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(Received for publication, May 8, 1995; and in revised form, August 10, 1995) From the
The
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ( The
Figure 1:
Tunicamycin inhibits GM-CSF binding
without affecting
Figure 3:
Tunicamycin leads to cell surface
expression of N-unglycosylated
To correlate the effect of tunicamycin on
GM-CSF binding with receptor function, we assessed glucose uptake and
protein tyrosine phosphorylation in HL60(eos). In the absence of
tunicamycin, GM-CSF stimulated deoxyglucose uptake in a dose-dependent
manner (Fig. 2A). An effect on deoxyglucose uptake was
first seen at 10 pM with maximal stimulation (1.9-fold) seen
at 10 nM GM-CSF. This dose response is consistent with our
previous observations(7, 12) . In tunicamycin-treated
cells, deoxyglucose uptake was stimulated only at concentrations of
GM-CSF greater than 1 nM (Fig. 2A). At 10
nM GM-CSF, deoxyglucose uptake was stimulated only 1.3-fold.
In untreated HL-60(eos), GM-CSF signaling resulted in tyrosine
phosphorylation of several proteins migrating at 75, 60, 58, 55, and 42
kDa (Fig. 2B and data not shown)(9) . The p42
protein comigrated with the p42 microtubule-associated protein kinase.
Induction of protein phosphorylation by GM-CSF was dose dependent and
was stimulated by GM-CSF concentrations as low as 10-30
pM. Treatment of HL-60(eos) with tunicamycin completely
blocked GM-CSF-induced tyrosine phosphorylation, suggesting that N-glycosylation of the receptor is necessary for intracellular
signaling.
Figure 2:
Tunicamycin inhibits GM-CSF signaling in
HL-60(eos) cells. A, dose response of GM-CSF-stimulated
deoxyglucose uptake. Cells were left untreated (
To examine whether the N-unglycosylated Because the plasma membrane preparation in the above
experiments may contain intracellular membrane fractions, we studied
Figure 4:
Tunicamycin completely blocks GM-CSF
binding in transfected COS cells. A, GM-CSF binding to
mock-transfected (
We sought to verify the
results with tunicamycin using N-glycosidase F digestion.
Although only a small amount of N-glycan was removed from the
N-glycosylation is a cotranslational modification
found in most cell surface proteins, but the precise function of the
carbohydrate on these proteins is not well understood(21) .
Evidence suggests that N-glycosylation may be required for
protein folding and
trafficking(22, 23, 24, 25) , ligand
binding(26, 27, 28, 29, 30) ,
or signaling (31, 32, 33, 34) . N-Glycosylation occurs on asparagine residues in the consensus
sequence Asn-X-Ser/Thr, where X is any amino acid
except proline or aspartic acid. Such glycosylation is initiated in the
endoplasmic reticulum with an oligosaccharide core linked to asparagine
via dolichol phosphate(35) . The antibiotic tunicamycin
inhibits the function of dolichol phosphate as an acceptor of N-acetyl glucosamine and thereby prevents N-glycosylation(35) . We examined the role of N-glycosylation in the GM-CSF receptor and found that
tunicamycin inhibited GM-CSF binding by decreasing the number of
binding sites 3-fold and the affinity 2-fold in HL-60(eos) cells
expressing the high affinity receptor. GM-CSF signal transduction as
measured by glucose uptake and protein tyrosine phosphorylation was
blocked by tunicamycin. In a previous report(36) , it was
concluded that tunicamycin treatment resulted in decreased cell surface
expression of the GM-CSF receptor proteins as evidenced by decreased
GM-CSF binding sites. Our data indicate that binding inhibition and
lack of ligand-induced signaling did not result from abrogation of cell
surface expression of We investigated
the role of N-glycosylation on the expression and function of
the isolated It is uncertain why N-glycosylation of Tunicamycin reduced the number of GM-CSF binding sites 3-fold and
decreased the receptor affinity 2-fold in HL-60(eos). Based on our data
in COS cells indicating that N-unglycosylated GM-CSF binding in GM-CSF is a glycoprotein in which glycosylation is
not required for its biologic activity. Unglycosylated GM-CSF produced
