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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3774-3784
Functional Differentiation Signals Mediated by Distinct Regions of the
Cytoplasmic Domain of the Granulocyte Colony-Stimulating Factor
Receptor
By
Debbie C. Koay and
Alan C. Sartorelli
From the Department of Pharmacology and Developmental Therapeutics
Program, Cancer Center, Yale University School of Medicine, New Haven,
CT.
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ABSTRACT |
Granulocyte colony-stimulating factor receptor (G-CSFR) regulates
the proliferation and differentiation of neutrophilic progenitor cells
through interaction with its cytokine. Exposure of WEHI-3B D+ myelomonocytic leukemia and myeloid LGM-1 cells
overexpressing the G-CSFR to G-CSF resulted in induction of
differentiation as measured by (1) the ability to reduce nitroblue
tetrazolium (NBT), (2) the expression of Mac-I antigen, and (3) the
expression of Fc II/III receptor. Mutational analyses indicated that
distinct regions of the cytoplasmic domain were critical for efficient induction of each functional marker. The membrane proximal region containing homology sequences of boxes 1 and 2 was important for the
activation of all three functional markers of mature neutrophils. Induction of the capacities to express Mac-I antigen or FcII/III receptor also required additional sequences in the membrane proximal region between amino acids 70 and 100 and may be dependent on the
phosphorylation of Tyr703. The findings suggest that
distinct sequences within the amino-terminal region of the cytoplasmic
domain of the receptor are sufficient to induce these functional
markers of differentiation, and receptor tyrosine phosphorylation may
be necessary.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
GRANULOCYTES FORM a major proportion of
the circulating cells of the blood, playing an important role in the
mammalian defense system. The formation of granulocytes from
pluripotent stem cells is a multi-stage process, which is achieved
through continuous proliferation and differentiation of stem cells. In the process of granulocytic maturation, a series of tightly regulated biochemical and morphological changes take place which result in
end-stage mature cells with specific functions and characteristic biochemical and cell-surface markers. Three cytokines, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and
granulocyte colony-stimulating factor (G-CSF), have been shown to be
involved in the generation of granulocytes. IL-3 and GM-CSF exert their
effects during the early stages of hematopoiesis, acting on both
macrophagic and granulocytic progenitor cells. G-CSF acts specifically
on granulocytic progenitor cells.
In the process of functional differentiation, the neutrophilic
progenitor cells pass through several stages of maturation during which
they acquire a number of functional properties that are necessary for
mature neutrophils to eradicate microbes.1 The capacity for
microbial killing requires oxidative processes that can be measured by
the ability to reduce nitroblue tetrazolium (NBT). The biochemical
basis of NBT reduction in neutrophils is superoxide generation, and
cytochemical NBT reduction is an expression of the respiratory
burst.2 The expression of both Mac-I antigen (CD11b/CD18)
and FcII/III receptor (CD32/CD16) on the cell surface enables
neutrophils to recognize microbes as foreign through opsonins, serum
components that coat microorganisms.1 Interaction of Mac-I
antigen or FcII/III receptors with opsonins promotes both phagocytosis and superoxide generation. The level of each of these three functional properties increases as the level of maturation increases.
The process of differentiation is mediated in part through the
interaction of G-CSF with the G-CSF receptor (G-CSFR), a member of the
cytokine receptor superfamily. Like most cytokine receptors, the G-CSFR
cytoplasmic domain is thought to be involved in the initiation of
signaling events from the receptor. Through expression of different
G-CSFR forms in various cells, distinct regions of the G-CSFR
cytoplasmic domain have been suggested to be critical for signal
transduction.3-5 Signal transduction by members of the
cytokine receptor superfamily involves activation of the JAK-STAT pathway.6-9 Upon cytokine stimulation, the receptor
dimerizes and associates with members of the JAK (Janus kinase) family
of protein tyrosine kinases through its cytoplasmic membrane proximal region, thereby leading to tyrosine phosphorylation and activation of
the catalytic activity of the JAK kinases. After their activation, the
JAK kinases induce tyrosine phosphorylation of the cytokine receptor,
as well as of the family of cytoplasmic transcription factors known as
STATs (signal transducers and activators of transcription). Tyrosine
phosphorylation of the receptor creates docking sites for the
recruitment of signaling proteins, which contain Src homology 2 (SH2)
domains, to the receptor complex. Tyrosine phosphorylated STAT proteins
undergo dimerization and translocate to the nucleus where they bind to
characteristic enhancer sequences and activate transcription of genes.
In the present studies, we investigated the role of the G-CSFR in
regulating the process of differentiation in WEHI-3B D+
murine myelomonocytic leukemia and murine myeloid LGM-1 cells overexpressing the G-CSFR. WEHI-3B D+ cells express a low
level of the G-CSFR and respond weakly to the differentiation inducing
properties of G-CSF.10-12 In contrast, LGM-1 cells do not
express the G-CSFR.3 However, exogenous expression of the
G-CSFR makes LGM-1 cells responsive to G-CSF both by proliferation and
by morphological differentiation into neutrophils.3
Overexpression of the G-CSFR markedly increased the degree of
differentiation in response to G-CSF as demonstrated by a significant
increase in three functional markers of mature neutrophils: (1) the
ability to reduce NBT, (2) the expression of Mac-I, and (3) the
expression of the FcII/III receptor. Introduction of mutant G-CSF
receptors into these cells indicated that functional differentiation
signals are mediated by distinct regions of the cytoplasmic domain. The
membrane proximal region containing homology sequences of boxes 1 and 2 was important for the activation of all three functional markers of
mature neutrophils. Induction of the capacities to express Mac-I
antigen or FcII/III receptor also required additional sequences in
the membrane proximal region between amino acids 70 and 100 and may be
dependent on the phosphorylation of Tyr703.
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MATERIALS AND METHODS |
Cell culture.
WEHI-3B D+ murine myelomonocytic leukemia cells, obtained
from Dr Malcohm A.S. Moore (Sloan-Kettering Institute for Cancer Research, New York, NY), and transfectants derived therefrom in our
laboratory were maintained in suspension culture in McCoy's 5A
modified medium supplemented with 15% fetal bovine serum (FBS) (GIBCO-BRL, Grand Island, NY) at 37°C in a 95% air/5%
CO2 humidified incubator. Murine myeloid LGM-1 cells,
obtained from Dr Tasuku Honjo (Kyoto University, Kyoto, Japan), and
transfectants derived therefrom in our laboratory were maintained in
suspension culture in RPMI 1640 medium supplemented with 10% FBS and
45 U/mL of recombinant mouse IL-3 at 37°C in a 95% air/5%
CO2 humidified incubator.
