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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 95 No. 11 (June 1), 2000:
pp. 3357-3362
HEMATOPOIESIS
High-affinity binding to the GM-CSF receptor requires intact
N-glycosylation sites in the extracellular domain of the  subunit
Linghao Niu,
Mark L. Heaney,
Juan Carlos Vera, and
David W. Golde
From the Program in Molecular Pharmacology and Therapeutics and
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New
York, NY.
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Abstract |
The human granulocyte-macrophage colony-stimulating factor (GM-CSF)
receptor consists of 2 glycoprotein subunits, GMR and GMR . GMR
in isolation binds to GM-CSF with low affinity. GMR does not bind
GM-CSF by itself, but forms a high-affinity receptor in association
with GMR. Previously, it was found that N-glycosylation of GMR is
essential for ligand binding. The present study investigated the role
of N-glycosylation of the subunit on GM-CSF receptor function.
GMR has 3 potential N-glycosylation sites in the extracellular domain at Asn58, Asn191, and Asn346. Single mutants and triple mutants
were constructed, converting asparagine in the target sites to aspartic
acid or alanine. A single mutation at any of the 3 consensus
N-glycosylation sites abolished high-affinity GM-CSF binding in
transfected COS cells. Immunofluorescence and subcellular fractionation
studies demonstrated that all of the GMR mutants were faithfully
expressed on the cell surface. Reduction of apparent molecular weight
of the triple mutant proteins was consistent with loss of
N-glycosylation. Intact N-glycosylation sites of GMR in the
extracellular domain are not required for cell surface targeting but
are essential for high-affinity GM-CSF binding.
(Blood. 2000;95:3357-3362)
© 2000 by The American Society of Hematology.
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Introduction |
Human granulocyte-macrophage colony-stimulating factor
(GM-CSF) is a hematopoietic cytokine that stimulates the proliferation of myeloid precursor cells and enhances the function of neutrophils, eosinophils, and monocytes.1 GM-CSF is secreted by a
variety of tissue types and its elaboration is regulated by mediators of inflammation.2 The biologic functions of GM-CSF are
initiated by interaction with its cell surface receptor that consists
of 2 subunits,  (GMR) and (GMR). The subunit is ligand
specific and binds to GM-CSF with low affinity (equilibrium
dissociation constant, kd = 1-10
nmol/L).3-6 The subunit does not bind
GM-CSF by itself, but forms a high-affinity receptor
(kd = 20-100 pmol/L) with the subunit.5,7-9 Both and subunits are members of a
cytokine receptor superfamily characterized by 4 spatially conserved
cysteine residues and a tryptophan-serine motif (WSXWS) in the
extracellular domain.10 GM-CSF receptors are found on myeloid progenitors and mature myeloid cells including neutrophils, eosinophils, mononuclear phagocytes, and monocytes.5,11-14
In addition, GM-CSF receptor subunits have also been found in normal nonhematopoietic tissues such as human placenta, endothelium, and
oligodendrocytes of the central nervous system.15-17
Both GMR and GMR are transmembrane glycoproteins. GMR has an
apparent molecular weight of 84 kd with all 11 potential
asparagine-linked glycosylation (N-glycosylation) sites located in the
extracellular domain of the receptor.3 The subunit is a
130-kd protein.8 Analysis of the complementary DNA
(cDNA)-deduced amino acid sequence of GMR suggests 7 potential
N-glycosylation sites, 3 of which are in the extracellular domain at
Asn58, Asn191, and Asn346. We previously showed that tunicamycin, an
N-glycosylation inhibitor, completely abolished GM-CSF binding in COS
cells expressing either low- or high-affinity GM-CSF receptor but did
not affect cell surface expression of the subunit.18
Because tunicamycin treatment inhibited N-glycosylation of both and
subunits in cotransfected COS cells, and unglycosylated GMR
subunit alone was unable to bind GM-CSF, the role of N-glycosylation of
GMR in high-affinity binding has not been defined.
