Binding of Leukemia Inhibitory Factor (LIF) to Mutants of Its
Low Affinity Receptor, gp190, Reveals a LIF Binding Site Outside and
Interactions between the Two Cytokine Binding Domains*
Jean-Luc
Taupin
§,
Véronique
Miossec
,
Vincent
Pitard
,
Frédéric
Blanchard¶,
Sophie
Daburon
,
Sylvie
Raher¶,
Yannick
Jacques¶,
Anne
Godard¶, and
Jean-François
Moreau
From
CNRS UMR 5540, Université de Bordeaux II,
Bâtiment 1b, 146 rue Léo-Saignat, 33076 Bordeaux Cedex and
¶ INSERM U463, Institut de Biologie, 9, quai Moncousu,
44035 Nantes Cedex, France
 |
ABSTRACT |
The gp190 transmembrane protein, the low affinity
receptor for the leukemia inhibitory factor (LIF), belongs to the
hematopoietin family of receptors characterized by the cytokine binding
domain (CBD). gp190 is one of the very few members of this family to contain two such domains. The membrane-proximal CBD (herein called D2)
is separated from the membrane-distal one (called D1) by an immunoglobulin-like (Ig) domain and is followed by three fibronectin type III repeats. We used truncated gp190 mutants and a blocking anti-gp190 monoclonal antibody to study the role of these repeats in
low affinity receptor function. Our results showed that the D1Ig region
was involved in LIF binding, while D2 appeared to be crucial for the
proper folding of D1, suggesting functionally important interactions
between the two CBDs in the wild-type protein. In addition, a point
mutation in the carboxyl terminus of the Ig region strongly impaired
ligand binding. These findings suggest that at least two distinct
sites, both located within the D1Ig region, are involved in LIF binding
to gp190, and more generally, that ligand binding sites on these
receptors may well be located outside the canonical CBDs.
 |
INTRODUCTION |
The receptor for the cytokine leukemia inhibitory factor
(LIF)1 comprises gp190, a
transmembrane protein with low affinity for this cytokine, and the
gp130 signal-transducing chain, which is the low affinity receptor for
oncostatin M (1), as well as the signal transducer for IL6, IL11,
ciliary neurotrophic factor (CNTF), OSM, and cardiotrophin-1 (CT-1)
(reviewed in Ref. 2). The last three cytokines also use gp190 as part
of their high affinity receptor complex (3-5), in conjunction with a
specific low affinity binding subunit in the case of CNTF (6) and
probably CT-1 (7). gp190 belongs to the large and growing family of hematopoietin-binding receptors, which is characterized by the presence
of the 200-amino acid-long cytokine binding domain (CBD), which
comprises two modules each of around 100 amino acids, containing 4 conserved cysteine residues in the amino-terminal one and a consensus
WSXWS motif in the carboxyl-terminal one. gp190 is unusual because it contains two CBDs (8), like a few other receptors, i.e. c-Mpl (9), Ob-R (10), and KH-97 (11), which are,
respectively, the thrombopoietin receptor, the leptin (Ob) receptor,
and the
-common signal transducing chain shared by IL3, IL5, and
granulocyte/monocyte-colony stimulating factor in humans. The murine
homologs of KH-97, AIC2A (12) and AIC2B (13), also contain two CBDs.
gp190 and other receptors of this family have additional domains in
their extracellular regions, such as an immunoglobulin-like (Ig) module
of around 100 amino acids situated between the two CBDs in gp190, and a membrane-proximal region encompassing 300 amino acids and similar in
structure to three repeats of type III fibronectin (FN region). Comparison of the primary structure of gp190 with those of other family
members showed that it was most homologous with a group including the
alternative OSM receptor OSM-R
(14), the signal-transducing chain
gp130, G-CSF-R, IL12-R
, and Ob-R, with the percentage of amino acid
identity ranging from 32% for the former to 20% for the latter
(14).
For several members of this family of receptors, the extracellular
region is composed only of one CBD, as is the case with the receptors
for erythropoietin, prolactin (PRL-R), and IL2 (
chain, IL2-R
),
for example. Therefore, the CBD was thought to be fully responsible for
the cytokine binding. This hypothesis was confirmed by deletion studies
and single-point mutagenesis analysis of many members of this family of
receptors, including growth hormone receptor (15), IL2-R
(16), AIC2A
(17), IL6-R gp80 (18), G-CSF-R (19), PRL-R (20), and CNTF-R
chain (21). Because gp190 contains two CBDs and is involved with four different cytokines, one could speculate that these two domains, as
well as the Ig region and the FN repeats, do not have the same importance for ligand binding. In this study, the relationships among
the different constitutive domains of gp190 and LIF and each other were
examined using deletion mutants of the receptor and a panel of
anti-gp190 monoclonal antibodies (mAbs).
 |
EXPERIMENTAL PROCEDURES |
Site-directed Mutagenesis of gp190--
The cDNA encoding
human gp190 was obtained from Dr. C. Wood (Genetics Institute, Boston,
MA). Soluble gp190 (sgp190) consisting of the extracellular region of
the receptor, was obtained by inserting in frame, using polymerase
chain reaction, an XbaI restriction site immediately
followed by a stop codon at the junction with the transmembrane domain
of the molecule, i.e. after nucleotide 2674 or amino acid
832 of the original gp190 sequence (8). This construct was cloned in
XhoI and NotI sites in a polylinker-modified pGEM-3Zf(
) plasmid. A synthesized double-stranded oligonucleotide, consisting of, from 5' to 3', an XbaI site, the sequence
encoding the c-Myc epitope recognized by the mAb 9E10 (22), a stop
codon, an EcoRI site, and a NotI site, was cloned
in frame in this plasmid digested with XbaI and
NotI, to obtain the sgp190myc chimera. The amino acid
sequence of the c-Myc epitope is as follows: SREQKLISEEDL (position of XbaI site is underlined). This chimera was
subcloned in XhoI and EcoRI sites in plasmid
pEDr, a pED4 derivative with an improved polylinker:
5'-BglII-PstI-SalI-XbaI-SmaI-EcoRI-3' (23).