Construction of expression plasmids.
The expression plasmid p75/15GR95, which contains the full-length
murine G-CSFR cDNA, was constructed as previously
described.11 Six mutant murine G-CSFR expression plasmids
were also constructed. The mutant G-CSFRs have intact extracellular and
transmembrane domains but have cytoplasmic domains truncated at
approximately every 30 amino acids from the C-terminus. The truncated
G-CSFR DNA fragments were generated through the polymerase chain
reaction (PCR) using the plasmid pBLJ17 (kindly provided by Dr
Shigekazu Nagata, Osaka Bioscience Institute, Osaka, Japan) as
template, primers containing restriction enzyme sites (synthesized by
the Program for Critical Technologies in Molecular Medicine, Yale University Department of Pathology, New Haven, CT), and
VentR DNA polymerase (New England Biolabs, Beverly, MA).
The forward primer, 5'-TCTAGAACTAGTGGATCCCC-3', was derived from the 5'
upstream region of the G-CSFR cDNA in the plasmid pBLJ17 and has a
BamHI site. The sequences of the six reverse primers, which
were derived from the sequence of the G-CSFR cytoplasmic domain and
have an XbaI site, as well as an in-frame termination
codon, are: reverse primer A:
5'-AGTTCTAGAGATCTTCCTGGTTTGGAGGTTG-3', reverse primer B:
5'-AGTTCTAGAGCAAGAGGGGCTGAGTGGAGTC-3', reverse primer C:
5'-AGTTCTAGAGCTGGTCACCAGTGCGAGAGGG-3', reverse primer D:
5'AGTTCTAGAGGCTACCATTCCCAGAGCTTTC-3', reverse primer E:
5'-AGTTCTAGAGGTCCCAGAAGCTGGGTAACTG-3', and reverse primer F:
5'-AGTTCTAGAGTGACCAGAAGGAAGTCTTTCC-3'. For each PCR, the same forward
primer and one of the reverse primers were used to generate mutant
forms A through F, which have truncations of 27, 59, 87, 117, 147, and
177 amino acids from the C-terminus of the G-CSFR cytoplasmic domain,
respectively. After amplification, the PCR products were digested with
XbaI and BamHI, and purified by gel electrophoresis.
The purified digested PCR products were subcloned into the same vector
that was used to construct the full-length G-CSFR expression plasmid.
Each of the truncated G-CSFR expression plasmids (designated as
p75/15GR-A, p75/15GR-B, p75/15GR-C, p75/15GR-D, p75/15GR-E, and
p75/15GR-F) was sequenced by the W.M. Keck Foundation Biotechnology
Resource Laboratory (Yale University) to confirm the sites of truncation.
Transfection, selection by antibiotic, and single-cell cloning.
Exponentially growing WEHI-3B D+ and LGM-1 cells were
transfected with linearized plasmids by electroporation as previously described.11 Transfected WEHI-3B D+ and LGM-1
cells were selected with 400 or 500 µg/mL of G-418 sulfate (Geneticin
from GIBCO-BRL), respectively, for 2 to 3 weeks.
Western blotting.
Cells, 3 × 106, were collected and washed with cold
serum-free McCoy's 5A modified medium. The washed cells were
resuspended in 250 µL of cold suspension buffer (100 mmol/L NaCl; 10 mmol/L Tris-HCl, pH 7.6; 1 mmol/L EDTA, pH 8.0) containing freshly
added protease inhibitors (100 µg/mL of phenylmethanesulfonyl
fluoride, 2 µg/mL of aprotinin, 2 µg/mL of leupeptin) followed by
the addition of 250 µL of 2X sample buffer (100 mmol/L Tris-HCl, pH
6.8; 4% sodium dodecyl sulfate [SDS]; 0.2% bromophenol blue; 20%
glycerol) containing freshly added 200 mmol/L dithiothreitol. This
sample was then passaged through a 22-gauge hypodermic needle four
times to shear the genomic DNA and boiled for 5 minutes. Twenty
microliters of sample was loaded onto a 7.5% polyacrylamide gel and
separated by gel electrophoresis in the presence of SDS. After
electrophoresis, the gel was transferred to a nitrocellulose membrane
(Schleicher and Schuell, Inc, Keene, NH).
Membranes were blocked with 2% dry milk in Tris-buffered saline (20 mmol/L Tris base, 137 mmol/L NaCl, pH 7.6) containing 0.01% Tween-20
(TBS-T) at room temperature for 1 hour. Membranes were then incubated
at 4°C for approximately 12 hours with primary antibody that
recognizes both the full-length and truncated G-CSFR forms (rabbit
polyclonal antibody raised against the extracellular domain of the
murine G-CSFR13) diluted 1:2,000 in TBS-T containing 2%
dry milk. After incubation with primary antibody, membranes were washed
4 × 15 minutes at room temperature with TBS-T. Then membranes were
incubated at room temperature for approximately 3 hours with secondary
antibody (horseradish peroxidase-conjugated donkey anti-rabbit IgG
from Amersham Corp, Arlington Heights, IL) diluted 1:4,000 in TBS-T
containing 2% dry milk and washed 4 × 15 minutes at room temperature
with TBS-T. The immunoreactive proteins were visualized by enhanced
chemiluminescence according to the protocol provided by Amersham.
Binding of phycoerythrin-conjugated recombinant human (rh) G-CSF to
cell-surface receptor.
Binding was performed according to the protocol provided with the human
G-CSF phycoerythrin conjugate kit from R&D Systems (Minneapolis, MN).
The kit supplied 11.9 µg/mL of phycoerythrin-conjugated rhG-CSF, 4.0 µg/mL of phycoerythrin-conjugated streptavidin, and 10X RDF1 wash buffer.