To investigate the function of N-glycosylation of the GMR subunit,
we performed site-directed mutagenesis on the 3 potential N-glycosylation sites located in the subunit extracellular domain. The asparagine residues Asn58, Asn191, and Asn346 in the
consensus N-glycosylation sequence of Asn-X-Ser/Thr were converted to
aspartic acid or alanine. Our results indicated that a single mutation in any of the 3 N-glycosylation sites, as well as triple mutations affecting all 3 sites, eliminated the activity of GMR in
high-affinity GM-CSF binding when coexpressed with wild-type GMR
in COS cells. Thus, N-glycosylation of the subunit is
required for high-affinity GM-CSF binding of the /
receptor complex.
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Materials and methods |
Construction of human GMR mutants
The cDNA encoding the wild-type human GMR was subcloned into
pBluescript KS (pBKSGMR). The single mutants converting the target
Asn to Asp were created by polymerase chain reaction (PCR) using the
wild-type DNA template and 3 mutagenic primers that convert asparagine
located at positions 58, 191, and 346 to aspartate:
 M1: 5'-CACCCGGCGAATGAGGGTCACGTCGACGAGCCGCTGGGCATC-3'
M2: 5'-ATCCTCCTCTCCGACACCTCCCAGGCC-3'
M3: 5'-GCTGTCTCCATCCTTGGTCACGTCGAGGGATGGAGGGGCCAT-3'
The other PCR primers were as follows:
P1: 5'-CCCCCTCGAGGTCGACGGTATCGATAAGCTT-3'
(pBluescript KS polylinker sequence covering restriction sites Xho and
EcoR V)
P2: 5'-CCACTTGCTGGGACGTCCTGAGAGCCG-3'
(nt.661-687, anti-sense)
P3: 5'-CACCTCCTTCCTCACCTCCCAGGA-3'
(nt.781-804, anti-sense)
P4: 5'-CGGCTCTCAGGACGTCCCAGCAAGTGG-3'
(nt.661-687, sense)
P5: 5'-GTGACCAAGGATGGAGACAGCTAC-3'
(nt.1039-1062, sense)
P6: 5'-CTCTGTGGGTAGATCTGAGGCAGCTGG-3'
(nt.1666-1692, anti-sense)
To construct GMR-Asp58, a PCR fragment carrying the target mutation
was made using primer M1 and primer P1 encoding the polylinker sequence
of pBluescript KS located upstream of the initiation codon in
pBKSGMR. The first PCR product was used as a primer to generate the
mutated DNA fragment with another gene-specific primer P2 encoding the
unique Aat II restriction site using the wild-type pBKSGMR as a
template. The mutated DNA fragment from the second PCR reaction was
digested with Xho I and Aat II and ligated into the cognate sites.
GMR-Asp191 was created in a manner similar to GMR-Asp58. The
first PCR reaction used mutagenic primer M2 and the gene-specific primer P3. The PCR product was then used as a primer together with
primer P1 in a second PCR reaction to synthesize a DNA fragment encoding Asp191. The mutated DNA fragment was subcloned into wild-type GMR sequences at the Xho I and Aat II restriction sites.
GMR-Asp346 was made by 3 PCR reactions. The first PCR was performed
with mutagenic primer M3 and primer P4 encoding the Aat II site to
generate a DNA fragment containing Asp346. In the second PCR, primer P5
was used with primer P6 containing the unique Bgl II restriction site
to generate a DNA fragment with a sequence overlapping the first PCR
product from nucleotide 1039 to nucleotide 1059. A DNA fragment
containing restriction sites Aat II, Bgl II, and Asp346 was generated
using the 2 PCR products from the previous steps as overlapping
templates together with primer P4 and the primer P6. The third PCR
product containing Asp346 was subcloned into the wild-type GMR gene
using the unique Aat II and Bgl II restriction sites.