Deletion mutants within sgp190 were prepared by site-directed
mutagenesis using the pALTER-1 phagemid system (Promega,
Charbonnières, France), and following the manufacturer's
recommendations. sgp190 digested from pGEM-3Zf(
) with XhoI
and XbaI, was subcloned in pALTER-1 cut with SalI
and XbaI. Oligonucleotides were synthesized that allowed
creation of an SpeI site or an XbaI site without insertion or deletion, respectively, at the very beginning of the
membrane-distal CBD D1 following the signal sequence (oligonucleotide 001), at the junctions between D1 and the Ig region (oligonucleotide 006), between Ig and the membrane-proximal CBD D2 (oligonucleotide 002), and at the junction between D2 and the FN region of the molecule
(oligonucleotide 003). They generated mutants sgp190(001), sgp190(002),
sgp190(003), and sgp190(006), which were verified by restriction
analysis and DNA sequencing. Taking advantage of a unique
PstI restriction site in the FN region of gp190 cDNA, all these mutants were subcloned in pEDr by exchanging the
PstI fragment from pEDr-sgp190myc, to generate
sgp190(001)myc, sgp190(002)myc, sgp190(003)- myc, and
sgp190(006)myc.
Since all the mutations were made in the same reading frame, this
strategy allowed us to easily obtain by subcloning deletion mutants
lacking one or several of these domains, all fused to the c-Myc
epitope. The following battery of truncated variants of gp190 were
directly constructed from these mutants in the pEDr plasmid: FNmyc,
D1IgD2myc, D1Igmyc, D1myc, D1IgFNmyc, D2myc, and IgD2FNmyc.
Expression and Metabolic Labeling of gp190 Mutants--
Two cell
lines were used. Mycoplasma-free simian COS cells and Chinese hamster
ovary (CHO) dehydrofolate reductase (DHFR)
/
cells were maintained
in Dulbecco's modified Eagle's medium (Life Technologies, Inc., Les
Ulis, France) supplemented with 8% fetal calf serum (Life
Technologies, Inc.). Culture medium for CHO cells also contained
nucleosides (adenosine, deoxyadenosine, thymidine) at 10 µg/ml each
(Sigma), to circumvent DHFR deficiency of these cells.
Transfections were performed as follows. COS cells were transiently
transfected using the DEAE-dextran method, with 5 µg of plasmid DNA.
CHO cells were stably transfected by electroporation at 300 V and 900 microfarads using an Easyject Plus apparatus (Eurogentec, Seraing,
Belgium), and selection was started the next day in medium without
nucleosides to allow emergence of DHFR-producing cells.
Metabolic labeling was performed 72 h after transfection of COS
cells, or on a confluent monolayer of CHO cells, 3-4 weeks after
transfection (around 107 cells/dish). Cells were starved
for 2 h in 4 ml of Dulbecco's modified Eagle's medium without
methionine and cysteine supplemented with 2 mM glutamine
and 5% dialyzed fetal calf serum; 200 µCi of
[35S]methionine/cysteine (Tran35S-label, ICN,
Orsay, France) were added per dish for 12 h. Then, supernatants
were harvested and stored at 4 °C until use.
Immunoprecipitations--
To check for the effective secretion
of the recombinant proteins, 1 ml of COS or CHO supernatant was
precleared with 0.05 ml of a 50% suspension of protein A-Sepharose
beads (Affi-Gel protein A; Bio-Rad, Ivry-sur-Seine, France) for 1 h at 4 °C under continuous rolling. Beads were eliminated by
centrifugation, and supernatants were incubated with 30 µg of the
anti-Myc mAb 9E10 for 2 h under similar conditions. Immune
complexes bound to protein A were sedimented by rapid centrifugation,
and beads were washed three times with 1 ml of washing buffer (50 mM Tris, 1 mM EDTA, 150 mM sodium
chloride, 0.2% Nonidet P-40, pH = 8.0). Bead pellets were
resuspended in 0.025 ml of sample loading buffer containing 0.1 M dithiothreitol and boiled for 5 min. Proteins were
separated by SDS-PAGE on 10% gels and visualized by fluorography.
To study the low affinity complex formation between human LIF and its
gp190 receptor or our deletion mutants, supernatants containing the
35S-labeled receptor component were first incubated for
1 h with 0.4 µg of CHO-derived LIF obtained as described
previously (24). Then the non-blocking anti-LIF mAb 1F10 (30 µg) (25)
was added for another 2 h. Complexes were immunoprecipitated with
protein A, as described above. An alternative protocol was also used; 35S-labeled LIF was produced as described above, and 0.3 ml
were incubated with 0.65 ml of supernatant containing the non-labeled gp190 mutant. Then the receptor was immunoprecipitated with 30 µg of
the specified mAb in 0.05 ml.
Preparation of Anti-human gp190 Monoclonal Antibodies--
The
production and the characterization of a first series of anti-human
gp190 mAbs have been described elsewhere (26). mAbs 12D3 and 2G3, which
have not been described so far, were obtained using the sgp190myc
protein as the immunogen. Domains recognized by the anti-gp190
antibodies were analyzed in a flow cytometric assay, using CHO cells
expressing membrane-bound glycosylphosphatidylinositol-linked forms of
the sgp190 deletion mutants D1IgD2 and D2FN as described previously
(26). Distinction among D1Ig, D2, and FN depended on the flow
cytometric profile obtained for these two cell lines. For example, mAbs
12D3 and 2G3 bound to both of these deletion mutants, demonstrating
that they recognized D2.