To analyze surface G-CSFR expression, cells were collected, washed
twice with cold phosphate-buffered saline (PBS), and resuspended in PBS
at a final concentration of 4 × 106 cells/mL. Ten
microliters of phycoerythrin-conjugated rhG-CSF was added to 25 µL of
the washed cell suspension (105 cells) in a 12 × 75-mm
borosilicate tube, and the cells were incubated on ice for 20 minutes.
As a negative staining control, an identical sample of cells was
incubated with 10 µL of phycoerythrin-conjugated streptavidin for 20 minutes on ice. After incubation, the cells were washed twice with 2 mL
of cold 1X RDF1 buffer to remove unbound phycoerythrin-conjugated
reagents and resuspended in 200 µL of cold 1X RDF1 buffer for flow
cytometric analysis.
To determine the specificity of rhG-CSF binding, cells were collected,
washed twice with cold PBS, and resuspended in PBS at a final
concentration of 4 × 106 cells/mL. Three 12 × 75-mm
borosilicate tubes (designated as I, II, and III) were prepared for
each cell line. Tube I served as the negative staining control, tube II
represented the binding of rhG-CSF to the cell surface, and tube III
demonstrated the specificity of rhG-CSF binding. Twenty-five
microliters of the washed cell suspension (105 cells) was
added to each tube followed by the addition of 25 µL of cold PBS to
each of tubes I and II and 25 µL of unconjugated rhG-CSF (gift from
Glaxo, Inc, Geneva, Switzerland) to tube III at a final concentration
that gave a 10-fold molar excess ratio of unconjugated rhG-CSF to
phycoerythrin-conjugated rhG-CSF. The three tubes were then incubated
at room temperature for 15 minutes. After this incubation, 10 µL of
phycoerythrin-conjugated streptavidin was added to tube I and 10 µL
of phycoerythrin-conjugated rhG-CSF was added to each of tubes II and
III. The tubes were then incubated on ice for 20 minutes. Each tube of
cells was washed twice with 2 mL of cold 1X RDF1 buffer to remove
unbound reagents and resuspended in 200 µL of cold 1X RDF1 buffer for
flow cytometric analysis.
Measurement of growth.
Exponentially growing parental WEHI-3B D+ cells and
transfected cells derived therefrom were seeded into fresh culture
medium at a density of 5 × 104 cells/mL in the presence
of vehicle or 10 ng/mL of rhG-CSF (Glaxo, Inc), and cell numbers were
determined daily for 3 days after exposure to rhG-CSF using a Coulter
model ZM particle counter (Coulter Electronics, Inc, Hialeah, FL)
connected to a Coulter model 256 Channelyzer. Exponentially growing
parental LGM-1 cells and transfected cells derived therefrom were
seeded into fresh culture medium at a density of 5 × 104
cells/mL in the presence of vehicle or 10 ng/mL of rhG-CSF, the medium
was replenished every 3 days to maintain the cell density at 5 × 104 cells/mL, and cell numbers were determined daily for 10 days after exposure to rhG-CSF.
Measurement of differentiation capacity.
Exponentially growing parental WEHI-3B D+ or LGM-1 cells
and transfected cells derived therefrom were seeded into fresh culture medium at a density of 5 × 104 cells/mL in the presence
of vehicle or 10 ng/mL of rhG-CSF (Glaxo, Inc). Three to 10 days after
exposure to rhG-CSF, the capacity to differentiate was analyzed by
three different functional markers of mature neutrophils: (1) the
ability of cells to reduce NBT, (2) the expression of Mac-I antigen
(CD11b/CD18) on the cell surface, and (3) the cell-surface expression
of the FcII/III receptor (CD32/CD16).
To examine the ability of cells to reduce NBT, 106 cells
were collected and resuspended in 1 mL of serum-free McCoy's 5A
modified medium containing 0.1% NBT (Sigma Chemical Co, St Louis, MO)
and 2 µmol/L 12-O-tetradecanoylphorbol 13-acetate (Sigma
Chemical Co). The suspension was incubated at 37°C for 30 minutes
with shaking, and the percentage of cells with blue-black formazan deposits was determined microscopically on 200 consecutive cells.
To analyze the cell surface expression of Mac-I antigen or FcII/III
receptor, 106 cells were collected and washed once with
cold PBS. The cells were then incubated on ice for 30 minutes with 100 µL of fluorescein-conjugated rat anti-mouse/human Mac-I antigen
monoclonal antibody (MoAb) (Boehringer Mannheim, Indianapolis, IN) at a
final concentration of 10 µg/mL or fluorescein
isothiocyanate-conjugated rat anti-mouse FcII/III receptor MoAb
(PharMingen, San Diego, CA) at a final concentration of 0.6 µg/mL in
PBS containing 0.1% bovine serum albumin (BSA). After incubation, the
stained cells were washed twice with cold PBS containing 0.1% BSA to
remove unbound antibody and resuspended in 200 µL of cold PBS
containing 0.1% BSA for flow cytometric analysis.
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RESULTS |
Establishment of cell lines.
Exponentially growing WEHI-3B D+ and LGM-1 cells were
transfected by electroporation with full-length G-CSFR expression
plasmid p75/15GR95 or mutant G-CSFR expression plasmids p75/15GR-A,
p75/15GR-B, p75/15GR-C, p75/15GR-D, p75/15GR-E, and p75/15GR-F with
C-terminal truncations of 27, 59, 87, 117, 147, and 177 amino acids,
respectively (Fig 1). The vector p75/15v,
which was the same as p75/15GR95 but devoid of the G-CSFR and the
bovine growth hormone polyadenylation signal, was also introduced into
these cells as a control. Transfected cells were enriched by selection
with 400 or 500 µg/mL of the antibiotic G-418 sulfate.
D+V and LGM V represent WEHI-3B D+ and LGM-1
cells, respectively, transfected with the control plasmid p75/15v;
D+GR95 and LGM GR95 represent WEHI-3B D+ and
LGM-1 cells, respectively, transfected with the full-length G-CSFR
expression plasmid p75/15GR95; D+GR-A and LGM GR-A,
D+GR-B and LGM GR-B, D+GR-C and LGM GR-C,
D+GR-D and LGM GR-D, D+GR-E and LGM GR-E, and
D+GR-F and LGM GR-F represent WEHI-3B D+ and
LGM-1 cells, respectively, transfected with truncated G-CSFR expression
plasmids p75/15GR-A, p75/15GR-B, p75/15GR-C, p75/15GR-D, p75/15GR-E,
and p75/15GR-F, respectively.