by E. coli is fully active(37) . On the other hand, we
have demonstrated that GM-CSF receptor
Volume 270,
Number 41,
Issue of October 13, 1995 pp. 24580-24584
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Is Essential for Ligand Binding and Signal Transduction (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
subunit of the receptor for human
granulocyte-macrophage colony-stimulating factor (GM-CSF) is a
glycoprotein containing 11 potential N-glycosylation sites in
the extracellular domain. We examined the role of N-glycosylation on subunit membrane localization and
function. Tunicamycin, an N-glycosylation inhibitor, markedly
inhibited GM-CSF binding, GM-CSF-induced deoxyglucose uptake, and
protein tyrosine phosphorylation in HL-60(eos) cells but did not affect
cell surface expression of the subunit as detected by an
anti- subunit monoclonal antibody. In COS cells expressing the
subunit and treated with tunicamycin, N-unglycosylated
subunit was expressed and transported to the cell surface but was
not capable of binding GM-CSF. High affinity binding in COS cells
expressing both and 
subunits was also blocked by
tunicamycin treatment. These studies indicate that N-linked
oligosaccharides are essential for subunit ligand binding and
signaling by the human GM-CSF receptor.
)is a hematopoietic growth factor that promotes the
proliferation and maturation of myeloid progenitor cells and enhances
the function of mature granulocytes and mononuclear
phagocytes(1) . GM-CSF exerts its effect via its cognate
receptor on the cell surface. The human GM-CSF receptor is composed of
an subunit that binds GM-CSF with low affinity (K1-10 nM) (2, 3, 4) and a
subunit that has no
intrinsic GM-CSF binding capacity but associates with the subunit
to form a high affinity receptor with a K of 10-50 pM(5, 6) . The high
affinity receptor signals for proliferation and functional activation
via protein phosphorylation
pathways(7, 8, 9, 10, 11) .
We recently found that the isolated
subunit signals for glucose
uptake through a protein phosphorylation-independent
pathway(12) . subunit of the human GM-CSF receptor
is an 84-kDa glycoprotein that readily binds lectins and has 11
potential N-glycosylation sites in the cDNA-deduced amino acid
sequence, all located in the extracellular domain(13) . The
calculated molecular mass of the subunit based on amino acid
sequence is 40 kDa. The difference between the apparent and calculated
molecular mass (44 kDa) is in part due to N-glycosylation. It
is not known what role, if any, the N-linked carbohydrates
present in the extracellular domain play in the function of the
subunit. We used the N-glycosylation inhibitor tunicamycin to
probe the role of N-glycosylation in the function and surface
expression of the GM-CSF receptor in the HL-60(eos) cell line, which
expresses a high affinity GM-CSF receptor, and in COS cells transfected
with subunit cDNA alone or both and subunit cDNA. Our
results indicate that N-glycosylation of the subunit is
essential for ligand binding and signaling by the human GM-CSF
receptor.
Cell Culture
A previously described
eosinophilic subline of HL-60, HL-60(eos)(14, 15) ,
was cultured in Iscove's modified Dulbecco's medium (IMDM)
(pH 7.6) supplemented with 10% heat-inactivated fetal bovine serum
(FBS), 0.5 mM -butyric acid, 1% glutamine, and
antibiotics. COS cells were cultured in IMDM supplemented with 10% FBS,
1% glutamine, and antibiotics.
GM-CSF Binding
COS cells were detached
from culture dishes by incubation with EDTA and chondroitin
sulfate(13) . HL-60(eos) or suspended COS cells were incubated
with increasing concentrations of I-labeled GM-CSF
(DuPont NEN) (13) in IMDM containing 0.3% bovine serum albumin
overnight at 4 °C, and nonspecific binding was determined by
addition to the incubation of 1.5 µM unlabeled human
recombinant GM-CSF produced in Escherichia coli (a gift from
Amgen, Inc., Thousand Oaks, CA). Cells were washed by centrifugation
through FBS and counted in a
spectrometer to determine GM-CSF
binding.