Functional Assays in Ba/F3 Cells--
Ba/F3 cells coexpressing
wild-type gp130 together with chimeric sgp190 or its mutants fused to
the transmembrane and intracellular region of gp130 were obtained as
follows. Like the strategy used for gp190, gp130 cDNA was first
mutated at the junction between the extracellular and the transmembrane
regions to create a unique XbaI restriction site, allowing
easy generation of chimeric gp190 mutants by one step subcloning in the
pEDr vector. The chimeric receptors were cotransfected into Ba/F3 in
combination with wild-type gp130 in the pRcglo vector which contains
the neomycin phosphotransferase resistance gene. Transfected cells were
selected with both LIF and G418 (Life Technologies, Inc.), as described
previously (27). The rationale for expressing these chimeric forms of
gp190 was the reported inability of the gp190 intracellular region to
transduce a proliferative signal in Ba/F3 cells in the absence of the
gp130 intracellular region (28). In that report, a chimera expressing the G-CSF extracellular region and the transmembrane and intracellular regions of gp190 did not proliferate upon dimerization in response to
G-CSF, in contrast to a G-CSF-R-gp130 chimera. Two benefits were
expected from this approach. First, co-transfection of the deleted
chimeric receptors fused to transmembrane and intracellular regions of
gp130 together with gp130 would not impair the emergence of
transfectants expressing the two types of receptors and showing dependence on LIF via dimerization of intracellular gp130. Second, transfection of the deleted chimeric receptor alone would easily allow
the emergence of cells spontaneously signaling through homodimerization independently of any cytokine stimulus, which would not be expected with truncated mutants of sgp190 fused to transmembrane and
intracellular regions of gp190, since they do not trigger proliferation
of Ba/F3 cells. The cell lines raised upon progressive replacement of
IL3 with LIF were then tested for their dependence on LIF or OSM (R&D Systems, Indianapolis, IN) and the expression of the receptors by flow
cytometry as described elsewhere (26), using the anti-gp190 mAbs raised
in the laboratory and the H1 anti-gp130 mAb kindly provided by Dr. J. Brochier (INSERM U291, Montpellier, France).
Radioiodination of LIF--
Escherichia
coli-derived human LIF (PeproTech Inc., Rocky Hill, NJ) was
iodinated according to the chloramine-T method (29). LIF was labeled at
a specific radioactivity of around 35,000 cpm/fmol. Binding experiments
were carried out in PBS containing 0.5% bovine serum albumin (PBS-BSA)
as described previously (29). The binding data was subjected to
regression analysis using a one- or two-site equilibrium-binding
equation (Grafit, Erathicus Software, Staines, United Kingdom). Binding
to gp190 mutants was performed either with the Ba/F3 transfectants
cultured for 3 days in the presence of IL3 instead of LIF, then washed
three times and resuspended in PBS-BSA, or with the mutants of the
soluble receptor, as follows. Sgp190myc or a myc-tagged mutant in CHO
or COS supernatants (0.5 µg, quantified with a sandwich enzyme-linked
immunosorbent assay specific for human gp190; Refs. 26 and 30), was
incubated with 10 µg of anti-Myc mAb 9E10 and 0.01 ml of a 50%
suspension of protein A-Sepharose beads, in 0.1 ml of PBS-BSA, for
2 h at 4 °C under continuous rolling. Free LIF was separated
from LIF bound to beads by centrifugation through a dibutylphthalate
cushion at 15,000 rpm for 10 min.
 |
RESULTS |
Production of sgp190myc and Its Deletion Mutants--
The
extracellular region of the low affinity LIF receptor, sgp190, was
subjected to site-directed mutagenesis as described under
"Experimental Procedures," to introduce a unique restriction site
at the boundaries between the different modules D1, Ig, D2, and FN
(Fig. 1A). The mutations
induced two amino acid changes at these positions, except for mutation
003, which induced only one change (Table
I). The mutants and native sgp190 were
fused COOH-terminally and in-frame to the nucleotide sequence encoding the c-Myc epitope recognized by mAb 9E10. From these fusions, a panel
of secreted gp190myc (sgp190myc) truncated mutants was obtained, which
included D1IgD2myc, FNmyc, and mutants lacking either the
membrane-proximal CBD D2 (D1IgFNmyc, D1Igmyc, and D1myc) or the
membrane-distal CBD D1 (IgD2FNmyc and D2myc).

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Fig. 1.
Design and production of the Myc-tagged
truncated mutants of human sgp190. A, mutations
introduced into the gp190 extracellular region to obtain the deletion
mutants. SP depicts the signal peptide. D1, Ig,
D2, and FN, respectively, represent the
membrane-distal CBD, the immunoglobulin-like region, the
membrane-proximal CBD, and the three type III fibronectin repeats.
B, COS cells transfected with plasmids encoding the
specified truncated mutants were labeled with
[35S]methionine/cysteine, and the supernatants were
immunoprecipitated with the anti-Myc mAb 9E10. Proteins were separated
by SDS-PAGE on 10% polyacrylamide gels.
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|
These constructs were transiently expressed in COS cells, and several
of them were stably expressed in CHO cells. Production of the
recombinant proteins was assessed using metabolic labeling of the
cells, followed by immunoprecipitation with the anti-Myc mAb 9E10.
