From the CNRS UMR 5540, Université de Bordeaux
2-146, rue Léo Saignat, 33076 Bordeaux, France, § IFR
66, Pathologie infectieuse et cancer-146, rue Léo Saignat, 33076 Bordeaux, France,
INSERM U463, 9 quai Moncousu, 44035 Nantes
Cedex, France, ** IFR 26, 9 quai Moncousu, 44035 Nantes
Cedex, France, and the
School of
Biosciences, University of Birmingham, Birmingham B15 2TT, United
Kingdom
Received for publication, July 18, 2002, and in revised form, February 12, 2003
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ABSTRACT |
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The leukemia inhibitory factor (LIF)
receptor comprises the low affinity binding chain gp190 and the
high affinity converter gp130. The ectodomain of gp190 is among the
most complex in the hematopoietin receptor family, because it contains
two typical cytokine receptor homology domains separated by an
immunoglobulin-like (Ig-like) domain. Human and murine gp190 proteins
share 76% homology, but murine gp190 binds human LIF with a much
higher affinity, a property attributed to the Ig-like domain. Using
alanine-scanning mutagenesis of the Ig-like domain, we mapped a LIF
binding site at its carboxyl terminus, mainly involving residue
Phe-328. Mutation of selected residues into their orthologs in the
murine receptor (Q251E and N321D) significantly increased the affinity
for human LIF. Interestingly, these residues, although localized at
both the amino and carboxyl terminus, make a spatially unique LIF
binding site in a structural model of the Ig-like module. These
results demonstrate definitively the role of the Ig-like domain in LIF binding and the potential to modulate receptor affinity in this family
with very limited amino acid changes.
The leukemia inhibitory factor
(LIF)1 low affinity receptor
gp190 belongs to the large family of the hematopoietin receptors, which
are characterized by a consensus cytokine receptor homology (CRH)
domain. The extracellular region of gp190 is unusual in that it has two
CRH domains, herein called D1 for the amino-terminal membrane distal
domain and D2 for the membrane proximal domain. D1 and D2 are separated
by an immunoglobulin-like (Ig-like) module of around 100 amino acids,
and D2 is followed by three type III fibronectin modules (1). The gp190
receptor participates in the high affinity receptor complex for 5 human
cytokines (reviewed in Ref. 2), namely LIF, oncostatin M (OSM), ciliary
neurotrophic factor (CNTF), cardiotrophin-1 (CT-1), and
neurotrophin-1/B cell-stimulating factor 3 (NNT-1/BSF-3). The gp190
also exists in a soluble form capable of binding to LIF, behaving as a
competitor of membrane gp190 and as an inhibitor of LIF's biological effects.
Little information relative to the function of each module of gp190 is
available to date. The CRH domain is usually implicated in binding to
the ligand, and for this reason has been called the cytokine binding
domain. The function of the Ig-like module is less clear. In some
cases, it has also directly participated in ligand binding. Indeed the
Kaposi's sarcoma-associated herpes virus-derived interleukin (IL)-6
(vIL-6) homolog directly binds to the Ig-like domain of its unique
receptor chain gp130 (3), this latter being also the high affinity
converting chain for the LIF/gp190 complex. Similarly, human OSM
interacts with both the CRH and the Ig-like domains of its low affinity
receptor chain gp130 (4). Alternatively, the Ig-like module of gp130
does not directly interact with the human cytokine IL-6, but is
required when two gp80/IL-6/gp130 complexes dimerize to form the signal transducing hexamer (5). As a final example, the Ig-like domain of the
granulocyte-colony stimulating factor (G-CSF) receptor has also been
reported to participate in the dimerization of two receptor chains
triggered by the ligand (6) and also directly to ligand binding
(7).
With respect to human gp190 (hgp190), we ascribed to the Ig-like
module the role of a hinge allowing the two CRH domains to interact
with each other (8, 9). On the other hand, murine gp190 (mLIFR), which
shares 76% sequence homology with hgp190, displays a much higher
affinity for human LIF (hLIF) than hgp190 does (10). This could be
reproduced by the replacement of the Ig-like module of hgp190 with its
murine homolog (11). In addition, when analyzing a series of deletion
mutants lacking one or several modules of the hgp190 ectodomain, we
identified a mutation in the carboxyl terminus of the Ig-like module
(mutation 002, i.e. F328T/A329S), which led to a dramatic
decrease of hLIF binding (8). Given these preliminary results, we
decided to investigate more precisely the role of the Ig-like module of
hgp190 in binding to hLIF. Site-directed mutagenesis was performed, and
the single point mutants generated were expressed as soluble and
membrane-bound forms. They were analyzed for their ability to bind hLIF
and trigger LIF-dependent proliferation. In a first series
of mutants the residues of the carboxyl terminus of the Ig-like module
were mutated into alanine to identify residues involved in hLIF
binding. In a second series of mutants, selected residues in the
Ig-like module of the human receptor were mutated into their orthologs
in the murine protein, in an attempt to identify residues responsible for the difference in affinity between both species for hLIF.
Cell Culture--
All eukaryotic cell lines were grown in 5%
CO2 at 37 °C in a water-saturated atmosphere either in
Dulbecco's modified Eagle's medium (Invitrogen) for COS and CHO DUCKX
cells or RPMI 1640 medium for Ba/F3 cells, in the presence of 8% fetal
calf serum (Invitrogen) and 2 mM L-glutamine.
For CHO DUCKX cells 10 µg/ml of adenosine, deoxyadenosine, and
thymidine were added to the medium. Ba/F3 transfectants were grown in
the presence of hLIF at 50 ng/ml. For functional studies, the cells
were first starved of hLIF for 3 days by culture in medium supplemented
with murine IL-3 (mIL-3) in the form of a COS cell supernatant diluted
1:200 and then washed three times to remove mIL-3.
Construction of the Human gp190 Mutants--
Site-directed
mutagenesis of the extracellular region of hgp190 (soluble hgp190 or
shgp190) was performed using the pAlter-1 phagemid system (Promega,
Charbonnières, France) following the manufacturer's
recommendations as previously described (8). Mutational
oligonucleotides were synthesized (Cybergene, Evry, France) that
allowed for the creation of the desired mutation together with a new
restriction site, or the inactivation of an existing one wherever
possible, in order to allow for easy screening of the mutated plasmids.
All punctual shgp190 mutants were subcloned in the pEDr vector, either
as a carboxyl-terminal c-Myc-tagged soluble receptor (shgp190myc) or
fused to the transmembrane and intracellular region of hgp130
(hgp190/130 chimeras) as previously described (8). The mutants created
were all verified by sequencing (ESGS, Evry, France).
