Interleukin-11 signals through the formation of a hexameric receptor complex
Victoria A. Barton*, Mark A. Hall*†, Keith R. Hudson§ and John K. Heath¶
From the Cancer Research Campaign Growth Factor Group, School of Biosciences,
University of Birmingham, Edgbaston, Birmingham B15 2TT.
† Current address: Walter and Elisa Hall Institute for Medical Research and The
Rotary Bone Marrow Research Laboratories, PO Royal Melbourne Hospital,
Parkville, Victoria, Australia 3050.
§ Current address: Genesis Research and Development Corporation, 1 Fox Street,
Parnell, Auckland, New Zealand.
¶ To whom correspondence should be addressed: School of Biosciences, University
of Birmingham, Edgbaston, Birmingham, B15 2TT. Tel.: 44-0-121-414-7533, Fax.:
44-0-121-414-3983, Email: [email protected]
* equal first authors
Running title: IL-11 forms a hexameric receptor complex
1
Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on August 17, 2000 as Manuscript M004648200 by guest on A
pril 13, 2018http://w
ww
.jbc.org/D
ownloaded from
Abstract
Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These
cytokines drive the assembly of multi-subunit receptor complexes, all of which
contain at least one molecule of the transmembrane signalling receptor gp130. IL-11
has been shown to induce gp130 dependent signalling through the formation of a
high affinity complex with the IL-11 receptor (IL-11R) and gp130. Site directed
mutagenesis studies have identified three distinct receptor binding sites of IL-11,
which enable it to form this high affinity receptor complex. Here we present data
from immunoprecipitation experiments, using differentially tagged forms of ligand
and soluble receptor components, which show that multiple copies of IL-11, IL-11R
and gp130 are present in the receptor complex. Furthermore, it is demonstrated that
sites II and III of IL-11 are independent gp130 binding epitopes and that both are
essential for gp130 dimerization. We also show that a stable high affinity complex
of IL-11, IL-11R and gp130 can be resolved by non-denaturing PAGE, and its
composition verified by second dimension denaturing PAGE. Results indicate that
the three receptor binding sites of IL-11 and the Ig-like domain of gp130 are all
essential for this stable receptor complex to be formed. We therefore propose that
IL-11 forms a hexameric receptor complex composed of two molecules each of IL-
11, IL-11R and gp130.
2
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Introduction
Interleukin-111 (IL-11) is a secreted polypeptide cytokine which has been shown to
exhibit in vitro biological effects on a diverse range of cell types including
haemopoietic cells, hepatocytes, adipocytes, neurones and osteoblasts (reviewed in
ref.1). In vivo administration of IL-11 results in the stimulation of megakaryopoeisis
and increased platelet counts (2). Recombinant human IL-11 is now used for the
treatment of chemotherapy induced thrombocytopenia (3). IL-11 also has clinical
potential for the treatment of several disorders including chemotherapy induced oral
mucositis (4), Crohn's disease (5) and rheumatoid arthritis (6). Transgenic deletion
of the gene encoding the specific IL-11 receptor (IL-11R) in mice has revealed an
important role for IL-11 in embryonic implantation. Female mice deficient in the IL-
11R are infertile because of defective decidualization, following implantation of the
embryo (7, 8).
IL-11 is a member of the gp130 family of cytokines. These cytokines drive the
assembly of multi-subunit receptor complexes which initiates intracellular signal
transduction pathways. In all cases, the receptor complexes contain at least one copy
of the signal transducer glycoprotein gp130 (9). Other cytokines belonging to this
family include; interleukin-6 (IL-6), leukaemia inhibitory factor (LIF), oncostatin M
(OSM), ciliary neurotrophic factor (CNTF), cardiotrophin-1 (CT-1) and a viral
homologue of IL-6 encoded by the Kaposi's Sarcoma associated Herpes virus
(KSHV-IL-6). Each of these cytokines exerts its action by either homo- or hetero-
dimerization of gp130, which leads to the stimulation of signalling cascades via
protein kinases belonging to the Janus kinase, mitogen-activated protein kinase and
Src families (10-13).
The gp130 cytokines exhibit both overlapping and unique biological activities in
vitro and in vivo (reviewed in ref.14). The signal exerted by a cytokine and therefore
3
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
the biological response depends on the exact composition of the signalling receptor
complex. Signalling specificity of the gp130 cytokines is conferred by the use of
ligand specific receptors. Specific receptors for IL-6 (15), IL-11 (16, 17) and CNTF
(18) have been identified. These receptors are not directly involved in cytoplasmic
signalling, but their function is to promote the formation of a high affinity complex
between the respective ligand and gp130. These ligand specific receptors and gp130
are all members of the haemopoietic family of receptors (reviewed in ref.19),
characterised by the presence of a cytokine binding homology domain (CHD).