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| Fig 1.
Structures of full-length and mutant G-CSFR introduced
into WEHI-3B D+ and LGM-1 cells by electroporation. The
mutant G-CSFRs are designated by the letters of the alphabet (A, B, C,
D, E, and F) and their degrees of truncation from the C-terminus of the
cytoplasmic domain are illustrated. The cytokine receptor superfamily
homology regions (Box 1, Box 2, and Box 3) are represented by boxes,
and the four tyrosine residues (Tyr703, Tyr728,
Tyr743, and Tyr763) in the murine G-CSFR
cytoplasmic domain are indicated by "Y." Amino acids are denoted
by aa.
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Expression of full-length and mutant G-CSF receptors in transfected
cells.
Western blotting was performed to confirm the expression of the
exogenous full-length or truncated G-CSFR proteins in transfected cells. The G-CSFR proteins were detected using a rabbit polyclonal antibody against the extracellular domain of the murine G-CSFR. Protein
was extracted from each of the cell lines, separated by polyacrylamide
gel electrophoresis in the presence of SDS, and transferred to a
nitrocellulose membrane. The immunoreactive proteins were detected by
the antibody and visualized by enhanced chemiluminescence. The
polyclonal antibody against the G-CSFR extracellular domain recognizes
both the full-length and the truncated G-CSFR proteins. As illustrated
in Fig 2, control D+V cells
expressed a low level of endogenous full-length G-CSFR protein and
D+GR95 cells overexpressed the full-length receptor
protein. WEHI-3B D+ cells transfected with the truncated
G-CSFR expression plasmids expressed high levels of truncated G-CSFR
proteins, and the molecular size of the truncated receptor proteins
decreased as the degree of truncation increased. Thus, all of the
transfected cell lines, except D+V cells, expressed a high
level of exogenous full-length or truncated G-CSFR proteins. The three
bands of highest molecular weight are believed to be different forms of
the G-CSFR with possibly different posttranslational modifications. The
lowest of these bands is not clearly visible in vector-transfected
cells because they express a low endogenous level of the G-CSFR.
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| Fig 2.
Expression of full-length and mutant G-CSFR proteins in
transfected WEHI-3B D+ cells. Full-length and mutant
G-CSFR protein expression in vector-transfected cells
(D+V), full-length G-CSFR-transfected cells
(D+GR95), and truncated G-CSFR-transfected cells
(D+GR-A, D+GR-B, D+GR-C,
D+GR-D, D+GR-E, and D+GR-F)
were detected by Western blotting using rabbit polyclonal antibody
against the murine G-CSFR extracellular domain.
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Specific binding of phycoerythrin-conjugated rhG-CSF to transfected
cells.
To determine whether the exogenous full-length or truncated G-CSFR
proteins were expressed on the cell surface and were capable of
specifically binding rhG-CSF, phycoerythrin-conjugated rhG-CSF binding
was measured. Cells, 1 × 105, from each transfected cell
line were incubated with phycoerythrin-conjugated rhG-CSF, and
cell-surface G-CSFR expression levels were then determined by flow
cytometric analysis (Tables 1 and
2). The
surface G-CSFR expression level observed in D+V and LGM V
cells represents binding by any endogenous cell-surface receptor
proteins that may be present. By comparison, the rest of the
transfected cell lines showed greater specific binding than
D+V cells. The D+GR95, D+GR-A,
D+GR-B, D+GR-C, D+GR-D,
D+GR-E, and D+GR-F cells exhibited 2.2-, 2.8-, 3.6-, 3.1-, 2.8-, 4.2-, and 13.0-fold, respectively, greater surface
receptor expression than D+V cells (Table 1). The LGM V,
LGM GR95, LGM GR-A, LGM GR-B, LGM GR-C, LGM GR-D, LGM GR-E, and LGM
GR-F cells exhibited 2.9-, 4.3-, 3.7-, 6.5-, 6.1-, 4.1-, and 2.4-fold,
respectively, greater surface receptor expression than LGM V cells,
which do not exhibit any surface G-CSFR expression (Table 2). The
specificity of binding in each of these transfected cell lines was
determined by preincubating the cells with a 10-fold molar excess of
unconjugated rhG-CSF and then exposing the cells to conjugated
cytokine. Because the unconjugated rhG-CSF served as an effective
competitor, the binding of conjugated rhG-CSF to the transfected cell
lines was determined to be specific. Therefore, the exogenous full
length as well as truncated G-CSFR proteins are expressed on the cell
surface and are capable of specifically binding rhG-CSF.
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Table 1.
Phycoerythrin-Conjugated rhG-CSF (PE-G-CSF) Binding to
WEHI-3B D+ Cells Transfected With Full-Length or Mutant
G-CSFR Expression Plasmids
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Table 2.
Phycoerythrin-Conjugated rhG-CSF (PE-G-CSF) Binding to
LGM-1 Cells Transfected With Full-Length or Mutant G-CSFR Expression
Plasmids
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Effects of expression of full-length and mutant G-CSF receptors on
cellular growth.
Growth of transfected WEHI-3B D+ and LGM-1 cells was
monitored daily for 3 or 10 days after exposure to 10 ng/mL of rhG-CSF (Figs 3 and
4). Untreated
D+GR95 or LGM GR95, D+GR-A or LGM GR-A,
D+GR-B or LGM GR-B, D+GR-C or LGM GR-C,
D+GR-D or LGM GR-D, D+GR-E or LGM GR-E, and
D+GR-F or LGM GR-F cells had growth rates similar to that
of D+V or LGM V cells, respectively, indicating that high
levels of expression of full-length and truncated G-CSF receptors
themselves neither promote nor inhibit the proliferation of these
cells. Treatment of D+V and LGM V, D+GR95 and
LGM GR95, D+GR-A and LGM GR-A, D+GR-B and LGM
GR-B, D+GR-D and LGM GR-D, D+GR-E and LGM GR-E,
and D+GR-F and LGM GR-F cells with rhG-CSF had no
significant effects on growth. However, rhG-CSF treatment significantly
decreased the growth rate of D+GR-C and LGM GR-C cells.