Transfection of Cells--
COS cells were transiently
transfected with 5 µg of plasmid DNA encoding the shgp190myc
constructs using the DEAE-dextran method as previously described (8).
Culture supernatants were collected at day 4, and the recombinant
receptor was quantitated by ELISA. Stable transfectants were obtained
by electroporating CHO DUCKX DHFR( Flow Cytometry Staining of Cells--
The membrane expression of
the hgp130 and of the hgp190 mutants on the Ba/F3 cell lines was
detected by flow cytometry using the anti-D1 mAb 6G8 and the anti-D2
mAbs 8C2 and 12D3 for hgp190 (12), and using the anti-hgp130 mAbs AM64
and H1 for hgp130. An IgG1 irrelevant isotype-matched mAb was used as a
negative control. The staining was performed as previously reported (8) and was analyzed with a three color FACScalibur flow cytometer (BD
PharMingen) equipped with the CellQuest software. To analyze the
turnover of the transmembrane form of the LIF receptor or its mutants
from the cell surface, the indicated Ba/F3 cell lines were incubated
with hLIF at 100 ng/ml or without any added cytokine for the indicated
times at 37 °C, before being stained with the anti-hgp190 8C2 mAb or
an isotype-matched negative control mAb.
ELISAs for the Quantitation of shgp190 and of the
LIF/shgp190 Complexes--
Secreted shgp190myc and its
mutants were quantitated using a sandwich ELISA specific for hgp190,
according to the previously reported procedure (12, 13). The capture
mAb was the anti-D1 mAb 6G8, and the biotinylated developing mAb was
the anti-D2 mAb 8C2. In vitro hLIF binding to shgp190myc or
its mutants was assessed as follows. All steps were performed at room
temperature, and plate washes were performed in PBS containing 0.05%
Tween 20. ELISA plates were coated with the anti-D1 mAb 6G8, and
saturated with PBS/BSA for 1 h. Plates were washed once and then
incubated with supernatants containing shgp190myc or its point mutants
adjusted at a constant and saturating concentration of 150 ng/ml for
2 h and washed three times. Some of the mutants with lower
secretion levels were concentrated to reach 150 ng/ml using
polyethylene glycol 35000 (Fluka, Sigma). CHO-derived hLIF ranging from
0.256 ng/ml to 4000 ng/ml was added for 2 h. After three washes,
plates were incubated with the biotinylated non-blocking anti-hLIF mAb 1F10 (14) at a final concentration of 2 µg/ml for 1.5 h and washed again three times. Subsequent steps, i.e. incubation
of peroxidase-labeled streptavidin and addition of substrate were as
described for the ELISA for quantification of shgp190.
Proliferation Assay of Ba/F3 Cells--
The
proliferative response to hLIF, mLIF, hOSM, and mIL-3 of the
transfected Ba/F3 cell line was assessed by a colorimetric proliferation assay using
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT,
Sigma), after 3 days of culture, as described previously (8). To
analyze the inhibitory activity of the soluble receptor toward hLIF
action, we used Ba/F3 cells expressing wild-type full-length hgp190 and
hgp130. A mixture of hLIF (1 ng/ml final concentration) and shgp190myc
or soluble mutant Q251E/N321D at the indicated final concentrations, or
murine serum as a source of mouse LIF receptor (mLIFR) at the indicated
final dilutions, was added to the cells. Cell proliferation was
analyzed 3 days later. Whenever specified, cell culture medium was also
supplemented with extra amounts of soluble receptors on day 1 and day 2.
Affinity Chromatography for the Purification of shgp190myc
Mutants--
The purified anti-D1 mAb 10B2 (5 mg) was coupled to a
1-ml Hi-Trap-NHS-activated column (Amersham Biosciences) according to the manufacturer's instructions. Culture supernatant (500 ml) of CHO
cells stably producing the recombinant soluble receptor was passed
through the column. After washing with 5 ml of PBS at pH 7.5 containing
1 M KCl and 0.02% Tween 20, bound receptor was eluted with
0.1 M glycine at pH 2.5 as 1-ml fractions, and acidity was
immediately neutralized with a one-third volume of Tris-HCl at pH 8.0. After extensive dialysis against PBS, the receptor was quantitated by
ELISA, and the fractions with the highest concentrations were pooled.
Radioiodination Experiments--
Stably transfected Ba/F3 cells
were cultured for 3 days in the presence of mIL-3 instead of hLIF.
After one PBS wash, cells were resuspended in PBS/BSA with or without
the blocking anti-hgp130 B-R3 mAb (20 µg/ml) and incubated at 4 °C
for 30 min. Binding experiments were carried out in PBS/BSA for
experiments using labeled Escherichia coli-derived hLIF
(PeproTech Inc., Rocky Hill, NJ), or in PBS/BSA containing 5 mM mannose-6-phosphate for experiments using CHO-derived
hLIF, to avoid binding to the mannose-6-phosphate/insulin-like growth
factor II receptor (Man 6-P/IGFII-R) (15, 16). hLIF was iodinated using
the chloramine T method as described (17), at a specific radioactivity
of around 35,000 cpm/fmol. Regression analysis of the binding data was
accomplished using a one- or two-site equilibrium binding equation
(Grafit, Erathicus Software, Staines, UK).
Surface Plasmon Resonance Analysis of LIF Binding--
These
experiments were performed with the BIACore 2000 optical biosensor
(BIACore, Uppsala, Sweden). hLIF purified from transfected CHO cells
was covalently coupled to a carboxymethyl dextran flow cell (CM5,
BIACore) as recommended by the manufacturer. The level of
immobilization obtained was 1,500 resonance units. Binding of purified
CHO-derived soluble receptors was assayed at concentrations ranging
from 0.67 to 33 nM in Hepes-buffered saline and at a
flow rate of 40 µl/min. Association was monitored for 5 min before initiating the dissociation phase for another 11 min with
Hepes-buffered saline. Regeneration of the flow cells was achieved with
5 mM glycine-HCl at pH 2.0. The resulting sensorgrams
were analyzed using the BIAEvaluation software (BIACore).
Analysis of STAT3 Phosphorylation--
Ba/F3 cells expressing
wild-type hgp190 and hgp130 were used. CHO-derived hLIF at a final
concentration of 10 ng/ml giving maximal STAT3 phosphorylation was
incubated with or without the purified soluble receptors, or with
normal mouse serum as a source of mLIFR, at twice the indicated
concentrations at 37 °C for 20 min, before the cells (2 × 105 per condition tested) were added in an identical
volume. After 5 min at 37 °C, the cells were lysed in 50 µl of
Triton X-100 lysis buffer as described previously (9). After
centrifugation, the supernatant was harvested, and the total protein
concentration was determined using the bicinchoninic acid method
(Sigma). The cell lysate (10 µg of protein/lane) was boiled 5 min and
separated by SDS-PAGE on 8% gels and then transferred to a
nitrocellulose membrane (Amersham Biosciences). The immunoblotting was
performed as previously reported (9) with the rabbit anti-phospho-STAT3 antibody (Cell Signaling Technology, Ozyme, Saint-Quentin-en-Yvelines, France) or with the rabbit anti-actin antibody (Sigma) as a control. The proteins were visualized using the chemiluminescence ECL system (Amersham Biosciences).