GMR-Asp58/191/346 was constructed using GMR-Asp191 as a basis. A
double mutant encoding Asp58 and Asp191 was generated by the technique
detailed above to create Asp58. A PCR product carrying the double
mutant Asp58/191 was digested with XhoI and AatII and subcloned into
the plasmid encoding the Asp346 mutation to generate GMR- Asp58/191/346.
The PCR reactions were carried out in a volume of 100 µL with Vent
DNA polymerase buffer with 200 µmol/L dNTPs, 20 units of Vent DNA
polymerase (New England BioLabs, Beverly, MA) and appropriate primers
and templates as described above. The reactions were incubated at
94°C for 1 minute, 55°C for 1.5 minutes, and 72°C for 3 minutes for 30 cycles. The identity and fidelity of all PCR-generated sequences for each mutant were confirmed by dideoxynucleotide sequencing.19 The cDNA of each mutant was excised from
pBluescript and subcloned into a eukaryotic expression vector pMX (a
gift from Genetics Institute, Inc, Cambridge, MA).18
To change the asparagine in the target N-glycosylation sites to
alanine, the GMR mutants, GMR-Ala58, GMR-Ala191,
GMR-Ala346, and GMR-Ala58/191/346 were constructed by PCR-based
mutagenesis and confirmed by DNA sequencing (Retrogen, Inc, San Diego, CA).
Cell culture
Monkey kidney COS-1 cells were maintained in Iscove's modified
Dulbecco's medium (IMDM) supplemented with 10% heat-inactivated fetal
bovine serum (FBS), 1% glutamine, and antibiotics.
Expression of membrane-bound human GM-CSF receptor in COS cells
Eukaryotic expression plasmids encoding the gene of human GMR,
the wild-type , or the mutated subunit were transfected into
COS cells using a DEAE-dextran method20 or the
LIPOFECTAMINE reagent (GIBCOBRL, Grand Island, NY) according to the
manufacturer's instructions. Transfected COS cells were cultured in
Dulbecco's modified Eagle's medium containing 10% FBS, 1%
glutamine, and antibiotics with or without 2.5 µg/mL tunicamycin
(Sigma-Aldrich, St Louis, MO). COS cells were harvested 40 to 60 hours
after transfection by incubation with 40 mmol/L EDTA in IMDM at
37°C for 30 minutes followed by adding an equal volume of IMDM
containing 200 µg/mL chondroitin sulfate and 10% FBS. The mixture
was then incubated at 37°C for a further 40 minutes.15
Immunoblotting of the subunit
Cell membrane fractions were prepared using methods previously
described.21 Equal amounts of protein obtained from
membrane fractions of -transfected COS cells were electrophoresed on
10% sodium dodecyl sulfate-acrylamide gels and immunoblotted with a
rabbit polyclonal antihuman GMR antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
Receptor-binding assay
Kinetic binding assays were performed on transfected COS cells that
were detached and disaggregated using 125I-labeled GM-CSF
(DuPont, NEN, Boston, MA) in IMDM containing 10% FBS, 20 mmol/L EDTA,
and 50 µg/mL chondroitin sulfate. Nonspecific binding was determined
by the addition of 3 µmol/L unlabeled human recombinant GM-CSF (a
gift from Amgen, Thousand Oaks, CA) to the assay mixtures. For
equilibrium-binding kinetics, aliquots of cells were incubated with
increasing concentrations of 125I-labeled GM-CSF at 4°C
overnight. The assay mixtures were then layered over 0.5 mL bovine
serum and centrifuged for 3 minutes at 10 000g. Radioactivity
of the cell pellets was measured in a gamma counter. Equilibrium
dissociation constants were determined by Scatchard analysis and
analyzed using GraphPad Prism (GraphPad Software, San Diego, CA).