The CHD, of approximately 200 amino acids, is comprised of two fibronectin type
III domains (D1 and D2), with four positionally conserved cysteine residues in the
first domain and a WSXWS motif (where X is any amino acid) in the second domain
(20). The crystal structure of the CHD of gp130 revealed that the two fibronectin
type III domains exhibit an approximate L-shape (21) and mutagenesis studies have
identified residues in the hinge region which are important for ligand binding (22-
24). In addition to the CHD, gp130 and all known receptors which bind to the gp130
family of cytokines also contain an amino terminal domain predicted to adopt a
seven β stranded immunoglobulin-like conformation in their extracellular region (Ig-
like domain).
The gp130 cytokines share a common 4 α helix bundle fold. Crystal structures have
been determined for LIF (25), CNTF (26), IL-6 (27) and OSM (28). Detailed
structural analysis and mutagenesis studies of the gp130 family of cytokines have
revealed clear patterns of receptor engagement (reviewed in ref.29). It is now
apparent that receptor binding epitopes are conserved among the gp130 cytokines.
Three receptor binding sites have been identified for IL-6 (30, 31), IL-11 (32) and
CNTF (33, 34) (termed sites I, II and III). Sites I and II are analogous to the two
receptor binding sites identified for human growth hormone (35). In contrast, LIF
4
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
and OSM have been shown to have two binding sites (sites II and III), which enable
them to form trimeric receptor complexes (28, 36). IL-6 is known to form a
hexameric receptor complex consisting of two molecules each of IL-6, IL-6R and
gp130 (37, 38).
IL-11 has been shown to have three distinct receptor binding sites analogous in
location to sites I, II and III of IL-6 (32). Site I enables IL-11 to bind to IL-11R,
whilst sites II and III both mediate binding to gp130. Taken together with the
finding that IL-11 signalling requires IL-11R and gp130, but not LIF receptor (LIFR)
or OSM receptor (OSMR) (16, 39), it is predicted that IL-11 forms a signalling
complex in a manner analogous to that of IL-6. However, published work regarding
the composition and stoichiometry of the IL-11 receptor complex has, as yet, not
been conclusive. Neddermann et al reported a pentameric IL-11 receptor complex
consisting of two IL-11, two IL-11R and one gp130 (40). They suggested that gp130
homodimerization is not involved in IL-11 mediated signalling and that another, as
yet unidentified, signalling receptor component is required. Furthermore, Grotzinger
et al have suggested that the IL-11 receptor complex may be a tetramer, consisting of
one IL-11, one IL-11R and two gp130 molecules (41). Here we report findings from
in vitro immunoprecipitation experiments and gel shift assays which clearly
demonstrate that the IL-11 receptor complex is a hexamer, consisting of two
molecules each of IL-11, IL-11R and gp130.
5
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Experimental Procedures
Plasmid constructs.
The design and construction of pIG/mIL-11R-Fc, pIG/mgp130-Fc and pGEX/mIL-
11 plasmids (IL-11 wild type, R111A/L115A and W147A) has been previously
described (32, 39). The pIG/mgp130(Ig-)Fc plasmid was derived from pIG/mgp130-
Fc (39) by a PCR overlap technique. Two DNA fragments upstream and
downstream of the region encoding the N terminal Ig-like domain of gp130 were
generated by PCR, using two external primers and two internal primers with
overlapping ends (the sequences of all primers used are available on request). The
two DNA fragments were then combined in a subsequent PCR reaction with the two
external primers, which amplified the fusion product of the two fragments. This
fusion product was then cloned back into pIG/mgp130-Fc, therefore replacing the
region encoding the Ig-like domain of gp130.
pIG/mIL-11R-myc and pIG/mgp130-myc, which encode IL-11R and gp130
ectodomains with C-terminal myc-tags, were derived from pIG/mIL-11R-Fc and
pIG/mgp130-Fc (39) by subcloning. For both, the region encoding the Fc region of
human IgG1, bounded by Eco4711 and Not I, was replaced with a 401bp
Eco4711/NotI fragment from pIG/mLIFR-polyAsn-myc (K.R. Hudson, unpublished)
that encodes six Asn residues followed by the myc antibody epitope
(EEQKLISEEDL).
pGEX/HA-IL-11 was constructed as follows. The sequence encoding the
Haemaglutanin (HA) tag (YPYDVPDYA) was added to the 5' end of the IL-11
coding region by PCR, using a 5' primer encoding a BamHI restriction site and the
HA tag, and a 3' primer encoding an EcoRI restriction site. The PCR product was
then sub-cloned as a 571bp BamHI/EcoRI fragment into pGEX-3C (42).