Design of a Three-dimensional Model of the Ig-like Domain of
hgp190--
A structural model of the Ig-like domain of hgp190 was
constructed using the program MODELLER (18) using a sequence alignment of the Ig-like domain of hgp130 with the corresponding region of hgp190
and the crystallographic coordinates of the Ig-like domain of hgp130
(PDB accession number: 1I1R, Ref. 3). The quality of the model was
assessed using ProsaII.
Production and Expression of shgp190myc and Its Single Point
Mutants--
In order to elucidate the role of the Ig domain in LIF
binding, the region of hgp190 around mutation 002 (8), which abrogates its binding, was subjected to alanine-scanning site-directed
mutagenesis (Fig. 1A). A total
of 12 mutations were carried out extending from residues 321 to 334, except for the existing alanine at position 329, which was mutated to
glycine (Fig. 1B). Since the junction between the Ig-like
module and D2 lies between residues 330 and 331, as inferred from the
analysis of intron-exon organization of the hgp190 gene (1),
8 mutations were therefore localized in the Ig-like module and 4 were
in the D2 domain (Fig. 1B). Additionally, we also replaced 6 amino acids in the human sequence of the Ig-like module by their murine
orthologs as deduced from the alignment of the sequences from both
species (Matcher® Program, Ref. 19). Among these, 4 affected residues
close to mutation 002 (i.e. N321D, I322V, F323Y, and I327V),
and 2 affected residues localized in the amino terminus of the Ig-like
module (i.e. Q251E and D266N) were chosen by virtue of
electrostatic charge changes between both species (Fig. 1B).
A double mutant Q251E/N321D was also constructed. The cDNAs for
shgp190 and its mutants were fused carboxyl-terminal to the sequence
encoding the c-Myc epitope recognized by mAb 9E10. To examine whether
the resulting proteins were correctly produced and released in the cell
culture supernatant, each cDNA construct was transiently expressed
in COS cells, and the secreted receptor was quantitated with a sandwich
ELISA specific for hgp190 using mAbs 6G8 and 8C2, which are directed
against D1 and D2, respectively. We used mAbs specific for epitopes
outside the Ig-like domain to ensure that their reactivity could not be
affected by the mutations. In addition, these mAbs recognize
conformation-dependent epitopes (12), giving information on
the ternary conformation of the secreted receptor. All the hgp190
mutants were expressed at levels grossly comparable to that of
shgp190myc, which was produced at 161 ± 42 ng/ml, albeit mutants
002, F328A, A329G, G330A, Y331A, P332A, P333A, and D334A were at
approximately 2-4-fold lower
levels.2
Binding of hLIF to shgp190myc Mutants as Determined by
ELISA--
To determine the relative hLIF binding abilities of
shgp190myc and its mutants, we developed an ELISA to detect the
ligand/low affinity receptor complex in solution. Each mutant at a
saturating concentration was trapped on a microtitration plate coated
with the mAb 6G8 directed at the D1 domain. Serial dilutions of hLIF were then added, and hLIF bound to the receptor was detected with biotinylated anti-hLIF mAb 1F10, a mAb that does not affect hLIF binding (20). Fig. 2A
(left panel) displays the dose-response curves obtained with
representative mutants V326A, F328A, G330A, and P332A. For shgp190myc,
50% of the maximal binding (C50) was reached with hLIF at 14.0 ± 3.2 ng/ml (mean ± S.D. of three experiments), corresponding to
0.35 ± 0.08 nM given that the molecular mass of
CHO-derived hLIF is 40 kDa. The C50 obtained for all the mutants are
listed in Fig. 2A (right panel) and compared (in
percent) to the C50 value obtained with shgp190myc. Alanine mutants
could be sorted into two groups. A first group contained mutants N321A, A329G, Y331A, P332A, P333A, and D334A, each displaying an ability to
bind hLIF close to that of shgp190myc (C50 ratio between 50 and 120%).
A second group consisted of F323A, T325A, V326A, I327A, F328A, 002, and
G330A, each showing a severely impaired ability to bind hLIF (C50 ratio
of 10% and lower). Among these, no hLIF binding could be detected at
all for mutants 002 and F328A (indicated by two asterisks in
Fig. 2A and shown with mutant F328A in the left
panel) even at the highest hLIF concentration tested (4000 ng/ml).
In contrast, a faint binding could be detected at high concentrations
of hLIF for mutants F323A, T325A, V326A, I327A, and G330A (indicated by
one asterisk in Fig. 2A and shown for mutant
G330A in the left panel). Mutations that impaired hLIF binding were all localized in the Ig-like domain, because none of the
mutations generated downstream of the Ig-like/D2 junction altered hLIF
binding.
The human-to-mouse mutants could be sorted into three groups (Fig.
2B). Mutant D266N displayed the same hLIF binding
ability as shgp190myc, whereas I322V, F323Y, and I327V showed a
decreased binding. A third group consisted of Q251E, N321D, and the
double mutant Q251E/N321D, which all possessed a significantly
increased hLIF binding ability, with respectively 7, 14, and 35 times
that of wild-type shgp190myc.
There was no correlation between the production levels of the mutants
in the cell culture supernatants and their ability to bind hLIF,
strongly suggesting that the mutations performed did not alter the
conformation of the receptors or not to an extent that could impair
their function.
hLIF, hOSM, and mLIF Binding to the Membrane-bound Form of the
Alanine-scanning hgp190 Mutants--
To analyze the ability of the
hgp190 single point mutants to trigger a LIF-dependent
signal, the c-Myc tag of the mutants was replaced with the
transmembrane and intracellular regions of hgp130, and
mIL-3-dependent Ba/F3 cells were transfected with wild-type
hgp130 and either each of the mutants or a chimeric non-mutated
hgp190/130 as a positive control. Stable cell lines were obtained with
all the mutants. In contrast, transfection of hgp130 or hgp190/130
alone never allowed the selection of cells in the presence of hLIF.
Membrane expression of the receptor chains was analyzed by flow
cytometry. Fig. 3A depicts the
staining of the parental Ba/F3 cell line, the hgp190/130 cell line, and
three representative Ba/F3 cell lines bearing hgp130 in combination
with either F328A, G330A, or P332A chimeric mutants of hgp190. The
expression level of the receptor chains may vary from one transfection
experiment to another, but the variations observed were not associated
with particular mutations.