Results of immunoprecipitations from COS cells supernatants are
depicted in Fig. 1B. Wild-type sgp190myc and all but the
D1Igmyc and the D1myc proteins could be detected in variable amounts in the culture supernatants, with molecular masses corresponding to what
was expected from the deletions performed, thereby showing that the
recombinant proteins were correctly processed and secreted. D1Igmyc and
D1myc constructs were stably transfected into CHO cells but, as for COS
cells, these recombinant proteins were not secreted into the
supernatant. In this study, sgp190myc, sgp190(002)myc, and D1IgD2myc
were derived from CHO cells, and FNmyc, D2myc, IgD2FNmyc, D1IgFNmyc, sgp190(001)myc, sgp190(003)myc, and sgp190-(006)myc were
obtained from COS cells. Despite repeated attempts, D1Igmyc and D1myc
could never be obtained in these two cell lines, and thus could not be
further analyzed.
Irrelevance of the FN Region for the Reconstitution of the
LIF-sgp190 Complex in Solution--
35S-Labeled sgp190myc
from CHO cells was incubated with 10 nM 40-kDa CHO-derived
human LIF (0.4 µg/ml). LIF was then immunoprecipitated using the
non-blocking anti-LIF mAb 1F10 (24). SDS-PAGE and autoradiography were
carried out to detect the coprecipitation of the low affinity LIF
receptor. The radiolabeled sgp190myc was immunoprecipitated by the
anti-LIF mAb only in the presence of LIF (Fig.
2), as no specific band was detected with
1F10 when LIF was omitted. We therefore concluded that sgp190 fully
retained its binding capacity when produced in CHO cells. The complex
was also immunoprecipitated using sgp190myc from COS cells, showing that COS cells were also capable of producing functional sgp190 (data
not shown), and that the recombinant proteins produced in this cell
line could be used as well. Preliminary experiments showed that LIF
binding to sgp190myc was dose-dependent, with a maximum
signal obtained with 10 nM LIF. At a higher concentration, an excess of free LIF may have saturated the 1F10 mAb, thereby decreasing the signal. Considering the signal intensity of labeled sgp190myc with as little as 0.2 nM LIF, this system
appeared to be suitable for the detection of sgp190 mutants with at
least a 50-fold decrease in their affinity for LIF (data not
shown).

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Fig. 2.
Reconstitution of the LIF-sgp190 complex in
solution. 35S-Labeled sgp190myc as supernatant from
transfected CHO cells was incubated with 10 nM cold LIF
(lane 1) or without LIF (lanes
2 and 3), and immunoprecipitated with the
anti-LIF mAb 1F10 (lanes 1 and 2) or
the anti-Myc mAb 9E10 (lane 3).
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|
The role of the FN segment of gp190 in LIF binding was investigated in
our immunoprecipitation assay with the truncated D1IgD2myc form of
sgp190myc lacking the FN domain, and the isolated FNmyc fragment. The
full-length point mutants sgp190(001)myc and sgp190(003)myc, which were
used to generate both of these truncated receptors, were also assayed.
Fig. 3 shows that sgp190(001)myc and
sgp190(003)myc were still able to bind LIF, as the anti-LIF mAb 1F10
immunoprecipitated the labeled protein only after preincubation with
LIF. This finding demonstrated that amino acid changes induced by
mutations 001 and 003 neither altered the sgp190 conformation nor
involved residues implicated in ligand binding, at least to a
significant extent. The truncated D1IgD2myc protein was also able to
bind LIF, whereas the FNmyc fragment was not (Fig. 3). In a binding
experiment with iodinated LIF followed by immunoprecipitation via
anti-Myc mAb, the affinity of LIF for D1IgD2myc was measured at 15 ± 7 nM, which was similar to that of sgp190myc in this
assay (see Fig. 7C). Therefore, the membrane-proximal FN
region of gp190 is not involved in LIF binding, a function that
appeared to be accorded to one or the two CBDs D1 and D2 separated by
the Ig region.

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Fig. 3.
Dispensability of the FN region for LIF
binding of gp190. 35S-Labeled mutants sgp190(001)myc
(lanes 1-3), sgp190(003)myc (lanes
4-6), D1IgD2myc (lanes 7-9), and
FNmyc (lanes 10-12) were incubated with 10 nM cold LIF (lanes 2, 5,
8, and 11) or without LIF (lanes
1, 3, 4, 6, 7,
9, 10, and 12). The labeled receptor
was then immunoprecipitated using the anti-Myc mAb (lanes
3, 6, 9, and 12) or the
anti-LIF mAb (lanes 1, 2,
4, 5, 7, 8, 10,
and 11).
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A Crucial Role for D2 in Maintaining a Functional D1--
To
determine the relative importance of each of the two CBDs for LIF
binding, we attempted to reconstitute the ligand-receptor complex with
sgp190 mutants lacking one of them. As described above, the proteins
encoded by the D1Igmyc and D1myc constructs were never detected in the
supernatants of transfected COS and CHO cells. However, using the
anti-Myc mAb, it was possible to immunoprecipitate, from cell lysates,
small amounts as several isoforms of different sizes probably
corresponding to various maturational steps of these proteins (data not
shown). This finding suggested protein instability impairing its
intracellular processing and leading to intracellular degradation prior
to secretion. Since the FN region was not able by itself to bind the
cytokine, the binding function of D1Ig was studied using the D1IgFNmyc
mutant whose FN region forced the secretion of fused D1Ig (Fig. 1). The D1IgFNmyc protein was recognized by the anti-Myc mAb 9E10, but it did
not bind LIF since it was not precipitated using the anti-LIF mAb in
the presence of LIF (Fig. 4A).
