F4/80 antigen, all M1/c/ cells containing at least
the 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.
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| 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.
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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 rhGM-CSF 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.
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| 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.
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Regulation of Jak phosphorylation by the GM-CSF subunit
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 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
thereafter) was similar to that seen with M1/c/-1
cells. Thus it is unlikely that the differences in phosphorylation
result from untimely sampling.
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| 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.
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| 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).
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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.
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| 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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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 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
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| 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.
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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.
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| 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.
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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
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).
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| 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.
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Discussion |
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.
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 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
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
C-terminal 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 membrane-proximal 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 likely, secondary) structure of the
distal domain, as much as its presence or absence, can modulate signal
transduction events and ligand-dependent responses.
View larger version (30K):
[in this window]
[in a new window]
| 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 (*).
|
|
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 µM, 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
rhGM-CSF 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.
| |
Footnotes |
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.).
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.
Reprints: Michael Lilly, Department of Medicine, Loma Linda
University, Loma Linda, CA 92354; e-mail: mlilly{at}som.llu.edu.
| |
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Abstract
Introduction