All the transfectants obtained were then tested for their ability to
proliferate in the presence of hLIF and hOSM. Dose-response proliferation curves are shown in Fig. 3B for the
representative mutants F328A, G330A, P332A, and the hgp190/130 control
cell line. Cytokine concentrations giving half-maximum proliferation
(EC50) are given in Table I.
All the cell lines proliferated in a dose-dependent manner
in the presence of hLIF (Table I and Fig. 3B,
left panel). The non-mutated hgp190/130 receptor supported
Ba/F3 cell proliferation with an EC50 of 0.18 ± 0.05 ng/ml, i.e. 4.5 ± 1.25 pM. As expected, the mutations leading to conserved hLIF binding ability in the ELISA
also behaved like the non-mutated receptor in the Ba/F3 proliferation
assay (see mutant P332A in Fig. 3B). Mutation F328A, which
led to complete abrogation of hLIF binding ability in the ELISA
resulted in a sharp (80-100-fold) decrease in Ba/F3 cells proliferation in the presence of hLIF (EC50 of 18.67 ± 6 ng/ml), and fully accounted for the decrease observed with
mutation 002 (EC50 of 16.38 ± 4.7 ng/ml). In
contrast, mutations that only partially impaired hLIF binding in the
ELISA had no influence on cellular proliferation, as shown for
mutant G330A in Fig. 3B. The proliferation curves presented
depict a representative experiment, and the apparent differences in
response to hLIF, which can be seen between the mutants, are not
biologically significant with the exception of mutant F328A (see also
Table I). The cell transfectants did not proliferate at all in the
absence of added cytokine and proliferated comparably in the presence
of mIL-3, demonstrating that their intrinsic ability to proliferate was
fully retained after transfection.2 All the Ba/F3 cell
lines also proliferated in a dose-dependent manner in the
presence of hOSM (Fig. 3B, middle panel for
representative mutants and Table I), and no difference was noted
between all the mutants and the hgp190/130 cell line (EC50
of 2.02 ± 0.37 ng/ml, i.e. 100 ± 18 pM). Of note, mutation F328A had no effect on the response
to hOSM. The response to mLIF was also analyzed for the F328A cell line
and the hgp190/130 cell line (Fig. 3B, right panel and Table I). As with hLIF, mutation F328A also
strongly impaired the response to mLIF, and the barely detectable
residual activity did not allow the calculation of the EC50
for mLIF. Therefore, residue Phe-328, which is conserved between the
human and mouse species, is crucial for binding both human and murine
LIF.
Using iodinated hLIF, we measured the high and low affinity
dissociation constants of the receptors expressed by Ba/F3 cell lines
harboring hgp130 in association with hgp190/130 or the F328A and G330A
mutants (Table II). The low affinity
binding component (representing hLIF binding to isolated hgp190) was
measured in the presence of the anti-hgp130 blocking mAb B-R3, which is
known to inhibit the heterodimerization of hgp130 with hgp190 (21, 22).
On the hgp190/130 cell line, the high and low affinity binding
components were characterized by equilibrium dissociation constants
(Kd) of 0.21 ± 0.03 nM and
2.4 ± 0.8 nM respectively, values which are
consistent with previous reports (17). For mutant G330A the
Kd for the high affinity binding was unchanged
(Kd, 0.23 ± 0.07 nM), whereas the
Kd for the low affinity binding was slightly higher
(Kd, 6 ± 1.4 nM), in agreement
with its lower hLIF binding ability in the ELISA. For mutants F328A and
002, a very low hLIF binding was observed, which did not permit the
determination of the high and low affinity binding components.
hLIF, hOSM, and mLIF Binding to the Membrane-bound Form of the
Human-to-Mouse hgp190 Mutants--
Membrane-bound forms of the
human-to-mouse mutants were also constructed and transfected with
hgp130 in Ba/F3 cells. We used as a control the native full-length
mLIFR. Cell lines were obtained with all these constructs. Membrane
expression of the mutated receptors and hgp130 was confirmed by flow
cytometry, and Fig. 4A depicts
the staining obtained for three representative cell lines Q251E, N321D,
and Q251E/N321D with the anti-hgp190 mAb 6G8 and the anti-hgp130 mAb
AM64.
All the cell lines raised were tested for their ability to proliferate
in the presence of hLIF, hOSM, and mLIF. Dose-response proliferation
curves are shown in Fig. 4B for mutant Q251E/N321D, and the
control cell lines expressing mLIFR or hgp190/130. The EC50
concentrations are given for all the mutants in Table
III. The Ba/F3 cell line bearing the
hgp190/130 chimera was as responsive to hLIF (EC50 of
0.18 ± 0.05 ng/ml) as the one bearing mLIFR (EC50 of
0.13 ± 0.03 ng/ml); was 40-fold more responsive to hOSM; and was
140-fold less sensitive to mLIF (EC50 of 0.26 ± 0.08 ng/ml versus EC50 of 37.00 ± 5.60 ng/ml)
(Table III and Fig. 4B). All the mutations performed
conferred dose-dependent proliferation with the three
cytokines, and all the cell lines responded identically to mIL-3 (data
not shown). None of the mutations performed altered the response to any
of the three cytokines, and the minimal differences that can be seen on
the figure are not biologically significant (p > 0.05 for all the cell lines tested in comparison to the hgp190/130 receptor,
see Table III), especially for the mutants with a higher affinity for
hLIF in the ELISA (EC50 around 0.10 ng/ml for Q251E, N321D,
and Q251E/N321D; p > 0.1 for all three).
The effect of the Q251E/N321D mutation on the high and low affinity
hLIF binding components was measured by the method of Scatchard, in
comparison with the non-mutated hgp190/130 chimeric receptor or the
mLIFR (Table II). In the context of low affinity binding (gp190
component), the Kd for hgp190/130 was around 15-fold
lower than for mLIFR (Kd of 2.4 ± 0.80 nM for hgp190 versus 0.16 ± 0.02 nM for mLIFR). The dramatic increase in hLIF binding
brought by the double mutation Q251E/N321D, as determined in the ELISA,
corresponded to a 3-fold increase in the affinity of the receptor for
hLIF (Kd of 0.77 ± 0.16 nM). In
the context of high affinity binding (in the presence of hgp130), hLIF
displayed a slightly higher affinity for the mLIFR/hgp130 complex than
for the human receptor complex. However, this could not be explained by
the Q251E/N321D mutation, which did not increase the affinity of hLIF
for the human receptor complex (Table II).