The D1Ig conformation in the D1IgFNmyc protein was assessed by
immunoprecipitating the 35S-labeled protein using a panel
of mAbs specific to the D1Ig region of human gp190 that we recently
characterized (26). Fig. 4B shows that none of six mAbs
recognizing different epitopes in the D1Ig region could
immunoprecipitate radiolabeled D1IgFNmyc, although the anti-Myc mAb
did. Among them, four recognized conformation-dependent epitopes (10B2, 1B4, 6G8, and 1C7) because they could not bind to
denatured sgp190myc in Western blot, unlike the 12D9 and 6C10 mAbs,
which apparently bound to linear epitopes (26). These observations
strongly suggested that the D1Ig spatial conformation was profoundly
altered, thus explaining why it could no longer bind to the ligand.
However, D1IgFNmyc could be immunoprecipitated by a polyclonal
anti-D1IgD2 antiserum (26), indicating that the protein was correctly
translated in the cells (data not shown). Therefore, as suspected for
D1Igmyc and D1myc, the absence of D2 seemed to markedly impair protein
conformation, which in turn might have abrogated the binding capacity
of these mutants.

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Fig. 4.
The truncated D1IgFNmyc receptor does not
bind LIF and does not fold properly. A,
35S-labeled sgp190myc (lanes 1-3) or
D1IgFNmyc (lanes 4-6) were incubated with 10 nM LIF (lanes 2 and 5) or
without LIF (lanes 1, 3, 4,
and 6). The receptor was immunoprecipitated with the
anti-LIF mAb 1F10 (lanes 1, 2,
4, and 5) or the anti-Myc mAb 9E10
(lanes 3 and 6). B,
35S-labeled D1IgFNmyc was immunoprecipitated
(arrowhead) with the anti-Myc mAb 9E10 (lane
1), an unrelated mAb (lane 2), or the
following anti-D1Ig mAbs: 1B4, 10B2, 6G8, 1C7 (lanes
3-6, conformation-dependent) and 6C10 and 12D9
(lanes 7 and 8,
conformation-independent).
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The CBD D2 Is Unable to Bind LIF in the Absence of D1--
To
investigate whether D2 could directly interact with LIF, the truncated
IgD2FNmyc receptor lacking D1 was assayed in the receptor-reconstitution assay (Fig.
5A). It was well secreted and
recognized by the anti-Myc antibody, but failed to bind LIF, since the
anti-LIF mAb 1F10 did not immunoprecipitate the radiolabeled truncated
receptor in the presence of LIF. In the binding experiment with
iodinated LIF, no affinity of LIF for this mutant could be measured
(see Fig. 7C). The spatial conformation of D2 was assessed using the two conformation-dependent anti-D2 mAbs we have
obtained so far, 8C2 and 2G3, which bind to two different epitopes on
D2. Both recognized the IgD2FNmyc deletion mutant in the
immunoprecipitation assay (Fig. 5B), whereas the anti-D1Ig
1C7 and 10B2 mAbs did not. This observation suggested that D2 is most
probably properly folded. Therefore, the absence of D1 did not seem to
substantially modify D2 conformation, in contrast to what was observed
for D1 with mutants lacking D2, but the absence of D1 impaired the
capacity of the truncated mutant to interact with LIF.

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Fig. 5.
The truncated IgD2FNmyc receptor does not
bind LIF despite its proper folding. A,
35S-labeled IgD2FNmyc (lanes 1-3)
was incubated with 10 nM cold LIF (lane
2) or without LIF (lanes 1 and
3). The receptor was immunoprecipitated
(arrowhead) with the anti-LIF mAb 1F10 (lanes
1 and 2) or the anti-Myc mAb 9E10
(lane 3). B, 35S-labeled
IgD2FNmyc was immunoprecipitated (arrowhead) with an
irrelevant mAb (lane 1), the anti-Myc mAb 9E10
(lane 2), the anti-D1Ig mAbs 10B2 and 1C7
(lanes 3 and 4), and the anti-D2 mAbs
8C2, 12D3, and 2G3 (lanes 5-7).
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LIF Binding Directly Involves the D1Ig Region--
We previously
reported that the anti-D1Ig mAb 1C7 specifically and
dose-dependently inhibited the LIF-induced proliferation of
Ba/F3 cells expressing wild-type human gp130 and gp190 (26). We
therefore investigated, using our immunoprecipitation assay, whether
the blocking activity of 1C7 was mediated through competition with LIF
for the binding to the low affinity receptor. In such a case, mAb 1C7
would interfere with the precipitation of the radiolabeled cytokine
bound to the non-labeled receptor. Fig. 6
shows that the labeled ligand was recognized by anti-LIF mAb 1F10, and
that the LIF-sgp190myc could be efficiently immunoprecipitated by the
non-blocking anti-gp190 10B2. Conversely, the blocking anti-D1Ig mAb
1C7 did not precipitate any LIF-sgp190 complexes. This failure was not
due to a lower ability of 1C7 to immunoprecipitate sgp190myc, since
both 10B2 and 1C7 bound equally well to the receptor in this assay
(data not shown, and Fig. 7B
with an sgp190 mutant). Therefore, the blocking effect of 1C7 was most
likely explained by competition with LIF for its receptor. This result
suggested that the D1Ig region was directly involved in the interaction with LIF.

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Fig. 6.
The anti-gp190 1C7 blocking antibody competes
with LIF for binding to gp190. 35S-Labeled LIF was
incubated with (lanes 3 and 5) or
without (lanes 1, 2, and 4)
cold sgp190myc, and immunoprecipitated with the non-blocking anti-gp190
mAb 10B2 (lanes 2 and 3) or the
blocking anti-gp190 mAb 1C7 (lanes 4 and
5). LIF was also precipitated directly with the anti-LIF mAb
1F10 (lane 1).
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Fig. 7.
Mutation 002 impairs LIF binding to the
soluble form of the receptor, without altering its conformation.