Therefore, only D+GR-C and LGM GR-C cells, which express
high levels of G-CSFR with a truncation of 87 amino acids from the
cytoplasmic domain, exhibited significant reductions of growth after
treatment with rhG-CSF. High levels of expression of the full-length
and the rest of the truncated receptors did not influence the growth
rate of the treated cells.
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| Fig 3.
Effects of the expression of full-length and mutant
G-CSFR on WEHI-3B D+ cell growth after treatment with 10 ng/mL of rhG-CSF. Untreated and G-CSF-treated cells were seeded at 5 × 104 cells/mL, and their cell densities were determined
daily for 3 days. I, D+V (vector-transfected cells); II,
D+GR95 (full-length G-CSFR-transfected cells); III-VIII,
D+GR-A, D+GR-B, D+GR-C,
D+GR-D, D+GR-E, and D+GR-F
(truncated G-CSFR-transfected cells). Each value is the average of
three independent experiments ± standard deviation. (Note: When
standard deviations are very small, the error bars may not be clearly
visible in the graphs.)
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| Fig 4.
Effects of the expression of full-length and mutant
G-CSFR on LGM-1 cell growth after treatment with 10 ng/mL of rhG-CSF.
Untreated and G-CSF-treated cells were seeded at 5 × 104
cells/mL, and their cell densities were determined daily for 10 days.
I, LGM V (vector-transfected cells); II, LGM GR95 (full-length
G-CSFR-transfected cells); III-VIII, LGM GR-A, LGM GR-B, LGM GR-C, LGM
GR-D, LGM GR-E, and LGM GR-F (truncated G-CSFR-transfected cells).
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Effects of overexpression of full-length G-CSF receptor on cellular
differentiation.
The effects of the overexpression of full-length G-CSFR on cellular
differentiation were investigated by exposing transfected cells to
rhG-CSF for 3 or 10 days. The degree of differentiation was determined
on day 3 or day 10 after treatment with 10 ng/mL of rhG-CSF. Three
functional markers of mature neutrophils were examined: (1) the ability
of cells to reduce NBT, (2) the expression of Mac-I antigen
(CD11b/CD18) on the cell surface, or (3) the cell-surface expression of
the FcII/III receptor (CD32/CD16). When the ability of cells to
reduce NBT was examined, rhG-CSF-treated D+GR95 cells
showed 7.8-fold higher NBT positivity than treated D+V
cells (Table 3). The increase in the
ability to reduce NBT was only observed in the D+GR95 cells
treated with rhG-CSF, indicating that overexpression of the G-CSFR
itself is not sufficient to initiate maturation and that treatment with
rhG-CSF is necessary for the cells to acquire the differentiated
phenotype. In contrast, neither rhG-CSF-treated LGM V nor LGM GR95
cells exhibited NBT positivity. To further analyze the differentiation
capacity of cells transfected with the full-length receptor after
treatment with rhG-CSF, immunofluorescent staining of the Mac-I antigen
and the FcII/III receptor on the cell surface was also performed.
The cell-surface expression of the Mac-I antigen and the FcII/III
receptor was 1.2-fold and 1.4-fold higher, respectively, in
rhG-CSF-treated D+GR95 cells than in treated
D+V cells (Tables 4 and
5).
Similarly, the cell-surface expression of the Mac-I antigen and the
FcII/III receptor was 1.5-fold and 2.1-fold higher, respectively, in
rhG-CSF-treated LGM GR95 cells than in treated LGM V cells (Tables 6
and 7). Thus,
after exposure to rhG-CSF, D+GR95 cells showed a higher
capacity to reduce NBT and a greater surface expression of Mac-I
antigen and FcII/III receptor than D+V cells, and
rhG-CSF-treated LGM GR95 cells showed a greater surface expression of
Mac-I antigen and FcII/III receptor than LGM V cells.
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Table 3.
Differentiation of WEHI-3B D+ Cells
Transfected With Full-Length or Mutant G-CSFR Expression Plasmids as
Determined by NBT Positivity After G-CSF Treatment
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Table 4.
Differentiation of WEHI-3B D+ Cells
Transfected With Full-Length or Mutant G-CSFR Expression Plasmids as
Determined by Mac-I Expression After G-CSF Treatment
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Table 5.
Differentiation of WEHI-3B D+ Cells
Transfected With Full-Length or Mutant G-CSFR Expression Plasmids as
Determined by FcII/III Receptor Expression After G-CSF Treatment
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Table 6.
Differentiation of LGM-1 Cells Transfected With
Full-Length or Mutant G-CSFR Expression Plasmids as Determined by Mac-I
Expression After G-CSF Treatment
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View this table:
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Table 7.
Differentiation of LGM-1 Cells Transfected With
Full-Length or Mutant G-CSFR Expression Plasmids as Determined by
FcII/III Receptor Expression After G-CSF Treatment
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|
As a control, the capacity of D+GR95 and LGM GR95 cells to
differentiate in response to retinoic acid (RA) was also analyzed (data
not shown). These G-CSFR overexpressing cells were treated with 7 µmol/L RA for 3 days, and their ability to reduce NBT was examined on
day 3. Like RA-treated D+V cells, D+GR95 cells
exposed to the retinoid exhibited a similar increase in NBT positivity.
In contrast, RA-treated LGM V and LGM GR95 cells did not express NBT
positivity. These results show that overexpression of the G-CSFR in
WEHI-3B D+ cells does not significantly increase the
differentiation response to RA, thereby confirming the specificity of
G-CSF to induce differentiation through occupancy of its receptor.
Effects of expression of mutant G-CSF receptors on cellular
differentiation.
Phycoerythrin-conjugated rhG-CSF binding has shown that the truncated
receptors are on the cell surface and are capable of specifically
binding rhG-CSF (Tables 1 and 2). Thus, it was appropriate to evaluate
the effects of truncation of the G-CSFR cytoplasmic domain on cellular
differentiation, thereby identifying regions that are critical for the
regulation of the maturation process.