Surface Plasmon Resonance Analysis of the Binding of hLIF to the
Q251E/N321D Mutant of hgp190--
The kinetic association
and dissociation constants kon and
koff for mutant Q251E/N321D in a soluble form
were determined by surface plasmon resonance toward immobilized hLIF
(Fig. 5). As a negative control, we used
mutant 002, which did not bind hLIF at all. Both shgp190myc and mutant
Q251E/N321D bound hLIF with a similar association rate (3.0 × 105 M Enhancement of Membrane Receptor Turnover by Mutations
Q251E/N321D--
The failure to observe an improved hLIF
responsiveness for mutant Q251E/N321D despite a significantly enhanced
binding to hLIF prompted us to analyze whether the improvement of the
binding could come along with a faster turnover of the receptor from
the cell surface (Fig. 6), as already
demonstrated for hgp190 and hgp130 (23, 24). Ba/F3 cells expressing
hgp130 and either hgp190/130, mutant 002 or mutant Q251E/N321D in their
transmembrane form were incubated with hLIF (100 ng/ml) or in the
absence of any added cytokine for up to 320 min. At the indicated
times, the level of membrane receptors was determined by flow cytometry and expressed as percentage of the amount measured at the beginning of
the incubation period. In the absence of hLIF, we observed that the
amount of each of the membrane receptors gradually increased until 160 min, before starting to decrease. In the presence of hLIF, mutant 002 slightly increased, whereas hgp190/130 and mutant Q251E/N321D were
diminished as early as 80 min after the addition of hLIF. However, at
320 min, only mutant Q251E/N321D was still decreasing whereas
hgp190/130 returned to the basal level.
Enhancement of the Blocking Activity of shgp190 on
hLIF-dependent Cell Proliferation by Mutations
Q251E/N321D--
Since soluble gp190 is an inhibitor of LIF
action by competing with the membrane-bound receptor for the binding to
LIF, we hypothesized that the mutant Q251E/N321D in a soluble form
should be a more potent inhibitor of LIF due to its higher affinity for the cytokine. We therefore analyzed its ability to impair hLIF-induced proliferation of the Ba/F3 cell line expressing wild-type hgp190 and
hgp130, with hLIF used at a saturating concentration of 1 ng/ml (Fig.
7, hatched histograms).
Soluble hgp190myc had only a marginal inhibitory effect at high
concentrations (15% at 1.67 µg/ml), whereas mutant 002 did not
impair hLIF-induced proliferation at all, as could be expected due to
its very low affinity for hLIF. We also used normal mouse serum as a
source of mLIFR since it is known to contain about 1-2 µg/ml of this
soluble receptor (11, 25). Mouse serum strongly inhibited the
proliferation of the Ba/F3 cells, although it exerted a nonspecific
trophic effect at low dilutions (1:50 and 1:100, see Fig. 7 and data
not shown), which counterbalanced the blocking effect. Half-maximal blockade was obtained with a serum dilution between 1:200 and 1:400,
which grossly corresponds to 5-10 ng/ml of soluble mLIFR. Mutant
Q251E/N321D displayed a stronger inhibitory activity than shgp190myc on
cell proliferation (30% inhibition versus 15%,
respectively, at 1.67 µg/ml), but was far less efficient than mouse
serum. ELISA measurement of soluble gp190 in culture medium maintained
at 37 °C showed that 40-50% of the shgp190myc and mutant
Q251E/N321D is no more detectable as early as day 1, suggesting that it
is unstable in the conditions of the proliferation assay (data not shown). Therefore, in order to try to improve the effect of the soluble
human receptors, we added in the cell culture medium the shgp190myc and
mutant Q251E/N321D every day over the 3-day-long proliferation assay
(Fig. 7, filled histograms). This treatment significantly
improved the activity of the Q251E/N321D mutant while it only had a
weak effect on shgp190myc (50% inhibition versus 20%,
respectively).
Enhancement of the Blocking Activity of shgp190 on STAT3
Phosphorylation by Mutations Q251E/N321D--
Because of
the quick disappearance of the added receptor in solution, we performed
a short term assay that employed hLIF-induced STAT3 phosphorylation in
the Ba/F3 hgp190+hgp130 cell line, in order to compare the ability of
shgp190myc and mutant Q251E/N321D to inhibit hLIF signaling, with
mutant 002 and mouse serum as controls. For this purpose, the soluble
receptors at three different concentrations were preincubated with a
saturating concentration of hLIF, before adding the cells. After 5 min,
the cells were lysed, and the phosphorylation status of STAT3 was
analyzed by immunoblotting with an antibody specific to the
tyrosine-phosphorylated form of STAT3 (Fig.
8). Phosphorylated STAT3 was not detected in the absence of hLIF, but was strongly induced by the cytokine. A
partial blockade of STAT3 phosphorylation was observed with 10 µg/ml
of shgp190myc. This inhibition was dose-dependent and specific since it was not observed with mutant 002 at the same concentration. In contrast, mutant Q251E/N321D almost completely blocked STAT3 phosphorylation at a concentration as low as 2 µg/ml, demonstrating that it was a much stronger inhibitor of hLIF action than
shgp190myc. In this assay, mouse serum was the most efficient since it
completely blocked STAT3 phosphorylation at a 1:400 dilution (i.e. at an estimated concentration of 5 ng/ml).
Model of the Three-dimensional Structure of the Ig-like
Domain--
The three-dimensional structure of the Ig-like domain
of the hgp190 has not yet been described. We therefore constructed a structural model of this domain based upon the coordinates of the
Ig-like domain of hgp130 (5). Placing the location of the mutations
described above on this model reveals that they are all predicted to be
clustered on a single face of the molecule (Fig.
9), which corresponds in location to the
site of interaction between the Ig-like domain of hgp130 and the
cognate ligand vIL-6 (5). This region corresponds to the
carboxyl-terminal G strand of the Our alanine-scanning analysis of the carboxyl terminus of the
Ig-like domain of hgp190 showed that mutation to alanine of 6 of 8 residues impaired hLIF binding in our ELISA assay, whereas in contrast
hLIF binding was not affected by the mutation of any of the 4 residues
at the very amino terminus of D2. Among the 6 residues thus identified
in ELISA five were hydrophobic (Phe-323, Val-326, Ile-327, Phe-328,
Gly-330) and one hydrophilic (Thr-325). However, in the Ba/F3
proliferation assay, only mutation F328A impaired receptor function.