A, 35S-labeled sgp190(006)myc (lanes
1-3) and sgp190(002)myc (lanes 4-6)
were incubated with 10 nM cold LIF (lanes
2 and 5) or without LIF (lanes
1, 3, 4, and 6). The
receptor was immunoprecipitated (arrowhead) with the
anti-LIF mAb 1F10 (lanes 1, 2,
4, and 5) or the anti-Myc mAb 9E10
(lanes 3 and 6). B,
35S-labeled sgp190(002)myc was immunoprecipitated
(arrowhead) with the anti-Myc mAb (lane
1), an unrelated mAb (lane 2), the
anti-D1Ig mAbs 1B4, 10B2, 6G8, and 1C7 (lanes
3-6), and the anti-D2 mAbs 8C2 and 12D3 (lanes
7 and 8). C, iodinated LIF was
incubated with 0.5 µg of sgp190myc ( ), sgp190(006)myc ( ),
D1IgD2myc ( ), IgD2FNmyc ( ), or sgp190(002)myc ( ), and
immunoprecipitated via anti-myc mAb and protein A beads, before
separating bound and free LIF. The curve depicts the average binding of
sgp190myc, D1IgD2myc, and sgp190(006)myc.
|
|
A LIF Binding Site in the Ig Region Close to the Junction with
D2--
Since the deletion mutants lacking D2 had been obtained by
subcloning from sgp190(002)myc and sgp190(006)myc point mutants, these
latter have also been assayed for their abilities to bind LIF (Fig.
7A). The sgp190(006)myc protein was fully capable of interacting with LIF, indicating that the amino acid changes induced by
this mutation at the junction between D1 and the Ig region did not
impinge on its function. Unexpectedly, the sgp190(002)myc mutant, whose
residues Phe328 and Ala329 in the carboxyl
terminus of the Ig region were mutated, respectively, to Thr and Ser,
was unable to bind LIF in our system. A possible explanation was that
mutation 002 disrupted the overall conformation of the molecule,
thereby affecting its ability to bind LIF, as demonstrated above for
D1IgFNmyc. We then immunoprecipitated the sgp190(002)myc mutant with a
panel of conformation-dependent anti-D1Ig mAbs, including
the blocking 1C7, and with anti-D2 mAbs. Because all these mAbs
recognized the sgp190(002)myc protein (Fig. 7B), we deduced
that the conformation of the protein was not significantly altered by
the mutation. In the equilibrium binding of radioiodinated LIF to
soluble receptor via anti-Myc mAb, no binding of LIF to sgp190(002)myc
could be measured (Fig. 7C), while the affinity of LIF for
sgp190(006)myc was 15 ± 7 nM, which was similar to that for the wild-type sgp190myc. This also suggested that LIF binding
to mutant 002 is of very low affinity. Therefore, the lack of binding
that we observed was most probably due to the disruption of a punctual
site exquisitely involved to some extent in the interaction with
LIF.
The sgp190(002) mutant receptor was fused to the transmembrane and
intracellular region of gp130, and transfected in Ba/F3 cells together
with wild-type gp130. Double-transfectants were selected based on their
capacity to grow in the absence of IL3 but in the presence of LIF, and
tested for: 1) dependence on LIF for proliferation, 2) membrane
expression of the receptor chains by flow cytometry, and 3) binding
characteristics of iodinated LIF. Results were compared with those
obtained with a cell line transfected with both a non-mutated
gp190-gp130 chimeric construct and wild-type gp130, which was raised
simultaneously. Both cell lines proliferated in a
dose-dependent manner in the presence of LIF, OSM, or IL3
as control. However, the cells bearing the mutated gp190(002) had
dramatically lower capability (80-100-fold) to grow in the presence of
subsaturating concentrations of LIF, whereas no difference could be
noted for OSM and IL3 (Fig.
8A). Both cell lines expressed
comparable surface levels of gp190 and gp130, as determined in flow
cytometry using the anti-gp190 10B2 and the anti-gp130 H1 (31) mAbs
(Fig. 8B). Therefore, the different responses of the two
cell lines to LIF and OSM could not be explained by a limiting amount
of gp190 or gp130 membrane receptors, and the unaltered function of
mutant 002 in response to OSM suggested that the intrinsic function of
this mutated receptor was not impaired by the mutation.

View larger version (20K):
[in this window]
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|
Fig. 8.
Mutation 002 impairs LIF binding and the
biological function of the membrane form of the receptor.
A, Ba/F3 cells were cotransfected with full-length gp130 and
wild-type extracellular gp190 (open symbols) or
mutant 002 (closed symbols) fused to
transmembrane and intracellular gp130. Cell lines were incubated with
serial dilutions of IL3-containing WEHI supernatant
(circles), 7 µg/ml LIF (squares), or 1 µg/ml
OSM (triangles). Values represent one of three experiments
in duplicates. The standard error for all points was less than 5% of
the mean. B, Ba/F3 cells expressing gp130 and chimeric
gp190-gp130 (left panel) or mutant
gp190(002)-gp130 (right panel) were stained with
the anti-gp130 mAb H1 (bold line), the anti-gp190
mAb 10B2 (thin line), or an isotype-matched
control (dotted line). C, LIF binding
to Ba/F3 cells expressing gp130 and chimeric gp190-gp130
(filled circles) or mutant gp190(002)-gp130
(open circles) was studied by Scatchard
analysis.