Like rhG-CSF-treated D+GR95 cells, D+GR-A,
D+GR-B, D+GR-C, and D+GR-D cells
exposed to the cytokine all showed higher capacities to reduce NBT than
treated D+V cells. Thus, after treatment with 10 ng/mL of
rhG-CSF, D+GR-A, D+GR-B, D+GR-C,
and D+GR-D cells exhibited 6.1-, 3.6-, 6.2-, and 5.1-fold,
respectively, higher NBT positivity than D+V cells (Table
3). The increase in the ability to reduce NBT was only observed with
D+GR-A, D+GR-B, D+GR-C, and
D+GR-D cells exposed to rhG-CSF, indicating that
overexpression of these forms of the truncated G-CSFR was not
sufficient to initiate maturation and that exposure to the cytokine was
necessary for these cell lines to acquire the differentiated phenotype.
With D+GR-E and D+GR-F cells, the increased
capacity to reduce NBT was lost when 147 or 177 amino acids,
respectively, were deleted from the C-terminus of the G-CSFR
cytoplasmic domain. Both of these cell lines responded to rhG-CSF with
NBT positivities lower than that of the D+GR95 cells (Table
3), suggesting that expression of the G-CSFR with a truncation of 147 or 177 amino acids from the cytoplasmic domain may have a dominant
negative effect on the ability of the endogenous full-length receptor
to reduce NBT in response to treatment with rhG-CSF.
To further study the differentiation capacity of cells transfected with
mutant G-CSF receptors after treatment with rhG-CSF, immunofluorescent
staining of cell-surface Mac-I antigen and FcII/III receptor was
performed. Flow cytometric analysis of the cell-surface expression of
these functional markers of differentiation in both WEHI-3B
D+ (which express a low level of endogenous G-CSFR) and
LGM-1 (which do not express G-CSFR) cells showed that induction of each
of these markers may be regulated by a common membrane proximal region. The transfected WEHI-3B D+ cell lines, which showed high
NBT positivity, also responded with high surface expression of Mac-I
antigen after rhG-CSF treatment. Like rhG-CSF-treated
D+GR95 cells, cytokine-treated D+GR-A,
D+GR-B, D+GR-C, and D+GR-D cells
all exhibited increased levels of Mac-I antigen expression, with
relative fluorescence intensity values of 1.38, 1.30, 2.02, and 1.31, respectively (Table 4). The high expression level of the Mac-I antigen
was also lost when 147 or 177 amino acids were deleted from the
C-terminus of the G-CSFR cytoplasmic domain. This loss of response,
observed in D+GR-E and D+GR-F cells, resulted
in levels of Mac-I antigen expression lower than that of the
D+GR95 cells after rhG-CSF treatment (Table 4), suggesting
that expression of G-CSF receptors with truncations of 147 or 177 amino acids from the cytoplasmic domain may also have a dominant negative effect on the capacity of endogenous full-length receptor to express Mac-I antigen in response to rhG-CSF treatment. In contrast, like rhG-CSF-treated LGM GR95 cells, cytokine-treated LGM GR-A, LGM GR-B,
and LGM GR-C cells all exhibited high levels of Mac-I antigen expression, with relative fluorescence intensity values of 1.77, 1.55, and 1.79, respectively (Table 6). The high expression level of the
Mac-I antigen was lost when 117, 147, or 177 amino acids were deleted
from the C-terminus of the G-CSFR cytoplasmic domain (Table 6). By
comparison, between the expression level of the Mac-I antigen in
transfected WEHI-3B D+ cells and that in transfected LGM-1
cells after rhG-CSF treatment, the membrane proximal region between
amino acids 70 and 100 was determined to be critical for the induction
of Mac-I antigen expression.
The relatively high surface expression of the FcII/III receptor,
which was observed in rhG-CSF-treated D+GR95 cells, was
lost when 27, 59, 117, 147, or 177 amino acids were deleted from the
C-terminus of the G-CSFR cytoplasmic domain. This loss of response,
observed in D+GR-A, D+GR-B, D+GR-D,
D+GR-E, and D+GR-F cells, led to levels of
FcII/III receptor expression lower than that of the
D+GR95 cells after rhG-CSF treatment (Table 5). Only
cytokine-treated D+GR-C cells, which expressed G-CSF
receptors with a truncation of 87 amino acids from the C-terminus,
exhibited surface expression of FcII/III receptor at a high level
similar to that seen in treated D+GR95 cells (Table 5). In
contrast, the relatively high surface expression of the FcII/III
receptor, which was observed in rhG-CSF-treated LGM GR95 cells, was
lost only when 117, 147, or 177 amino acids were deleted from the
C-terminus of the G-CSFR cytoplasmic domain (Table 7). The
cytokine-treated LGM-1 cells, which expressed G-CSF receptors with a
truncation of 27, 59, or 87 amino acids from the C-terminus, exhibited
surface expression of FcII/III receptor at a high level similar to
that seen in treated LGM GR95 cells. By comparison between the
expression level of the FcII/III receptor in transfected WEHI-3B
D+ cells and that in transfected LGM-1 cells after rhG-CSF
treatment, like that occurring with the Mac-I antigen, the membrane
proximal region between amino acids 70 and 100 was determined to be
critical for the activation of FcII/III receptor expression in both
cell lines.
| |
DISCUSSION |
G-CSF has been reported to be an inducer of the differentiation of
WEHI-3B D+ cells, assayed by culture in semisolid
agar.14,15 Under these conditions, granulocytic colonies
were formed and the clonogenic activity of the cells was greatly
reduced by G-CSF. In contrast, Böhmer and Burgess16
have reported that G-CSF is not an inducer of the differentiation of
WEHI-3B D+ cells but is required for the survival of mature
progeny in suspension culture at low cell density. Our laboratory has
also found that G-CSF is not an exceedingly effective initiator of the
maturation of WEHI-3B D+ cells, and only a small percentage
of cells exhibited a differentiated phenotype when exposed to a
concentration of the cytokine as high as 50 ng/mL in suspension
culture.17 In contrast to WEHI-3B D+ cells that
express a low level of the G-CSFR, LGM-1 cells do not express the
G-CSFR. However, exogenous expression of the G-CSFR makes LGM-1 cells
respond to G-CSF by both proliferation and morphological differentiation into neutrophils.3 To gain a further
understanding of the effects of G-CSF, the role of its receptor in the
maturation process has been examined. This was accomplished by the
introduction of full-length as well as of truncated G-CSFR cDNAs into
WEHI-3B D+ and LGM-1 cells and evaluation of their effects
on differentiation.
