(Received for publication, May 8, 1995; and in revised form, August 10, 1995)
From the
The 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.
Granulocyte-macrophage colony-stimulating factor (GM-CSF) ()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 (K
1-10 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) .
The 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.
Figure 1:
Tunicamycin inhibits GM-CSF binding
without affecting 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.
Figure 3:
Tunicamycin leads to cell surface
expression of N-unglycosylated 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.
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 () 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).
To examine whether the N-unglycosylated 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.
Because the plasma membrane preparation in the above
experiments may contain intracellular membrane fractions, we studied
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.
Figure 4:
Tunicamycin completely blocks GM-CSF
binding in transfected COS cells. A, GM-CSF binding to
mock-transfected (),
-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.
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
subunit by N-glycosidase F as assessed by immunoblotting
using the anti-
subunit antiserum, GM-CSF binding was decreased
(data not shown).
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 subunit, since
subunit expression on
the cell surface was not affected by tunicamycin.
We investigated
the role of N-glycosylation on the expression and function of
the isolated 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.
It is uncertain why N-glycosylation of
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.
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 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.
GM-CSF binding in - 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.
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 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.