Immunofluorescence staining of transfected COS cells expressing
membrane-bound GMR
The COS-1 cells were grown and transfected with the plasmids
encoding the gene of wild-type or mutated GMR on chamber slides. Forty-eight hours after transfection, slides were washed twice with PBS
and air-dried at room temperature for 10 minutes. Slides were then
fixed with cold acetone for 5 minutes and washed with PBS. The fixed
cells were incubated with a rabbit polyclonal antihuman GMR antibody
(Santa Cruz Biotechnology) at room temperature for 1 hour and washed
twice with PBS. Binding of the primary antibody was detected by
incubation with a fluorescein-conjugated antirabbit Ig antibody for 60 minutes at room temperature. The stained cells were examined and
photographed with confocal fluorescence microscopy.
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Results |
GMR N-glycosylation mutants are expressed on the surface of
COS cells
To determine whether GMR subunits with mutated extracellular
N-glycosylation sites could be faithfully expressed on the cell surface, plasmids encoding the mutated subunits were transfected into COS cells and the cell membranes were isolated. Membrane fractions
enriched in plasma membrane were analyzed by Western blot using an
anti- subunit antibody. As shown in Figure
1, a dominant band was detected at
approximately 130 kd in the membrane fractions obtained from COS cells
transfected with wild-type GMR and in each of the single mutations
encoding Ala58, Ala191, Ala346 (Figure 1A) and the single mutations
encoding Asp58, Asp191, Asp346 (Figure 1B) with similar levels of
protein expression. The 130-kd band corresponded to the subunit
with complete posttranslational modification.8 The
wild-type GMR and each of the GMR proteins altered at a single
N-glycosylation site comigrated electrophoretically.
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| Fig 1.
Expression of GMR N-glycosylation mutants in COS cell
membranes.
Cells were transfected with expression plasmids encoding wild-type or
mutated GMR. Proteins isolated from the membrane fractions of COS
cells 48 hours after transfection were loaded onto 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis and immunoblotted with a
rabbit antihuman GMR subunit antibody. (Panel A) One microgram of
membrane protein from cells transfected with plasmids encoding
wild-type or mutated GMR was loaded in each lane: 1, wild-type
GMR; 2, GMR-Ala58; 3, GMR-Ala191; 4, GMR-Ala346; 5, mock
transfectant. The immunoreactive bands were detected by enhanced
chemiluminescence (ECL) with 1-minute exposure. (Panel B)
Two micrograms of membrane protein from cells transfected with plasmids
encoding wild-type or mutated GMR were loaded in each lane: 1, wild
type GMR; 2, GMR-Asp58; 3, GMR-Asp191; 4, GMR-Asp346; 5, mock transfectant. The immunoreactive bands were detected by ECL with
1-minute exposure. (Panel C) One microgram of membrane protein obtained
from cells transfected with plasmid encoding wild-type or
GMR-Ala58/191/346 was loaded in each lane: 1, wild-type GMR
without tunicamycin treatment; 2, wild-type GMR treated with 2.5 µg/mL tunicamycin; 3, GMR-Ala58/191/346. The immunoreactive bands
were detected by ECL with 1-minute exposure. (Panel D) Twenty
micrograms of membrane protein obtained from cells transfected with
plasmid encoding GMR-Asp58/191/346 (lane 1) and mock transfectant
(lane 2) were loaded. The immunoreactive bands were detected by ECL
with 15 minutes of exposure.
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Treatment with tunicamycin resulted in a subunit with an apparent
molecular weight approximately 9 kd less than the fully N-glycosylated
protein (Figure 1C, lane 2). The triple mutant GMR-Ala58/191/346 (Figure 1C, lane 3) comigrated with
the subunit expressed in tunicamycin-treated cells, suggesting that
mutations in all 3 target sites disrupted N-glycosylation on GMR and
that only the extracellular N-glycosylation sites of the subunit are subject to modification. The results also suggest that, in contrast
to the GMR subunit, N-glycosylation accounts for less than 7% of
the total molecular mass of the subunit.18 These results are also consistent with a study using N-glycosidase F to
deglycosylate GMR.22 Because the overall N-glycosylation level of GMR is small, the electrophoretic mobility of the subunit was not detectibly affected by mutation at a single site.