The high level of expression of exogenously introduced full-length as
well as of truncated G-CSFR cDNAs was confirmed by Western blotting and
phycoerythrin-conjugated rhG-CSF binding. Western blotting showed that
the full-length and truncated G-CSFR proteins had the correct molecular
sizes. Furthermore, phycoerythrin-conjugated rhG-CSF binding
demonstrated that both the full-length and truncated G-CSFR proteins
were expressed on the cell surface and were capable of specifically
binding rhG-CSF. These results indicated that truncation of 177 amino
acids from the G-CSFR cytoplasmic domain did not prevent cells from
processing the mutant receptors and expressing these receptors on the
cell surface in a manner that did not decrease their capacities to
specifically bind rhG-CSF.
WEHI-3B D+ and LGM-1 cells that overexpressed the
full-length G-CSFR exhibited a significant degree of differentiation in
response to rhG-CSF. Thus, rhG-CSF-treated D+GR95 cells
showed 7.8-fold higher NBT positivity than treated D+V
cells, and the cell-surface expression of the Mac-I antigen and the
FcII/III receptor was 1.2-fold and 1.4-fold higher, respectively, in
cytokine-treated D+GR95 cells than in treated
D+V cells. Furthermore, the cell-surface expression of the
Mac-I antigen and the FcII/III receptor was 1.5-fold and 2.1-fold
higher, respectively, in cytokine-treated LGM GR95 cells than in
treated LGM V cells. These results support the significant role of
G-CSF and its receptor in the initiation of the differentiation process.
Introduction of full-length G-CSFR into various cell lines generates
cell lines with different capacities to differentiate in response to
G-CSF. WEHI-3B D+ cells that express the G-CSFR
(D+GR95) respond to G-CSF by increased expression of Mac-I
antigen and FcII/III receptor as well as higher NBT positivity. In
contrast, LGM-1 cells that express the G-CSFR (LGM GR95) show only high expression of Mac-I antigen and FcII/III receptor but no NBT positivity after G-CSF treatment. These findings indicate that expression of G-CSFR in WEHI-3B D+ and LGM-1 cells leads to
different mature phenotypes after induction of differentiation by
G-CSF. Similar results have also been obtained from previous studies.
Thus, murine myeloid FDC-P1 cells expressing the G-CSFR respond to
G-CSF by induction of the myeloperoxidase gene.4 In
contrast, the pro-B cell line BAF-B03 that expresses the G-CSFR does
not show myeloperoxidase gene induction after G-CSF
treatment.4 Therefore, the interaction of G-CSF with its
receptor in different cell lines appears to only activate the signaling
pathways that exist in the cell line.
To identify the regions of the cytoplasmic domain of the G-CSFR that
are involved in the differentiation process, the cytoplasmic domain of
the G-CSFR cDNA was systematically truncated at approximately every 30 amino acids from the C-terminus and these truncated G-CSFR cDNAs were
introduced into WEHI-3B D+ and LGM-1 cells. The G-CSFR
cytoplasmic domain is thought to be involved in the initiation of
signaling events from the receptor. Through expression of different
G-CSFR forms in various cells, distinct regions of the G-CSFR
cytoplasmic domain have been suggested to be critical for transduction
of neutrophilic maturation signals and gene induction.3-5
The 98 carboxy-terminal amino acids of the G-CSFR cytoplasmic domain
appeared to be involved in the development of murine L-GM myeloid cells
into morphologically mature granulocytes when cultured with
G-CSF.3 The induction of the myeloperoxidase gene in
IL-3-dependent mouse myeloid precursor FDC-P1 cells required both the
amino- and carboxy-terminal regions of the cytoplasmic domain.4 The membrane proximal region between amino acids
57 and 96 of the cytoplasmic domain was shown to be capable of inducing acute-phase plasma protein gene expression in human hepatoma cell lines.5 Our results demonstrate that functional
differentiation signals (induction of the abilities to reduce NBT, to
express Mac-I antigen, and/or to express the FcII/III receptor) may
be mediated by distinct regions within the G-CSFR cytoplasmic domain. The functional regions that we have identified to be critical to the
process of terminal differentiation are not precisely the same as those
regions suggested by previous studies.3-5
Located within the membrane proximal region of the cytoplasmic domain
are cytokine receptor superfamily homology regions boxes 1 and
218,19 that are required for G-CSF-induced tyrosine
phosphorylation and activation of the catalytic activity of the JAK
protein tyrosine kinases.20,21 After their activation, the
JAK kinases induce tyrosine phosphorylation of the G-CSFR, as well as
of the STAT family of cytoplasmic transcription factors. Activation of
JAK kinases appears to be necessary for tyrosine phosphorylation of cytokine receptors and STAT proteins.6,7,9,22 The
cytoplasmic domain of the G-CSFR contains four tyrosine residues. In
the murine G-CSFR, all four tyrosine residues (Tyr703,
Tyr728, Tyr743, and Tyr763) are
phosphorylated upon stimulation with G-CSF, and phosphorylation of
Tyr703 is the most prominent.23,24 The
phosphotyrosine residues on the G-CSFR create potential docking sites
for recruitment of signaling proteins that contain SH2 domains, such as
STATs.25,26 The G-CSFR may thus regulate the process of
differentiation by selectively activating various functional markers of
mature neutrophils through association with different signaling molecules.
WEHI-3B D+ and LGM-1 cells that expressed high levels of
the truncated G-CSFRs were examined for their capacities to enter a
differentiation pathway under identical conditions as those used for
WEHI-3B D+ and LGM-1 cells that overexpressed the
full-length G-CSFR. The differentiation capacities of WEHI-3B
D+ and LGM-1 cells expressing truncated G-CSF receptors
were determined in multiple independent experiments. Thus, in
transfected WEHI-3B D+ cells, NBT positivity was determined
in three independent assays, and the expression of Mac-I antigen as
well as FcII/III receptor was analyzed in two independent
experiments. In transfected LGM-1 cells, expression of Mac-I antigen as
well as FcII/III receptor was measured every 3 days for a period of
10 days or more, and the differentiation capacities of each of the
transfected cell lines showed the same pattern of differentiation from
day to day.