Deoxyglucose Uptake
HL-60(eos) cells were
glucose starved for 2 h, treated with GM-CSF for 1 h, and incubated
with 2 µCi/ml [1,2-
H]deoxyglucose (DuPont
NEN) and unlabeled 200 µM deoxyglucose (Sigma) for 10 min.
Cells were washed, lysed, and counted by liquid
scintillation(16, 17) .Phosphotyrosine Immunoblotting
HL-60(eos)
cells were serum starved for 24 h, incubated with 0.01-10 nM GM-CSF for 5 min in IMDM containing 0.3% bovine serum albumin,
lysed by ultrasonication, and immunoblotted with an antiphosphotyrosine
monoclonal antibody (Upstate Biotechnology Inc.) using enhanced
chemiluminescence (ECL) reagents (Amersham, Buckinghamshire, United
Kingdom)(12) .GM-CSF Receptor Expression in COS
Cells
Complementary DNA encoding the or subunit
subcloned into the eukaryotic expression vector PMX (a gift from
Genetics Institute, Inc., Boston) was transfected into COS cells using
a DEAE-dextran method(18) . Transfected COS cells were cultured
in Dulbecco's Modified Eagle's Medium supplemented with 10%
FBS, 1% glutamine, and antibiotics with or without varying
concentrations of tunicamycin (Sigma) for 2 days. Medium was replaced
after 24 h.Immunoblotting of the
Whole
cell lysates obtained by ultrasonication and membrane proteins obtained
by ultracentrifugation were separated on 10% SDS-acrylamide gels and
immunoblotted using the ECL protocol with a polyclonal antibody we
developed against a bacterial fusion protein composing the C terminus
of the Subunit subunit.Immunologic Detection of Cell Surface
COS cells were detached with EDTA and chondroitin
sulfate and incubated with varying amounts of monoclonal anti- Subunit
subunit antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h
followed by incubation with I-protein A (DuPont NEN) for
1 h at 23 °C. Cells were washed by centrifugation through FBS and
counted in a
spectrometer.
Tunicamycin Inhibits GM-CSF Binding and Signaling
without Affecting
To determine whether N-glycosylation plays a
role in the function of the GM-CSF receptor, we treated HL-60(eos)
cells with tunicamycin for 24 h and assessed GM-CSF binding.
Tunicamycin treatment inhibited GM-CSF binding in a dose-dependent
manner with maximal inhibition (85%) seen at 3 µg/ml (Fig. 1A). At 3 µg/ml tunicamycin, GM-CSF binding
was markedly inhibited over the entire range of GM-CSF concentrations
examined (Fig. 1B). Scatchard analysis of GM-CSF
binding in untreated HL-60(eos) revealed a single class of binding
sites with an affinity of 230 pM and a density of 650
sites/cell (Fig. 1C). Tunicamycin treatment induced a
2-fold decrease in binding affinity to 470 pM and a 3-fold
decrease in the number of binding sites to 160 sites/cell. To exclude
the possibility that inhibition of ligand binding by tunicamycin
treatment was a result of decreased Subunit Expression in HL-60(eos)
Cells subunit expression on the
cell surface, we assessed the cell surface expression of protein
with an anti- subunit monoclonal antibody. This is a blocking
antibody that reacts with an extracellular epitope in the
subunit, and therefore it can be used to determine if the protein is
located on the cell surface. Absence of or decreased binding of the
antibody to the cell surface of tunicamycin-treated cells must be
interpreted with caution due to the monoclonal nature of the antibody.
Tunicamycin did not alter the binding of the monoclonal anti-
antibody to the cell surface (Fig. 1D). Control
experiments demonstrated absence of antibody binding to the surface of
cells that do not express GM-CSF receptor subunit (data not
shown, see also Fig. 3, C and D). The data can
be interpreted as indicating that tunicamycin treatment, although
greatly affecting GM-CSF binding, did not alter the level of cell
surface subunit.
subunit expression in HL-60(eos) cells. A, dose response of tunicamycin effect on GM-CSF binding.