|
|
The LIF binding capacities of the two cell lines were also evaluated
using iodinated LIF (Fig. 8C). The Ba/F3 cell line
coexpressing gp190-gp130 and gp130 displayed significant numbers of low
and high affinity receptors (461 ± 96 and 387 ± 35, respectively), with respective affinities of 1.1 ± 0.65 nM and 32 ± 3.4 pM, comparable to those
previously reported (29, 31). In sharp contrast, the Ba/F3 cell line
bearing gp190(002)-gp130 and gp130 displayed receptors with very low
affinity for iodinated LIF (Kd impossible to
determine accurately), explaining why much higher concentrations of LIF
were required to induce the proliferation of this cell line. As a
whole, a selective loss of binding affinity for LIF due to the
disruption of a LIF binding site at the carboxyl terminus of the Ig
domain impaired the function of gp190.
 |
DISCUSSION |
We demonstrated that the 300-amino acid-long membrane-proximal FN
region was not able by itself to bind LIF and that its deletion did not
impair the capacity of the remaining upstream fragment to interact
normally with the cytokine. Therefore, the binding site(s) lie(s)
within the two CBDs D1 and D2 separated by the Ig-like region. A
homologous FN region is also found in the G-CSF-R (33), the IL12-R
chain (34), the Ob-R (10), and the IL6 signal transducer gp130 (35).
Similar deletions have also been made in G-CSF-R and gp130, and led to
the same conclusions (36, 37). Therefore, it appears to be a general
feature in this family of receptors that the FN region, when present,
has no direct or indirect function in the binding to the specific ligand.
The respective deletion of either D1 or D2 in the truncated IgD2FNmyc
or the D1IgFNmyc receptors abolished ligand binding in our
immunoprecipitation systems. This observation suggested either that
both missing CBDs were necessary for the interaction between LIF and
gp190, or that these truncated molecules had an altered tertiary
structure responsible for the incapacity to bind LIF. Indeed, it is
well known that the correct folding of the protein is absolutely
essential for its exportation outside the cell and its ability to bind
to its ligand(s), as has been shown with the growth hormone receptor
(15, 38), the IL2-R
chain (39), the PRL-R (20), the AIC2A (17), the
IL6-R
chain (18), and the erythropoietin receptor (40). The
truncated D1myc or D1Igmyc receptors were suspected of folding
improperly since they could not be recovered from cell supernatants.
Despite being well secreted, the D1IgFNmyc was not recognized by
anti-D1Ig mAbs, attesting to the profound alterations in the folding of this part of the molecule. As a consequence, no definitive conclusion could be drawn as to the function of D1Ig in the binding to LIF, at
this step. In contrast, the IgD2FNmyc protein was immunoprecipitated by
conformation-dependent anti-D2 mAbs, but was not capable of binding LIF in our two binding assays. Isolated D2myc could also be
recovered from cell supernatants and was recognized by anti-D2 antibodies (data not shown). Although these two mutants strongly suggested that D2 folding was not significantly altered in the absence
of D1, it also argued against a direct involvement of D2 in LIF
binding. Conversely, the proper folding of D1 seemed to depend on the
presence of D2, and the minimal truncated mutant with detectable LIF
binding capability was D1IgD2myc, which as expected harbored a
correctly folded D1Ig region, as demonstrated with a panel of anti-D1Ig
mAbs (data not shown). In experiments not shown, we also replaced D2 by
the homologous CBD from human gp130, which does not bind LIF directly,
but the chimeric protein produced was also unable to bind the cytokine
either, and still bore an improperly folded D1Ig region. Therefore, in
the wild-type gp190 receptor, direct interactions seem to exist between
the two CBDs, which are crucial for receptor conformation and
consequently for ligand binding.
In this regard, the behavior of gp190 appears to be different from that
of human KH-97 and its murine counterparts AIC2A and AIC2B. These
receptors also contain two CBDs, but they immediately follow each other
without an intercalated Ig region. Residues important for cytokine
binding have been located in the membrane-proximal CBD of AIC2A and
KH-97 (17, 41), and a truncated mutant of KH-97 lacking the
membrane-distal CBD was correctly expressed on the cell surface, and
remained functional and dependent on IL3 for signal transduction (42).
This observation demonstrates that, for this particular receptor, one
domain involved in ligand binding is sufficient to achieve its own
proper folding. This situation contrasts with the results described
here for gp190. The Ig domain lying between D1 and D2 could help
reconcile these data; its persistence as a hinge would help maintaining
biological function of the gp190 protein by allowing interactions
between the two CBDs.
The role of the D1Ig region in LIF binding was also studied using the
anti-D1Ig mAb 1C7, which inhibits LIF-induced proliferation of Ba/F3
cells coexpressing gp130 and gp190. Blocking mAb 1C7 was able to
compete with LIF for sgp190 in our immunoprecipitation assay. Although
the 1C7 did not bind to the IgD2FNmyc protein, and in the absence of
available isolated D1 for mapping experiments, we cannot exclude that
it recognizes a conformational epitope in the Ig region. Overall, these
results, in agreement with the immunoprecipitations performed with the
truncated receptors lacking D1, emphasize a major role for D1 and/or Ig
in LIF binding.