Scatchard determination of the affinity for hLIF confirmed that mutant
F328A had a dramatically decreased ability to bind hLIF, whereas mutant
G330A displayed only a slight affinity decrease (3-fold). To explain
the apparent discrepancy between the ELISA and the Scatchard
determination, one should consider that the former does not measure
binding at equilibrium in contrast to the latter. In the ELISA, all the
steps occurring after the removal of the excess hLIF are performed in
the absence of the ligand. Part of the hLIF/hgp190 complexes can
dissociate during this 1.5 h time lapse and the loss in the ELISA
signal as compared with equilibrium conditions will be exponentially related to the kinetic dissociation constant of these complexes. Nevertheless, the ELISA helped to discriminate among the mutants with a
strongly decreased ability to bind hLIF, those with a residual binding
ability (i.e. Phe-323, Thr-325, Val-326, Ile-327, and Gly-330) at high hLIF concentrations, and mutant F328A for which no
binding to hLIF could be detected even at the highest concentration of
ligand. Therefore, the ELISA screening assay is more sensitive to
minute variations in binding ability in comparison with the Scatchard
determination. Residue Phe-328 is conserved in the mLIFR protein
sequence, and we found that it was also important for mLIF-induced
proliferation of Ba/F3 cell lines bearing the mutated human receptor.
Therefore, this aromatic amino acid is crucial for ligand binding, as
was previously reported for such residues in other cytokine receptors
(7, 26, 27).
The importance of the carboxyl terminus of the Ig-like region of hgp190
in hLIF binding was confirmed with the analysis of the human-to-mouse
mutants. Interestingly, the stretch of twelve residues that was
analyzed by alanine-scanning presents a higher degree of interspecies
homology than the overall Ig-like domain, since only four amino acids
are different. Three of the four mutants generated displayed a strongly
impaired ability to bind hLIF in the ELISA (I322V, F323Y, and I327V),
as was found with the corresponding alanine mutations for two of them
(Phe-323 and Ile-327). In contrast, mutation N321D increased hLIF
binding 14-fold in the ELISA, while mutation to alanine had no effect.
The amino terminus of the Ig-like domain also displays a high homology
between both species, and we identified mutation Q251E in this region,
which increased hLIF binding by 7-fold. Combining mutations Q251E and
N321D led to a 35-fold increase in hLIF binding by ELISA. Scatchard and
surface plasmon resonance analysis showed that this increased ability to bind hLIF corresponded to a 3-fold increase in affinity for hLIF,
which could be fully attributed to a slower dissociation rate of the
ligand/receptor complex. This is consistent with our interpretation of
the higher sensitivity of the ELISA assay in detecting changes in hLIF
binding ability, as discussed earlier. In addition, the 6G8 and 8C2
anti-hgp190 mAbs used in ELISA to measure the soluble receptor or the
hLIF/receptor complexes, bound with comparable affinities to the
gp190/130 chimera and to the membrane forms of the F328A and
Q251E/N321D mutants, as determined by Scatchard analysis (data not
shown). It becomes therefore evident that the results obtained with the
ELISA cannot be attributed to fluctuations of the affinities of the
anti-gp190 mAbs used toward the different mutated receptors.
Our results also show that the affinity for LIF of the mutants as
determined by the Scatchard analysis does not strictly correlate with
their potency to trigger cell proliferation. Indeed, only mutant F328A
displayed an impaired ability to trigger cell proliferation. We suggest
two possible explanations for this phenomenon. First, in the Ba/F3
proliferation assay, hgp130 may compensate at least partially for a
loss of affinity of hLIF for hgp190, and this may occur via the
stabilization of labile hLIF/hgp190 complexes. Indeed, we previously
reported using a Ba/F3 cell line co-expressing hgp130 and mutant 002 that a few receptors with a high affinity still formed in the presence
of hLIF, probably accounting for the residual proliferation effect (8).
Second, a very low number of functional receptors is sufficient to
trigger the activation signal, since it has been reported previously
for the LIF-responsive murine DA1a that the occupancy of less than 5%
of all 50 membrane receptors was sufficient to trigger half-maximal
proliferation (17), similar results having also been obtained with the
murine M1 cell line (28). Therefore, it may be rather difficult to alter the response to hLIF, unless the affinity for hgp190 is drastically diminished as it is in the case for mutant F328A. For the
mutants with moderately decreased affinity for hLIF, no consequences on
the function of the receptor should be expected. The same reasoning may
be applied to mutant Q251E/N321D, i.e. the increase in
affinity obtained may not be sufficient to have functional
consequences. However, our results also show that the Ba/F3 cell line
expressing mLIFR does not display a higher sensitivity to hLIF since
the EC50 are comparable (see Table III), despite the much
higher affinity for hLIF of the mLIFR. Therefore, it is possible that
the signal triggered by the activation of the receptor cannot be
improved beyond that given by the wild-type receptor simply via an
increase in affinity for the ligand. Alternatively, the mLIFR may not
allow us to form a definitive conclusion, because we used it in
combination with the hgp130 chain, and this heterodimer may not behave
like the human high affinity receptor complex in this respect. As
another explanation for the lack of functional effect of mutant
Q251E/N321D, we noticed that the enhanced binding to hLIF caused a
faster turnover of this mutant in comparison to the non-mutated
hgp190/130 receptor. Therefore, such a decrease in the pool of membrane
receptor could also explain the failure to improve hLIF responsiveness,
via a decrease in cellular hLIF binding capacity and/or a shortening in
the duration of signal transduction through the activated receptors.
Although transmembrane mutant Q251E/N321D did not enhance the
sensitivity to hLIF in comparison to the wild-type receptor, the
soluble form was a more potent inhibitor of hLIF action on hLIF-sensitive cells than shgp190myc. This was more striking with the
short term STAT3 phosphorylation assay than with the 3-day-long MTT
proliferation assay, during which the integrity (via degradation) or
activity of the soluble receptor was deeply affected. These findings
demonstrate that an increase in hLIF binding ability may indeed have
functional consequences, as shown with the soluble receptor, and
reinforce our hypothesis that the lack of enhancing effect of the
membrane form may be intrinsic to the functioning of the high affinity
receptor complex.