6
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
pCDNA3/IL-11R-HA(3C)Fc was generated by the addition of the HA-tag coding
sequence at the 3' end of IL-11R by PCR. The IL-11R-ectodomain coding region
was amplified using a 5' primer encoding an EcoRI restriction site, and a 3' primer
encoding the HA tag and a BamHI restriction site. The PCR fragment was then sub-
cloned as a 1137bp EcoRI/BamHI fragment into pcDNA3-3C-Fc (M.A. Hall,
unpublished).
Expression and purification of proteins.
Murine IL-11R-Fc, gp130-Fc, gp130(Ig-)Fc, IL-11R-HA-Fc, IL-11R-myc and
gp130-myc were expressed in human embryonic kidney 293T cells (43) by transient
transfection, as described previously (39). Conditioned media containing Fc fusion
receptors were then subjected to Protein A affinity chromatography and receptor
ectodomains were released by on-column cleavage with the Human Rhinovirus
protease 3C (44). Conditioned media containing myc-tagged proteins were stored at
-20° C until required. IL-11, wild type and mutants, and HA tagged IL-11 were
expressed as glutathione S-transferase fusion proteins in E. coli (strain JM109).
Details of induction, purification using Glutathione Sepharose (Pharmacia) and
cleavage using Human Rhinovirus protease 3C, are as described previously for
Leukaemia Inhibitory Factor (36).
Proteins were analysed by SDS-PAGE followed by staining with Coomassie brilliant
blue R250, or Western blotting and detection with antisera. For Western blotting,
proteins were transferred onto PVDF (Millipore) using a standard protocol (45).
Membranes were then blocked overnight in PBS/3% BSA and subjected to
immunodetection using antisera diluted in blocking buffer. Blots were developed
using Super Signal West-Pico Enhanced Chemiluminescence (ECL) (Pierce).
7
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Biotinylation of proteins.
IL-11 was modified by biotinylation on ε-amino groups of lysine residues using
biotin amido-caproate N-hydroxysuccinimide (Sigma) following a published
protocol (32). gp130 was biotinylated on oxidised oligosaccharides using biotin-
hydrazide (Pierce) as follows: gp130 was first buffer exchanged into
phosphate/EDTA buffer (100 mM sodium phosphate pH 6.0, 5 mM EDTA) and
treated with 20 mM sodium m-periodate for 20 minutes at 4° C in the dark. gp130
was then buffer exchanged into fresh phosphate/EDTA buffer and reacted with an
equimolar quantity of biotin-hydrazide for 16-18 hr at 4°. Dialysis against PBS was
carried out to remove unbound biotin. Biotinylated proteins were examined by SDS-
PAGE followed by Western blotting and detection with Streptavidin-HRPO
conjugate (Amersham) and ECL.
Co-immunoprecipitation of differentially tagged cytokine-receptor complexes.
Slightly different strategies were adopted for co-immunoprecipitation of the different
components of the IL-11 receptor complex. For immunoprecipitation of bIL-11/HA-
IL-11 complexes, equimolar concentrations (100nM) of HA-IL-11, bIL-11, gp130
and IL-11R were mixed together in various combinations, in a total volume of 500µl
binding buffer (PBS/1% BSA/0.05% Tween 20). Mixtures were incubated for 3hr at
room temperature at which point, Neutravidin Agarose (Promega) (10µl) was added
and then agitated for 16-18hr at 4° C.
For immunoprecipitation of complexes containing myc-tagged components, 5µl anti-
myc monoclonal antibody (Clone 9E10, BABCo, USA) was first immobilized on 8µl
Protein G-Sepharose (Pharmacia), in a final volume of 500µl binding buffer. The
resin was then used to immunoprecipitate gp130-myc from 500µl 293T conditioned
medium. This 'loaded' resin was then added to 500µl binding buffer containing
equimolar concentrations (100nM) of IL-11, IL-11R and bgp130, in various
8
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
combinations, and incubated for 16-18hr at 4°C with agitation. Similarly, IL-11R-
myc was immunoprecpitated using resin coated with anti-myc monoclonal antibody
and then added to 500µl binding buffer containing equimolar concentrations
(100nM) of IL-11, IL-11R-HA and gp130 or bgp130, in various combinations, and
incubated for 16-18hr at 4°C with agitation.