The gp190(002) mutant, which bore a punctual mutation at the junction
between the Ig region and the membrane-proximal CBD D2, also shed light
on the LIF-gp190 molecular interactions. First, despite a proper
folding as assessed with anti-gp190 mAbs, sgp190(002)myc did not bind
LIF in the immunoprecipitation assay. This finding also implied that
mutation 002 could not be held responsible for the altered conformation
of the D1Ig region in the D2-deleted D1IgFNmyc truncation mutant, which
was derived from mutant 002. Second, when expressed in Ba/F3 cells, the
chimeric gp190(002)-gp130 receptor remained able to trigger LIF-induced
proliferation, only in the presence of gp130, which indicates the
reconstitution of a high affinity functional tripartite receptor
complex. Although the mutant's biological activity was 80-100 times
lower than that of the non-mutated gp190-gp130 chimeric receptor, this
experiment demonstrated that it was still able to bind LIF. This
difference could not be explained by significantly different amounts of
membrane low affinity receptors and high affinity converters, which
were similar in both cell lines as assessed by flow cytometry. The intrinsic binding ability and signal transduction capacity of mutant
002 was preserved, since the response to OSM was not altered. Scatchard
analysis of LIF binding to Ba/F3 cells expressing gp130 and the mutant,
or to soluble sgp190(002)myc, revealed a markedly decreased affinity
for LIF. Taking into account that at a concentration of 8.5 nM LIF there is less than 5% binding of LIF to
sgp190(002)myc as compared with non-mutated sgp190myc, it can be
calculated that the Kd of mutant 002 for LIF is
higher than 450 nM, corresponding to at least a 30-fold
loss in affinity. On Ba/F3 cells surface, a few receptors with higher
affinity still remained, which were thought to be responsible for the
residual ability to trigger proliferation of Ba/F3 cells. Therefore,
mutation 002 was introduced in an area that is directly and selectively
involved in the binding to LIF, thereby substantially decreasing the
affinity of the receptor for LIF. This could be the consequence of a
faster off-rate, explaining why binding is not detectable at the level
of the low affinity receptor, i.e. in the absence of gp130,
in the experiments performed with sgp190(002)myc. On the contrary, on
the Ba/F3 cell surface, the gp130 could stabilize the interaction
between LIF and mutant 002, allowing signal transduction to occur and
detection of higher affinity receptors, although in small numbers.
The only partial loss of binding and function of mutant 002 could mean
that at least one other LIF binding site exists on gp190. Consistent
with this hypothesis, previous reports involved two distinct binding
sites for LIF on gp190 (28, 43-45). The anti-D1Ig blocking mAb 1C7
impaired LIF binding to gp190 but still recognized sgp190(002)myc.
Therefore, in addition to the site at the carboxyl terminus of the Ig
region, the second LIF binding site is most probably located upstream
within D1Ig. Moreover, since the IgD2FNmyc deletion mutant, which does
not bear the deleterious mutation 002, was unable to bind LIF in the
immunoprecipitation assay, this second LIF binding site could well lie
within D1, and not in the Ig region. This possibility is supported by
other experiments not shown in Ba/F3 cells where the IgD2FN deletion mutant fused to the transmembrane and intracellular region of gp130 and
transfected together with gp130 did not allow the emergence of
LIF-dependent transfected cell lines. However, a residual
activity of the IgD2FN receptor could be expected in Ba/F3 cells, since it does not bear the deleterious mutation 002 and therefore should bind
LIF weakly. If, as proposed above, direct interactions occur between D1
and D2, then the Ig region would be expected to lose any mobility
toward D2 in the wild-type receptor. Such spatial constraints would
stiffen the junction between Ig and D2, and facilitate the interaction
with LIF. It is conceivable that the deletion of D1 in the IgD2FN
molecule frees the Ig region from its spatial constraints toward D2,
thereby masking the LIF binding site and leading to the observed loss
of LIF binding.
Mutation 002 lies one amino acid upstream from the 3' end of the
sequence encoded by exon 7 of the gp190 gene, which encompasses the
full Ig region (46). Recently, it has been reported, based on
experiments using chimeric receptors between murine and human gp190
domains, that the Ig loop is involved in LIF binding (47). In that
study, the downstream boundary of the Ig region was considered to be 7 amino acids downstream from our mutation 002. Therefore, the binding
site we describe at mutation 002 position would still remain within Ig
as defined by those authors. It is noteworthy that mutation 002 substitutes a phenylalanine with a threonine. Phenylalanine and other
hydrophobic aromatic amino acids have often been involved in
interactions with ligands, but so far, they have always been located
within the CBDs (reviewed in Ref. 48). To the best of our knowledge, no
such deleterious mutations have been described in any of the other
receptors belonging to this family. Of note, deletion of the Ig region
in the IL6-R
chain did not decrease the binding of the cytokine
(18), whereas a similar deletion in G-CSF-R only impaired the
reconstitution of the high affinity complex (49). For these two
receptors, the Ig region was proposed to play a role in the
oligomerization of the receptor chains in high affinity complexes (49,
50). From the experiments described herein, the Ig region, at least in
gp190, could be involved in other or additional roles, first as a LIF
binding module, and second as a structurally constrained hinge
sandwiched between the two CBDs whose direct interactions seem
necessary for receptor conformation and function.
 |
ACKNOWLEDGEMENT |
We thank Dr. Eugene Bosmans (Eurogenetics,
Tessenderlo, Belgium) for purification of anti-gp190 antibodies.
 |
FOOTNOTES |
*
This work was supported by the French Association pour la
Recherche sur le Cancer and the Ligue contre le Cancer de Gironde.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.
§
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{at}umr5540.ubordeaux2.fr.
 |
ABBREVIATIONS |
The abbreviations used are:
LIF, leukemia
inhibitory factor;
BSA, bovine serum albumin;
CBD, cytokine binding
domain;
CHO, Chinese hamster ovary;
CNTF, ciliary neurotrophic factor;
CT-1, cardiotrophin-1;
D1, membrane distal CBD;
D2, membrane-proximal
CBD;
DHFR, dihydrofolate reductase;
FN, type III fibronectin repeats;
G-CSF, granulocyte-colony stimulating factor;
Ig, immunoglobulin-like
domain;
IL, interleukin;
mAb, monoclonal antibody;
OSM, oncostatin M;
PAGE, polyacrylamide gel electrophoresis;
PBS, phosphate-buffered
saline;
PRL, prolactin;
R, receptor;
sgp190, soluble extracellular
region of the low affinity LIF receptor.
 |
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