The substitution of asparagine with aspartic acid of the 3 extracellular glycosylation sites resulted in a protein that, although presented on the cell surface (Figure 2I),
was unstable and subject to degradation (arrows, Figure 1D, lane 1).
Presumably, the alteration in charge introduced by the aspartate triple
mutation affected protein stability.
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| Fig 2.
Immunofluorescence of GMR N-glycosylation mutants in
COS cells.
The COS cells grown on chamber slides were transfected with plasmids
encoding wild-type or mutated GMR subunits and examined by
immunofluorescence staining with a rabbit antihuman GMR subunit
polyclonal antibody. (A) Wild type GMR; (B) GMR-Ala58; (C)
GMR-Ala191; (D) GMR-Ala 346; (E) GMR-Ala58/191/346; (F)
GMR-Asp58; (G) GMR-Asp191; (H) GMR-Asp346; (I)
GMR-Asp58/191/346; (J) mock-transfectant.
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To verify that mutation of the N-glycosylation sites of the GMR
extracellular domain did not affect localization of the mutated GMR
proteins to the cell membrane, COS cells transfected with plasmids
encoding wild-type and mutated subunits were examined by
immunofluorescence staining with a polyclonal anti-GMR
antibody (Figure 2). Plasma membranes of cells expressing wild-type
(Figure 2A) and mutated subunits (Figure 2B-I) demonstrated
high-intensity fluorescence confirming the expression of GMR
and all of the mutants on the cell surface. No fluorescence
was observed in mock-transfected COS cells (Figure 2J).
Expression of wild-type GM-CSF receptor in COS cells
To investigate the effect of N-glycosylation on the function of
GMR, we first measured the GM-CSF binding activity of reconstituted wild-type receptor. COS cells were cotransfected with expression plasmids encoding wild-type GMR and plasmids encoding GMR at different ratios. Equilibrium kinetic analyses were performed 48 hours
after transfection. COS cells cotransfected with and subunits
at a ratio of 2:1 expressed both high- (kd = 229.9 pmol/L) and low- (kd = 9.01 nmol/L) affinity binding
sites (Figure 3A). When cotransfected with
/ at a ratio of 1:6, the transfected cells exclusively displayed
high-affinity GM-CSF binding activity with a kd of 216.9 pmol/L (Figure 3B). COS cells expressing only wild-type GMR
displayed low-affinity binding with kd = 9.10 nmol/L (Figure 3C).
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| Fig 3.
GM-CSF binding activity of wild-type receptors.
The COS cells were either transfected with GMR alone or
cotransfected with plasmids encoding GMR and plasmids encoding
GMR at the indicated ratios. 125I-labeled GM-CSF binding
of the transfected cells was determined at 48 hours after transfection
by Scatchard analysis. (A) / = 2:1, high-affinity
kd = 229.9 pmol/L, low-affinity
kd = 9.01nmol/L. (B) / = 1:6,
kd = 216.9 pmol/L. (C) GMR alone,
kd = 9.10 nmol/L.
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N-glycosylation of the extracellular domain of GMR is
essential for high-affinity ligand binding activity of the
GMR/ complex
Having established that GMR mutants at N-glycosylation sites were
expressed on the cell surface and having defined transfection conditions yielding high-affinity binding, we examined the functional contribution of subunits altered at N-glycosylation sites to GM-CSF
binding. To ensure the reconstitution of a GMR/ complex with the
potential for high-affinity binding, plasmids encoding each mutated subunit were cotransfected with plasmids encoding wild-type GMR into
COS cells at an / ratio of 1:6 and the GM-CSF binding activity
was analyzed (Figure 4). Cells expressing
subunits that had been altered at any single site with either
alanine or aspartate substitution (Ala58, Ala191, Ala346, or Asp58,
Asp191, Asp346) as well as the triple mutants of
GMR (GMR-Ala58/191/346, GMR-Asp58/191/346) exhibited
low-affinity GM-CSF binding activity exclusively, identical to COS
cells transfected with GMR alone (Figure 3C). No high-affinity
binding was detected in cells expressing any of the mutated GMR
proteins. Equilibrium binding kinetics of cells transfected with each
mutant and the wild-type receptor were measured by at least 3 independent experiments (Table 1). The data
indicate that mutation of any 1 of the N-glycosylation sites in the
GMR extracellular domain abolishes the ability of GMR to form a
high-affinity receptor with GMR. Thus, N-glycosylation of the subunit is critical for high-affinity GM-CSF
binding.