Following all immunoprecipitation reactions, complexes were harvested by
centrifugation, extensively washed with PBS/0.1% Tween 20 and resuspended in
20µl SDS-PAGE loading buffer (300mM Tris pH6.8, 600mM DTT, 12% SDS ,0.6%
bromophenol blue, 30% glycerol). The immunoprecipitated proteins were then
resolved by SDS-PAGE, Western blotted and detected using a mouse anti-HA
monoclonal antibody (Clone 12CA5, Boehringer-Mannheim) or streptavidin-HRPO
conjugate, followed by ECL.
Non-denaturing PAGE and second dimension denaturing PAGE
Equimolar concentrations of IL-11 and soluble receptor components were mixed
together, in various combinations, in a total volume of 16µl PBS/0.05% Tween 20.
Complexes were allowed to form for a minimum of 4hrs at 18-22˚C. 4µl of native
gel loading buffer (120mM Tris pH 6.8, 745mM glycine, 50% glycerol, 0.5%
bromophenol blue) was then added and each sample loaded onto a 4-20% Tris-
glycine gel (Novex). Electrophoresis was then carried out at 15mA for 2hrs in native
running buffer (24 mM Tris, 149 mM Glycine). Proteins were detected using either
Coomassie brilliant blue R250 or silver staining (46).
For second dimension SDS-PAGE, Coomassie stained bands were excised from the
gel, soaked in SDS loading buffer (62.5mM Tris pH6.8, 5% 2-mercaptoethanol, 2%
SDS, 0.1% bromophenol blue, 10% glycerol) for 5 minutes. The proteins were then
9
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
resolved by SDS-PAGE using a 12% polyacrylamide gel and detected by silver
staining (46).
10
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Results
Multiple copies of gp130, IL-11R and IL-11 are present in the IL-11 receptor
complex
IL-11 mediated signalling has been shown to require both IL-11R and gp130 (16, 32,
39). A specific interaction between IL-11 and the IL-11R has been demonstrated
and a soluble form of IL-11R has been shown to promote the formation of a high
affinity complex between IL-11 and gp130 (39). To assess the stoichiometry of this
high affinity IL-11 receptor complex immunoprecipitation experiments using
differentially tagged receptor components, similar to the assays used by Paonessa et
al (38), were carried out. Each component of the receptor complex was labelled with
two different tags. Receptor complexes were then immunoprecipitated using one tag
and examined by Western blot analysis to determine whether the second tag could be
detected. The ability to co-precipitate differentially tagged forms of a protein
indicated the presence of two copies of that protein in the receptor complex.
To examine the number of gp130 molecules in the IL-11 receptor complex, myc-
tagged gp130 was first immobilized on sepharose beads coated with anti-myc
monoclonal antibody. Immunoprecipitations were then carried out using various
combinations of IL-11, IL-11R and biotinylated gp130 (bgp130). Following SDS-
PAGE and Western blotting, immunoprecipitated bgp130 was detected using
streptavidin HRPO conjugate. The results, as shown in Figure 1, indicate that
bgp130 (which migrates with an approximate molecular mass of 97kDa) was co-
precipitated with gp130-myc, in the presence of IL-11 and IL-11R (see lane 1). In
the absence of either IL-11R or IL-11, immunoprecipitated bgp130 could not be
detected (see lanes 2 and 4). These results show that at least two copies of gp130 are
present in the IL-11 receptor complex and that both IL-11 and IL-11R are required
for gp130 dimerization.
11
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
A similar approach, using myc-tagged IL-11R and HA-tagged IL-11R, was used to
investigate the number of IL-11R molecules present in the IL-11 receptor complex.
Following immunoprecipitations, using immobilised IL-11R-myc and various
combinations of IL-11, gp130 and IL-11R-HA, the presence of the latter was
detected using anti-HA antiserum. The results, as shown in Figure 2, show that IL-
11R-HA (which migrates with an approximate molecular mass of 45kDa) was co-
precipitated with IL-11R-myc, but only in the presence of both IL-11 and gp130 (see
lane 3). This indicates that the high affinity IL-11 receptor complex contains at least
two copies of IL-11R.
To examine the number of IL-11 molecules in the IL-11 receptor complex,
biotinylated IL-11 (bIL-11) was first immobilized on Neutravidin-agarose.