Crystallographic and mutagenesis studies have shown that the A to D
four-helix cytokine LIF interacts with gp190 via two epitope sites
called site I and site III (10, 29, 30, 37). Site III consists
of the B-C loop, the C-D loop, and the second half of the D helix, and
contributes the majority of the free energy for the binding (10, 29,
30). The cognate recognition epitopes on the gp190 ligands OSM and CNTF
have also been defined. For these three cytokines, the most prominent
feature of the site III epitope is a conserved solvent-exposed
tryptophan residue in the amino terminus of the D helix (reviewed in
Refs. 2 and 31). In the case of ligands such as IL-11, which interact
with gp130 via site III, the interaction involves an analogous
solvent-exposed non-polar phenylalanine residue (32). Inspection of the
structure of the complex between vIL-6 and hgp130 reveals that this
residue interacts with a glycine residue and the preceding
phenylalanine residue via a pi-stacking mechanism (3). This glycine
residue is conserved in the Ig-like domains of both hgp190 and
OSM-specific receptor chain, as is the presence of an adjacent
non-polar residue (Phe-323 in the case of gp190, Fig. 9). We therefore
suggest that the site III interaction between LIF, OSM and CNTF with
the hgp190 involves burying the exposed tryptophan of the ligand on the
glycine core (Gly-324) with additional interactions occurring with the side chain of Phe-323. This arrangement is essentially similar to the
site II interaction between OSM and hgp130 (31) except in this case the
donor glycine core is on the ligand, and exposed hydrophobic residue is
provided by the receptor. Following the principles established for
other examples of this recognition strategy (2) we would expect that
additional specificity would be conferred by the identity of residues
surrounding the glycine core. The identity of such residues is also
suggested from the mutagenesis studies and the model. Thus Phe-328 is
predicted to form an exposed surface for interaction with ligand as is
Thr-325 adjacent to the glycine core. Collectively these results
suggest that the interaction between hLIF and the Ig-like domain of
hgp190 conforms to a common pattern of ligand/receptor
recognition: a non-polar core interaction is modified by residues in
the immediate vicinity. In this respect we can therefore account for
the increase in affinity observed in the Q251E and N321D mutants: the
predicted fold of the Ig-like domain places these residues in the
vicinity of the core (Fig. 9) and the consequence of the mutation is to introduce, directly or indirectly, additional interaction points with
the ligand. Mutant Q251E/N321D did not increase the response of the
Ba/F3 cells to mLIF. This suggests that by themselves these residues
are not sufficient to improve mLIF binding, although mutant F328A
showed that mLIF binds in the vicinity.
Interestingly, hLIF site III contains three positively charged lysine
residues in a row in the C-D loop, whereas the carboxyl terminus of the
Ig-like domain of mLIFR contains three negatively charged aspartic acid
residues in a row, to which correspond one glutamic acid, one aspartic
acid, and one positively charged asparagine (Asn-321) in the human
receptor at positions 319-321. Therefore, it is tempting to speculate
that these acidic amino acids in the Ig-like domain could participate
in the binding of gp190 to hLIF site III by engaging electrostatic
interactions. If so, the higher affinity of mLIFR for hLIF could be
explained by the difference at position 321, since mutation N321D could
increase the affinity of hgp190 for hLIF via the creation of a stretch
of three consecutive negatively charged amino acids as found in
mLIFR.
This work illustrates a new approach for modulating the action of
cytokines. Mutations of cytokines have been made that either enhanced
their affinity for a low affinity binding receptor, or impaired their
binding to the high affinity transducing chain, providing superagonists
or antagonists, respectively (33-35). Here for the first time, we
bring evidence that the ability to bind a cytokine can also be improved
by mutating a very limited number of residues in the receptor sequence.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) cells with the cDNAs encoding
the soluble receptors and selecting the transfectants in
nucleoside-free medium based on the DHFR gene carried by the
pEDr plasmid. For stable expression of the transmembrane forms of
hgp190 mutants in Ba/F3 cells, the chimeric receptors were
cotransfected by electroporation into Ba/F3 cells in the presence of
wild-type hgp130 cDNA. Transfected cells were selected in the
presence of G418 (Invitrogen) and by progressive replacement of mIL-3
by hLIF as previously described (8).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Punctual mutations in human gp190
ectodomain. Panel A, schematic representation of the
ectodomain of human gp190 showing domain composition. Arrow
indicates mutation 002 at the carboxyl terminus of the Ig-like domain.
Panel B, alignment (Matcher® Program) of the amino acid
sequences of human and murine Ig-like domains of gp190, with the
conserved residues highlighted with double dots. The
delineation of the domains was defined according to the intron-exon
junctions as determined by Tomida and Gotoh (36). Residues targeted in
mutation 002 are in bold type. The box depicts
the residues mutated to alanine. Human-to mouse mutations are
underlined. Residue numbering is for hgp190, according to
Gearing et al. (1).
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Fig. 2.
ELISA measurement of the LIF binding
capability of shgp190myc mutants. LIF binding to shgp190myc
mutants was assessed using a sandwich ELISA. Calculated C50 mean values
are specified on the right side of the figure. Left
panels depict binding curves for representative mutants with hLIF
binding expressed as the ratio B/Bmax with B representing
the amount (in OD) bound at a given hLIF concentration and
Bmax the maximal binding obtained with shgp190myc at
saturating concentrations of hLIF. Right panels depict the
LIF binding capability for all the mutants expressed in percent of that
of shgp190myc. The dashed line and the open
arrowhead highlight the 100% binding value. Neg
control is a supernatant of COS cells transfected with the empty
vector. Panel A, alanine mutants. Binding curves for
shgp190myc ( ), Neg control (
), and representative mutants V326A
(
), F328A (
), G330A (
), and P332A (
) are shown. Panel
B, human-to-mouse mutants. Binding curves for shgp190myc (
),
Neg control (
), and representative mutants D266N (
), I322V (
),
F323Y (
), and Q251E/N321D (
) are shown. One asterisk
indicates mutants for which C50 could not be calculated because of
faint binding, and two asterisks indicate no binding at all.
Results are mean ± S.D. of at least three independent
experiments.
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Fig. 3.
Expression of the alanine
mutants in a membrane-bound form in Ba/F3 cells, and analysis of hLIF-,
hOSM- and mLIF-dependent proliferation. Panel
A, flow cytometry staining of the parent Ba/F3 cell line and the
Ba/F3 cell lines transfected with human wild-type gp130 and either the
non-mutated gp190/130 chimera or the mutants F328A, G330A, and P332A in
a transmembrane form. The following antibodies were used: anti-gp130
mAb AM64 (thin line), anti-gp190 mAb 6G8 (bold
line), and isotype-matched irrelevant antibody (dotted
line). Anti-gp130 mAb H1 and anti-gp190 8C2 and 12D3 mAbs gave
comparable results but are not shown for the sake of clarity.
Panel B, cytokine-dependent proliferation
measured by the MTT assay. Transfected Ba/F3 cell lines were incubated
with serial dilutions of CHO-derived hLIF (50 ng/ml) (graph
on the left), COS-derived hOSM (500 ng/ml) (middle
graph), or E. coli-derived mLIF (10 µg/ml)
(graph on the right). Shown are cell lines
expressing wild-type hgp130 and either the non-mutated hgp190/130
chimera ( ) or the mutants F328A (
), G330A (
), and P332A (
)
fused to the transmembrane and intracellular region of hgp130. The
graphs depict one representative of three experiments performed in
duplicates.