Cells (1 
10
) were treated with the indicated
concentrations of tunicamycin for 24 h, and GM-CSF (0.8 nM)
binding was measured. B, GM-CSF binding curve ranging from
0.02 to 15 nM GM-CSF. HL-60(eos) cells (1
10) were left untreated (
) or incubated with 3
µg/ml tunicamycin (
) for 24 h. C, Scatchard analysis
of data in B. D, cell surface binding of increasing
concentrations of anti- subunit monoclonal antibody. Symbols are as in panelB.
subunit in transfected
COS cells. A, effect of increasing concentrations of
tunicamycin on the apparent M
of subunit.
Cells were incubated for 2 days with or without tunicamycin, and total
cell lysates from mock- or -transfected COS cells were
immunoblotted with anti- subunit antiserum. B, effect of
tunicamycin on the -subunit content of COS cell plasma membranes.
The anti- subunit antiserum was used to immunoblot total cell
lysates, and membrane preparations of COS cells mock or subunit
cDNA transfected were treated with or without 0.1 µg/ml tunicamycin
for 2 days. M standards (
10) for both panelsA and B are marked. C, dose-dependent binding of monoclonal
anti-
antibody to the surface of unfixed COS cells. ,
mock-transfected COS cells; , untreated -transfected COS
cells; 
, -transfected COS cells treated with 0.3 µg/ml
tunicamycin. D, time course of cell surface subunit
expression as detected by monoclonal anti- antibody (0.2
µg/ml) binding. Symbols are as in panelC.
) or pretreated
with 3 µg/ml tunicamycin () for 24 h. B, dose
response of GM-CSF-induced protein tyrosine phosphorylation. Cells were
left untreated or pretreated with 3 µg/ml tunicamycin for 24 h and
incubated with GM-CSF for 5 min; total cell lysates were then
immunoblotted with an anti-phosphotyrosine antibody. M standards ( 10) are marked. The arrows indicate positions of the major tyrosine
phosphorylation products in untreated cells. microtubule-associated
protein kinase was positioned by reprobing the blot with an
anti-microtubule-associated protein kinase antibody (not
shown).
Tunicamycin Treatment Causes Cell Surface Expression
of N-Unglycosylated
Since HL-60(eos) cells express a small number of high
affinity GM-CSF receptors composed of both Subunit in Transfected COS
Cells and subunits, we
sought to distinguish the effects of tunicamycin on the two subunits.
Using a COS cell expression system, we generated high level selective
expression of isolated subunit without the subunit. Whole
cell lysates of -transfected COS cells revealed a spectrum of
anti- subunit immunoreactive bands ranging from 40 to 90 kDa (Fig. 3A). No anti- immunoreactive bands were
observed in mock-transfected cells. Treatment of the cells with
increasing concentrations of tunicamycin (0.01-30 µg/ml)
decreased the amount of anti- immunoreactive proteins of higher
molecular mass (50-90 kDa) and increased those of lower molecular
mass (42 and 44 kDa). The simplest interpretation of these results is
that the 42-kDa band corresponds to the totally N-unglycosylated subunit. The 44-kDa band does not
simply reflect the presence of residual N-glycosylation
because the relative abundance of both bands did not change in cells
treated with concentrations of tunicamycin from 0.1 to 30 µg/ml (Fig. 3A). It may result from other post-translational
modifications such as O-glycosylation or
phosphorylation(19) . None of the large anti-
immunoreactive proteins were present at tunicamycin concentrations of
0.1 µg/ml or higher, and only two bands of 42 and 44 kDa were
present in these samples. protein, synthesized in
tunicamycin-treated cells, was able to be transported to the cell
surface, membrane fractions enriched in plasma membrane were prepared
and immunoblotted with anti- subunit serum. The membrane fraction
from untreated subunit-transfected COS cells revealed a spectrum
of anti- immunoreactive proteins ranging from 50 to 80 kDa (Fig. 3B, lane5). Membrane
anti- immunoreactive proteins detected were only smaller species
(40-46 kDa) (lane6) in tunicamycin-treated
cells. No membrane anti- immunoreactivity was detected in
mock-transfected cells (lane4). The two major