Immunoprecipitations were then carried out using various combinations of IL-11R,
gp130 and HA-tagged IL-11. The presence of HA-IL-11 in the immunoprecipitates
was detected using anti-HA antiserum. The results, as shown in Figure 3, show that
HA-IL-11 (which migrates with an approximate molecular mass of 22kDa) was co-
precipitated with bIL-11, but only in the presence of both IL-11R and gp130 (see
lane 1). This indicates that at least two copies of IL-11 are present in the high
affinity IL-11 receptor complex. The complex thus contains multiple copies of IL-
11, IL-11R and gp130.
IL-11 site II and site III mutants are unable to dimerize gp130
Immunoprecipitation experiments, similar to those described above, were also used
to examine the ability of IL-11 mutants to form high affinity receptor complexes. It
has previously been shown that both the site II mutant, R111A/L115A, and the site
III mutant, W147A, exhibit reduced binding to gp130 and hence reduced biological
activity, whilst maintaining normal affinity for IL-11R (32). Immunoprecipitation
assays were performed using immobilized gp130-myc and various combinations of
12
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
bgp130, IL-11R and wild type or mutant bIL-11. By using both biotinylated ligand
and bgp130, this enabled us to examine the ability of the IL-11 mutants to bind to
gp130-myc in the presence of IL-11R, and also the ability of the mutants to co-
precipitate bgp130. The results, as shown in Figure 4A, confirm those described
earlier; that is, bgp130 is only co-precipitated with gp130-myc in the presence of IL-
11R and wild type IL-11 (see lane 1). Neither the site II mutant, R111A/L115A, nor
the site III mutant, W147A, were able to dimerize gp130, as bgp130 was not co-
precipitated with gp130-myc in the presence of IL-11R (see lanes 5 and 6, Figure
4A). However, the fact that biotinylated mutant ligand was detected (see lanes 5
and 6) indicates that both of the IL-11 mutants were co-precipitated with gp130-myc,
in the presence of IL-11R, even though a second molecule of gp130 was not
detected.
These results provide evidence that sites II and III of IL-11 are independent gp130
binding sites and that both sites are required for the dimerization of gp130. The
results suggest that both the site II mutant and the site III mutant, although unable to
dimerize gp130, can bind a single molecule of gp130 in the presence of IL-11R. This
was confirmed by the co-precipitation of bgp130 with IL-11R-myc by the two
mutants, as shown in Figure 4B. These results indicate that mutation of one gp130
binding site (either site II or site III) does not affect the other gp130 binding site,
which remains free and intact to bind a single molecule of gp130. If IL-11 binds to
IL-11R with a 1:1 stoichiometry, this indicates that the two mutants formed trimeric
complexes, consisting of one molecule each of IL-11, IL-11R and gp130.
A stable complex of IL-11, IL-11R and gp130 can be resolved by non-denaturing
PAGE
The results described above, together with the fact that a complex of IL-11 and
soluble IL-11R can mediate signalling by association with gp130 (39) suggests that
13
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
interactions between IL-11 and the extracellular regions of gp130 and IL-11R are
sufficient for the formation of a high affinity receptor complex. A stable complex
formed between IL-11 and soluble forms of IL-11R and gp130 can be resolved by
non-denaturing PAGE. Equimolar quantities (1µM) of IL-11, soluble IL-11R and
soluble gp130 were mixed together in various combinations, incubated to allow
complexes to form, and subjected to non-denaturing PAGE. The results, as shown
in Figure 5A, show that a complex of IL-11, IL-11R and gp130 can be resolved as a
discrete band (see lane 1), which was not be detected if any one of the three
components was absent. IL-11R and gp130 alone were detected as single bands
following non-denaturing PAGE, as indicated in Figure 5A. Free IL-11 does not
migrate into the gel, because of its high isoelectric value (predicted to be 11.7). A
complex of IL-11 and IL-11R was also detected as a faint band which had migrated
further into the gel compared to the IL-11/IL-11R/gp130 complex (see lane 2, Figure
5A). However, this IL-11/IL-11R complex was only observed if high concentrations
(1µM) of recombinant protein were used. If lower concentrations (in the nanomolar
range) of recombinant protein were used, only the high affinity IL-11/IL-11R/gp130
complex could be detected (see Figure 6). This is probably because complexes of
IL-11 and IL-11R dissociate more easily compared to ternary complexes containing
IL-11, IL-11R and gp130. IL-11 has been previously shown to bind gp130 in the
presence of IL-11R with higher affinity compared to binding IL-11R alone (16, 39).