WEHI-3B D+ and LGM-1 cells that expressed the mutant G-CSFR
proteins with deletions of 147 or 177 amino acids from the C-terminus exhibited differentiation capacities similar to or lower than those
observed with vector-transfected WEHI-3B D+ and LGM-1
cells. These findings indicate that the membrane proximal region, which
contains boxes 1 and 2, is important for the activation of all three
functional markers of mature neutrophils (Fig
5). Boxes 1 and 2 were expected to be
critical because G-CSF-induced tyrosine phosphorylation and activation
of the catalytic activity of the JAK kinases are necessary for the
initiation of signaling events from the receptor. The dominant negative
effect exerted in WEHI-3B D+ cells by these mutant G-CSFR
proteins with deletions of 147 or 177 amino acids from the C-terminus
may be due to their disruption of the homodimerization of endogenous
full-length receptors and thereby the effective initiation of signaling
pathways that activate the differentiation markers. Comparison of the
sequences necessary for the activation of the capacities to express
Mac-I antigen and FcII/III receptor in WEHI-3B D+ and
LGM-1 cells indicates that additional sequences in the membrane proximal region between amino acids 70 and 100 are required for the
induction of these two functional markers. These additional sequences
are necessary for the activation of the ability to express Mac-I
antigen in LGM-1 cells because LGM-1 cells do not express endogenous
full-length G-CSFR that may help compensate for the effect exerted by
exogenously expressed truncated receptors. Whether additional sequences
in the amino-terminal region are required for the induction of the
ability to reduce NBT is unclear because WEHI-3B D+ cells
express a low level of endogenous G-CSFR and transfected LGM-1 cells do
not show NBT positivity after rhG-CSF treatment.
View larger version (27K):
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| Fig 5.
Summary of the effects of the expression of full-length
and mutant G-CSFRs on the capacity to differentiate in response to
G-CSF in WEHI-3B D+ or LGM-1 (indicated by brackets)
cells. The critical regions of the G-CSFR cytoplasmic domain that are
involved in the induction of three functional markers of mature
neutrophils after G-CSF treatment are illustrated. The mutant G-CSFRs
with deletions of 27, 59, 87, 117, 147, and 177 amino acids from the
C-terminus of the cytoplasmic domain are designated by the letters of
the alphabet A, B, C, D, E, and F, respectively. The cytokine receptor
superfamily homology regions (Box 1, Box 2, and Box 3) are represented
by boxes, and the four tyrosine residues (Tyr703,
Tyr728, Tyr743, and Tyr763) in the
murine G-CSFR cytoplasmic domain are indicated by "Y."
|
|
Examination of the sequences of the mutant receptors suggests that
induction of the capacities to express Mac-I antigen and FcII/III
receptor may be dependent on tyrosine phosphorylation of the G-CSFR
(Fig 5). Thus, the capacities of transfected LGM-1 cells to express
Mac-I antigen and FcII/III receptor were lost when the membrane
proximal region containing Tyr703 was deleted. Signaling
proteins, such as STATs, may interact with the region containing
Tyr703 and subsequently increase the sensitivity of the
transfected cells to rhG-CSF through activation of functional
differentiation markers and reduction in growth rate as observed in
cells expressing mutant G-CSF receptors with a truncation of 87 amino
acids. In contrast, induction of the capacity to reduce NBT in
transfected WEHI-3B D+ cells does not seem to be dependent
on tyrosine phosphorylation (Fig 5). However, because WEHI-3B
D+ cells express a low level of endogenous G-CSFR and
transfected LGM-1 cells do not show NBT positivity after rhG-CSF
treatment, whether tyrosine phosphorylation is necessary for the
induction of the capacity to reduce NBT is unclear.
Like induction of Mac-I antigen and FcII/III receptor expression,
induction of myeloperoxidase gene expression has been reported to
require tyrosine phosphorylation of the G-CSF receptor.24 Tyrosine to phenylalanine substitutions of Tyr703 or
Tyr728 resulted in receptors that were unable to induce
myeloperoxidase gene expression in murine myeloid LGM-1
cells.24 In contrast to activation of Mac-I antigen and
FcII/III receptor expression that requires only sequences in the
amino-terminal region, induction of myeloperoxidase gene expression
requires additional sequences in the carboxy-terminal
region.4 FDC-P1 cells that expressed mutant G-CSFRs with
deletions of 58, 111, 159, and 182 amino acids from the C-terminal did
not induce the myeloperoxidase gene.4 However, FDC-P1 cells
that expressed a mutant G-CSFR with a truncation of 88 amino acids
partially induced the myeloperoxidase gene.4 Similarly,
WEHI-3B D+ cells that expressed a mutant receptor with a
truncation of 87 amino acids showed increased sensitivity to rhG-CSF
through induction of a high level of Mac-I antigen or FcII/III
receptor expression and reduction in growth rate. Therefore, these
findings imply that differentiation signals are not mediated by a
single region but by distinct regions within the receptor cytoplasmic domain.
The three measured functional markers of mature neutrophils require
distinct regions of the G-CSFR cytoplasmic domain for efficient
induction (Fig 5). The membrane proximal region, which contains the
homology sequences of boxes 1 and 2, is important for the activation of
all three functional markers of mature neutrophils. Induction of the
capacities to express Mac-I antigen or FcII/III receptor also
requires additional sequences in the membrane proximal region between
amino acids 70 and 100 and may be dependent on the phosphorylation of
Tyr703. Therefore, these findings indicate that distinct
sequences within the amino-terminal region of the cytoplasmic domain of
the receptor are sufficient to induce these functional markers of
differentiation and receptor tyrosine phosphorylation may be necessary.
The G-CSFR may regulate the attainment of different stages of
differentiation, as measured by the expression of various functional
markers, through association of distinct portions of its cytoplasmic
domain with different signal transducing molecules.
| |
ACKNOWLEDGMENT |
We thank Jianming Li, Rick A. Finch, and Rocco Carbone for their
helpful discussions and technical assistance.
| |
FOOTNOTES |
Submitted May 11, 1998; accepted February 3, 1999.
Supported by US Public Health Service Grant No. CA-02817 from the
National Cancer Institute.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Alan C. Sartorelli, PhD,
Department of Pharmacology, Yale University School of Medicine, 333 Cedar St, New Haven, CT 06520; e-mail: ALAN.SARTORELLI{at}YALE.EDU.
| |
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ABSTRACT
INTRODUCTION