immunoreactive 70-kDa species present in total cell lysates (lanes1-3) but not in membrane fractions (lanes4-6) may be cytosolic proteins nonspecifically
bound by the antiserum, and their intensity of staining varied in
different preparations. Since tunicamycin treatment resulted in a
decreased size of the subunits from 47-90 kDa to
40-46 kDa, we consider the former to be N-glycosylated
forms and the latter N-unglycosylated forms. Densitometric
analysis of the blot revealed that almost all of the anti-
immunoreactive proteins present in total cell lysates of untreated
cells were N-glycosylated. Tunicamycin treatment drastically
decreased the amount of N-glycosylated protein and
increased the N-unglycosylated form. Similarly, all of the
anti- immunoreactive proteins detected in the cell membrane were N-glycosylated; however, more than 95% were N-unglycosylated after tunicamycin treatment. Thus,
tunicamycin profoundly reduced N-glycosylation of the
subunit. subunit expression in unfixed transfected COS cells using a
monoclonal anti- subunit antibody, which allows detection of
surface protein only. 2 days after transfection,
-transfected COS cells bound the antibody in the same manner in
the presence or absence of tunicamycin (Fig. 3C).
Antibody binding increased with increasing concentration of antibody
and was saturated at 0.3 µg/ml. In contrast, mock-transfected cells
did not bind the antibody. Using a sub-saturating concentration (0.2
µg/ml) of antibody allowed us to track the time course of
subunit expression on the cell membrane. In untreated cells, antibody
binding increased at 20 h, reached a maximum at 30 h, and declined
slightly at 40 h after subunit transfection (Fig. 3D). Tunicamycin delayed the appearance of
antibody binding for 10 h; however, antibody binding at 40 h was
equivalent to the maximal binding in untreated cells at 30 and 40 h.
These experiments revealed that despite a slight alteration in the
kinetics of cell surface expression, N-unglycosylated
subunit protein is efficiently transported to the cell surface.Tunicamycin Abolishes GM-CSF Binding in COS Cells
Expressing either Low or High Affinity GM-CSF
Receptor
Having established that tunicamycin inhibited N-glycosylation but did not affect cell surface expression of
subunit in COS cells, we examined whether such N-unglycosylated forms of the subunit expressed on the
COS cell surface bound ligand. Untreated subunit-transfected COS
cells bound I-labeled GM-CSF in a dose-dependent manner
without saturation even up to 8 nM (Fig. 4A).
Cotransfection of both
and subunits gave higher binding
over the entire range of GM-CSF concentrations tested. In contrast,
mock-transfected cells only bound small amounts of GM-CSF and only at
high concentrations. This base-line binding coincides with our previous
finding that COS cells express a low level of monkey GM-CSF receptor
subunit(20) . Scatchard analysis of GM-CSF binding data
from subunit-transfected cells revealed a single class of GM-CSF
binding sites with a K
of 9 nM and 3
10
sites per cell (Fig. 4B). This
low nanomolar dissociation constant is characteristic of the low
affinity GM-CSF receptor(4, 5) . The high number of
binding sites per cell is consistent with our ability to detect the
protein in immunoblots of total cell lysates. Scatchard analysis
of and cotransfected cells (Fig. 4C)
revealed two binding components, one with a dissociation constant (K) of 280 pM and a density of 9
10 sites/cell and the other with a K of 8 nM and a density of 3 10
sites/cell. The binding sites with a K of
280 pM are consistent with the affinity of the GM-CSF binding
sites present in HL-60(eos) and correspond to the high affinity GM-CSF
receptor, although the K is 5-10-fold
greater than the K measured on mature myeloid
cells and their progenitors(7) . Tunicamycin treatment (0.3
µg/ml) completely abolished GM-CSF binding in COS cells transfected
with the subunit alone and in cells cotransfected with both
and subunits (Fig. 4A), suggesting that the N-unglycosylated subunit is unable to bind to ligand
even in the presence of the subunit.