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| Fig 4.
N-glycosylation site mutations in the extracellular
domain of GMR prevent high-affinity GM-CSF binding in transfected
COS cells.
The COS cells were cotransfected with plasmids encoding wild-type
GMR and plasmids encoding mutated GMR at a ratio
/ = 1:6. Representative Scatchard analyses of GM-CSF binding
data obtained from cotransfected cells are shown. (A) GMR-Ala58,
kd = 4.31nmol/L. (B) GMR-Ala191,
kd = 5.92 nmol/L. (C) GMR-Ala346,
kd = 6.33 nmol/L. (D) GMR-Ala58/191/346,
kd = 9.52 nmol/L. (E) GMR-Asp58,
kd = 5.75 nmol/L. (F) GMR-Asp191,
kd = 6.62 nmol/L. (G) GMR-Asp346,
kd = 10.10 nmol/L. (H) GMR-Asp58/191/346,
kd = 5.68 nmol/L.
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Discussion |
Asn-linked glycosylation is a major form of cotranslational
modification in eukaryotic protein synthesis. N-glycosylation is
observed in many proteins containing the sequence Asn-X-Ser/Thr in an
appropriate context for recognition by oligosaccharyltransferases present in the endoplasmic reticulum (ER) and Golgi
apparatus.23 The first step of N-glycosylation involves the
transfer of a core oligosaccharide moiety,
Glc3Man9GlcNAc2, from a lipid
carrier to the asparagine residue in the nascent polypeptide chain in the rough ER. A series of trimming and modification steps are catalyzed
subsequently by exoglycosidases and glycosyltransferases in the ER and,
in some cases, in the Golgi compartments. The oligosaccharide processing reactions lead to the generation of different carbohydrate structures on the protein surface. High mannose oligosaccharides have a
core of 3 to 9 mannose residues linked to the 2 N-acetylglucosamine residues (Man3-9GlcNAc2)
attached to the asparagine residue in the consensus glycosylation motif
of the protein. Complex oligosaccharides contain GlcNAc, galactose, or
sialic acid additions to a Man3GlcNAc2 core,
and hybrid oligosaccharides possess at least one of these additions on
1 branch of the core and mannose residues on the other branch of the
core.23
The majority of membrane-bound and extracellular proteins in animals
are N-glycosylated. The solubility and stability of the protein can be
significantly affected by the carbohydrate groups on the outer surface
of the protein molecule.24 The oligosaccharide moieties can
also have dramatic effects on the biologic properties of glycoproteins
such as ligand-binding affinity,25-29 signal
transduction,30-33 immunogenicity,34,35 and
clearance rate.36-40 In addition, N-glycosylation may be
essential for proper protein folding and trafficking in the
cells.41-44
In the case of the GM-CSF receptor, the molecular weights of the and the subunits calculated on the basis of the deduced amino acid
sequence are 44 kd and 96 kd, respectively. The substantially higher
apparent molecular weights observed for GMR (84 kd) and GMR (130 kd) are in part due to N-glycosylation of these
molecules.18 Previously, using the N-glycosylation
inhibitor tunicamycin, we showed that N-glycosylation is essential for
the ligand binding activity of GMR.18 Because the
formation of high-affinity GM-CSF receptors requires the expression of
both GMR and GMR, and tunicamycin treatment blocks
N-glycosylation in both receptor subunits, it was necessary to mutate
the subunit to evaluate the effect of N-glycosylation on the action
of the subunit. We therefore mutated the potential N-glycosylation
sites in the extracellular portion of the subunit. In 1 set of
GMR mutants, the asparagines in the target sites were replaced by
corresponding aspartic acids. To address the possibility that the
negative charge introduced by the aspartate substitution might affect
the biology of the subunit irrespective of effects on
N-glycosylation, the target asparagines were also converted to
uncharged alanines. Nonetheless, we found that mutation of asparagine
by either aspartate or alanine substitution in any of the potential
N-glycosylation sites of the extracellular domain of GMR prevented
high-affinity GM-CSF binding when the mutated GMR was coexpressed
with the wild-type subunit in COS cells.