Ability of the alanine mutants of gp190 in a membrane form to trigger
hLIF-,hOSM-, and mLIF-dependent proliferation of
Ba/F3 cell lines
Dissociation constants for representative gp190 mutants as determined
by Scatchard analysis using iodinated hLIF
View larger version (31K):
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Fig. 4.
Expression of the human-to-mouse mutants in a
membrane-bound form in Ba/F3 cells, and analysis of hLIF-, hOSM- and
mLIF-dependent proliferation. Panel A, flow
cytometry staining of the Ba/F3 cell lines transfected with wild-type
hgp130 and the mutants Q251E, N321D, or Q251E/N321D fused to the
transmembrane and intracellular region of hgp130. The following
antibodies were used: anti-hgp130 mAb AM64 (thin line),
anti-hgp190 mAb 6G8 (bold line), and isotype-matched
irrelevant antibody (dotted line). Panel B,
cytokine-dependent proliferation measured by the MTT assay.
Transfected Ba/F3 cell lines were incubated with serial dilutions of
CHO-derived hLIF (50 ng/ml) (graph on the left),
COS-derived hOSM (500 ng/ml) (middle graph), or E. coli-derived mLIF (10 µg/ml) (graph on the
right). Shown are cell lines expressing wild-type hgp130 and
either the non-mutated hgp190/130 chimera ( ) or the mutant
Q251E/N321D (
) fused to the transmembrane and intracellular region
of hgp130 or mLIFR (
). The graphs depict one representative of three
experiments performed in duplicates.
Ability of the human-to-mouse mutants of gp190 in a membrane form to
trigger hLIF-, hOSM-, and mLIF-dependent proliferation of
Ba/F3 cell lines
1 s
1
versus 2.3 × 105
M
1 s
1). However, the
dissociation rate was around three times lower for the mutated receptor
(1.3 × 10
3 s
1 versus
4.2 × 10
3 s
1), leading to a 2.5 times
lower Kd (5.7 nM versus 14.1 nM for shgp190myc). In addition to confirming the result
obtained by the Scatchard method, this experiment demonstrated that
mutations Q251E/N321D increased the affinity of the receptor for hLIF
via an improvement of the stability of the ligand/receptor complex.
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Fig. 5.
Analysis of the association and dissociation
rates of hLIF to the double mutant Q251E/N321D in a soluble form.
CHO-derived shgp190myc, soluble mutant 002, and soluble mutant
Q251E/N321D were purified by affinity chromatography, and the
characteristics of their binding to immobilized CHO-derived hLIF were
assessed with surface plasmon resonance. The association and
dissociation (kon and
koff) rates and the dissociation constant
(Kd) were determined for shgp190myc and soluble
mutant Q251E/N321D.
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Fig. 6.
Increased down-regulation of transmembrane
gp190 from the cell surface by mutations Q251E/N321D. Ba/F3 cells
expressing hgp130 and either the chimeric non-mutated hgp190/130 ( )
or the chimeric mutants gp190(002)/130 (
) or gp190(Q251E/N321D)/130
(
) were starved of hLIF for 3 days in IL-3-supplemented medium,
washed, and incubated without (top panel) or with
(lower panel) 100 ng/ml hLIF at 37 °C. At the indicated
times, membrane hgp190 receptor was detected on the cell surface by
flow cytometry using the non-blocking 8C2 mAb. The expression level of
the receptor is expressed as the percentage of the level found at the
beginning of the experiment (t = 0). Results are
mean ± S.D. of four independent experiments.
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Fig. 7.
Enhancement of the inhibitory action of
soluble hgp190 receptor by mutations Q251E/N321D. Ba/F3 cells
expressing wild-type full-length hgp190 and hgp130 were starved of hLIF
for 3 days in IL-3-supplemented medium, washed, and incubated with hLIF
(1 ng/ml) in the presence of purified shgp190myc (wild type), soluble
mutant 002, or soluble mutant Q251E/N321D at the indicated
concentrations, or mouse serum at the indicated dilutions. The receptor
was added at day 0 only (hatched histograms) or every day
(filled histograms). Cell proliferation was analyzed 3 days
later by the MTT assay. Results are expressed as a percentage of
maximal proliferation obtained in the presence of hLIF alone.
Histograms represent the mean ± S.D. of three experiments.
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Fig. 8.
Effect of soluble mutant Q251E/N321D on
hLIF-induced STAT3 phosphorylation. Ba/F3 cells expressing
wild-type full-length hgp190 and hgp130 were starved of hLIF for 3 days
in IL-3-supplemented medium, washed, and incubated with (+) 10 ng/ml
hLIF or without hLIF ( ) in the presence of purified shgp190myc (wild
type), soluble mutant 002, or soluble mutant Q251E/N321D at the
indicated concentrations, or mouse serum at the indicated dilutions
before the phosphorylation status of STAT3 was analyzed as follows. The
cell lysate (10 µg of protein/lane) was separated on 8% SDS-PAGE and
immunoblotted with an anti-phosphoSTAT3 antibody or an anti-actin
antibody as a control. One representative out of three experiments is
shown.
sandwich and some elements of the
physically adjacent A strand including Gln-251.
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Fig. 9.
Model of the three-dimensional structure of
the Ig-like domain. A structural model of the Ig-like domain of
gp190 was generated and the surface rendered in GRASP. The location of
the core glycine 324 is colored in red, and residues
identified in this study to have an impact on ligand recognition
are colored in blue. Residues whose side chains are
predicted to exhibit solvent exposure in this model include Phe-323,
Phe-328, and Gln-251. The amino and carboxyl termini of the Ig-like
domain are indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
---|
* This work was supported by the Ligue Nationale contre le Cancer (Comités de la Dordogne et des Pyrénées-Atlantiques).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by a grant from the Ligue Nationale contre le Cancer.
§§ To whom correspondence should be addressed. Tel.: 33-5-57-57-14-71; Fax: 33-5-57-57-14-72; E-mail: jean-luc.taupin@umr5540.u-bordeaux2.fr.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M207193200
2 J. Bitard and J.-L. Taupin, unpublished data.
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ABBREVIATIONS |
---|
The abbreviations used are: LIF, leukemia inhibitory factor; CRH, cytokine receptor homology domain; D1, membrane-distal cytokine receptor homology domain; D2, membrane-proximal cytokine receptor homology domain; Ig-like, immunoglobulin-like domain; OSM, oncostatin-M; CNTF, ciliary neurotrophic factor; shgp190, soluble hgp190; IL, interleukin; hgp190, human gp190; mLIFR, murine LIF receptor; STAT3, signal transducer and activator of transcription 3; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; mAb, monoclonal antibody; PBS, phosphate-buffered saline; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; CHO, Chinese hamster ovary.
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