), -transfected (, ), or
- and -cotransfected (
, ) COS cells. Cells were
incubated with (
,
) or without (
, , )
0.3 µg/ml tunicamycin for 2 days. B, Scatchard analysis of
binding data for -transfected COS cells. C, Scatchard
analysis of binding data for - and -cotransfected COS
cells.
subunit by N-glycosidase F as assessed by immunoblotting
using the anti- subunit antiserum, GM-CSF binding was decreased
(data not shown).
subunit, since subunit expression on
the cell surface was not affected by tunicamycin. subunit in COS cells. subunit-transfected COS
cells expressed abundant N-glycosylated subunit on the
cell surface. Tunicamycin reduced the molecular weight of the
subunits as shown by immunoblotting but did not affect their cell
surface expression as measured by immunoblotting and antibody binding.
These results indicate that N-glycosylation does not play a
crucial role in the biosynthesis, stability, or cell surface targeting
of the GM-CSF receptor subunit. Tunicamycin, however, abolished
GM-CSF binding in COS cells transfected with subunit cDNA alone
or cotransfected with both and subunit cDNA. Thus, N-unglycosylated subunits present on the cell surface
were unable to bind GM-CSF. Removal of oligosaccharides from
subunits in the membrane fraction by N-glycosidase F led to a
decrease in both the molecular weight and GM-CSF binding capacity of
the subunit. Taken together, the results with N-glycosylation inhibition and N-endoglycosidase F
digestion in cells either endogenously or exogenously expressing
subunit indicate that N-glycosylation of the subunit is
essential for ligand binding and signaling by the human GM-CSF
receptor.
subunit is essential for ligand binding and signaling. N-Glycosylation may stabilize a conformation required for
binding, or oligosaccharides may themselves be an essential part of the
binding site. Our observations that subunit devoid of N-glycosylation was still expressed on the cell surface and
was recognized by a monoclonal antibody suggest no major conformational
difference between the N-glycosylated and N-unglycosylated forms. Therefore, we propose that N-glycosylation does not function to support the overall
conformation of subunit but rather plays a critical role in
maintaining the appropriate conformation of the binding site. subunit
does not bind GM-CSF, we reason that in HL-60(eos), tunicamycin blocked N-glycosylation and led to synthesis of N-unglycosylated subunits, which did not bind GM-CSF.
The turnover of previously synthesized N-glycosylated
subunit led to a decreased number of GM-CSF binding sites. The 85%
decrease in binding sites caused by tunicamycin over 24 h suggests that
the half-life of subunit on the membrane is less than 10 h. The
decreased affinity in HL-60(eos) resulting from tunicamycin treatment
may be due to partially N-glycosylated forms of subunit.
This concept is supported by our observation that partial removal of N-glycosylation from subunit by N-glycosidase F
led to inhibition but not complete abrogation of GM-CSF binding. - and -cotransfected COS cells was
abolished by tunicamycin, as it was in COS cells transfected with
subunit alone. The subunit is a 120-kDa glycoprotein with an
apparent molecular mass substantially larger than that (96 kDa)
calculated on the basis of the amino acid sequence deduced from its
cDNA(6) . The subunit contains three consensus N-glycosylation sites in the extracellular domain(6) .
Our experiments did not assess the contribution of N-glycosylation of subunit in high affinity GM-CSF
binding because unglycosylation of subunit alone abolished all
GM-CSF binding. subunit requires N-glycosylation for binding and signaling. Given that
glycosylation may vary in pattern and extent among cells of different
types(38, 39, 40, 41) , N-glycosylation of the subunit may serve as a means to
modulate cellular responsiveness to GM-CSF. Variations in binding
affinity of GM-CSF receptors in different cells (42) could be
explained by differences in glycosylation. Finally, the subunits
of interleukin-3 and interleukin-5 receptors, which share a common
subunit with GM-CSF receptor(43, 44) , are also
glycoproteins; the importance of N-glycosylation in GM-CSF
receptor function may have implication for interleukin-3 and
interleukin-5 receptors.
)
We thank Rong-Hua Zhang for excellent technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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