In some cases, N-glycosylation is necessary for proper processing and
intracellular transport of the protein. For example, mutation of
N-glycosylation sites in the insulin receptor subunit impairs cell
surface expression of the receptor.43 Because
immunofluorescence and Western blot studies showed that mutations in
GMR did not affect cell surface expression of the subunit, it
was apparent that N-glycosylation of these sites is not required for
plasma membrane targeting of GMR. Our previous studies indicated
that the unglycosylated GMR subunit can also be expressed on the
cell surface.18
In contrast to GMR, the contribution of N-glycosylation to the
posttranslational modification of GMR is small, comprising approximately 25% of added molecular mass. The discrepancy between the
apparent (130 kd) and the amino acid sequence deduced (96 kd) molecular
weights of GMR may also arise from other posttranslational modifications, such as phosphorylation and O-glycosylation. The finding
that tunicamycin treatment yields a subunit of the same size as
GMR mutated at the 3 extracellular N-glycosylation sites is a strong
indication that only the extracellular sites are N-glycosylated. Although the size of the glycosylated moiety is small, the result that
alteration of any of the 3 extracellular N-glycosylation sites
abrogates high-affinity binding demonstrates that N-glycosylation is as
important in the formation of the high-affinity receptor as it is in
ligand binding to the low-affinity receptor.
The subunit of the GM-CSF receptor alone does not bind ligand, but
confers high-affinity ligand binding activity to the receptor in the
presence of the ligand-specific GMR subunit. Our findings indicate
that N-glycosylation of the subunit plays a crucial role in ligand
binding by the high-affinity GM-CSF receptor complex. It is likely that
the oligosaccharide moieties on the extracellular domain of GMR are
essential for proper folding of the chain and therefore development
of the necessary conformation of the high-affinity ligand binding site.
Removal of carbohydrate structures on the chain prevents the
interaction with GMR that leads to high-binding energy. The data do
not indicate, however, whether intact N-glycosylation is required for
association of and subunits or whether the glycosylated
moieties play a more direct role in ligand binding. Our recent
observations regarding the role of the subunit in ligand
acquisition and release by the high-affinity receptor suggest a dynamic
role for the extracellular domain of the subunit.45
Other receptor systems have variable requirements for glycosylation.
For example, the lutropin/choriogonadotropin receptor does not require
glycosylation to bind its ligand human chorionic gonadotropin, whereas
a closely related receptor, the follitropin receptor,
does.46
Human GM-CSF is a 22-kd glycoprotein with a 4-helical bundle
structure.47 Unglycosylated GM-CSF resulting from mutation of N- and O-glycosylation sites is biologically active.48
In contrast, both the and subunits of the GM-CSF receptor
require N-glycosylation to function. Thus, the carbohydrates present on the extracellular domains of the GM-CSF receptor appear to be essential
for both ligand acquisition and high-affinity binding.
| |
Acknowledgments |
We thank Rong-Hua Zhang for excellent technical assistance and
Elizabeth P. Koers for editing the manuscript.
| |
Footnotes |
Submitted December 10, 1999; accepted January 31, 2000.
Supported by grants RO1 CA30388, RO1 HL42107, P30 CA08748 from
the National Institutes of Health, the Leukemia Society of America, and
the Schultz Foundation.
Reprints: David W. Golde, Memorial Sloan-Kettering Cancer
Center, 1275 York Ave, New York, NY 10021.
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.
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Abstract
Introduction