The high affinity complex resolved by non-denaturing PAGE (labelled as band 1* in
Figure 5A) was predicted to contain IL-11, IL-11R and gp130 because in the absence
of either one of the components it could not be detected. The composition of the
complex was confirmed by second dimension SDS-PAGE. The results, as shown in
Figure 5B, indicate that IL-11, IL-11R and gp130 were all present in the ternary
complex which resolved as a discrete band during non-denaturing PAGE. Similarly,
14
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
the composition of the IL-11/IL-11R complex (labelled as band 2* in Figure 5A) was
confirmed using this method (see Figure 5B).
IL-11 site II and site III mutants are unable to form a stable ternary receptor
complex
To further investigate the stoichiometry of the ternary receptor complex, observed as
a discrete band following non-denaturing PAGE, the ability of IL-11 mutants to form
such a complex was examined. The results described earlier indicate that the site II
mutant, R111A/L115A, and the site III mutant, W147A, are both unable to dimerize
gp130, although they can bind a single molecule of gp130 in the presence of IL-11R.
The ability of these two mutants to form a stable receptor complex was therefore
examined using non-denaturing PAGE. Equimolar concentrations (300nM) of IL-
11, IL-11R, gp130 were mixed together, incubated and subjected to non-denaturing
PAGE. Receptor complexes were then visualized using silver staining (46). The
results, as shown in Figure 6, were consistent with those described above; that is, a
complex of IL-11 wild type, IL-11R and gp130 was observed as a single discrete
band (see lane 1), which could not be detected if either IL-11R or gp130 were absent.
A complex of IL-11 and IL-11R was not detectable using these concentrations of
recombinant protein (300nM).
The results in Figure 6 also show that both the site II mutant, R111A/L115A, and the
site III mutant, W147A, were unable to efficiently form a stable receptor complex
co-migrating with that of IL-11 wild type. The receptor complexes formed by the
site II mutant (see lane 5) include a very faint band co-migrating with the ternary
complex formed by IL-11 wild type, but also a second complex which has migrated
further into the gel. This second complex appears to co-migrate with a dimer of IL-
11 R111A/L115A and IL-11R, observed in lane 6. However, the intensity of the
band is stronger in the presence of gp130 (compare lanes 5 and 6), which, together
15
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
with the earlier results from immunoprecipitation experiments, indicate that this band
is a trimer. The fact that a dimer of IL-11 R111A/L115A and IL-11R was detected,
whilst a dimer of IL-11 wild type and IL-11R was not detectable, correlates with the
fact that the site II mutant has a 4 fold increase in affinity for IL-11R compared to
IL-11 wild type, as previously described (32). The results in Figure 6 also show that
the site III mutant, W147A, was unable to form a stable receptor complex co-
migrating with that of IL-11 wild type during non-denaturing PAGE. Instead, a
single band which migrated further into the gel was detected (see lane 7). This band
was not detected in the absence of gp130 (see lane 8) which suggests that it
represents a trimeric receptor complex, as opposed to a dimer of IL-11 W147A and
IL-11R.
The results described here from non-denaturing PAGE, and the immunoprecipitation
experiments described earlier, suggest that the single discrete band formed by IL-11
wild type, IL-11R and gp130 (as seen in lanes 1 of Figures 5A and 6) represents a
hexamer. Trimeric receptor complexes can also be detected as single bands although
they co-migrate with dimers of IL-11/IL-11R and, like the dimers, they appear to be
less stable than the hexamer. The fact that the site II mutant exhibited a significant
reduction in its ability to form a hexamer, compared to IL-11 wild type (i.e small
amounts of hexamer were detected), whilst the site III mutant was unable to form a
hexamer correlates with the observed biological activities of these two mutants.
That is, R111A/L115A shows more than a ten fold reduction in biological activity,
compared to IL-11 wild type, whilst the activity of W147A is completely abolished,
as described previously (32).
The Ig-like domain of gp130 is required for a stable ternary complex of IL-11, IL-
11R and gp130 to be formed
16
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
The ability of a gp130 mutant, lacking the Ig-like domain, to form a stable receptor
complex was also examined using non-denaturing PAGE. Various combinations of
IL-11 wild type, IL-11R and either wild type or the Ig deletion mutant of gp130 were
mixed together, incubated and subjected to non-denaturing PAGE. Receptor
complexes were then visualized using silver staining (46). The results, as shown in
Figure 7, show that a complex of IL-11, IL-11R and the Ig deletion mutant of gp130
was detectable as a band which migrated further into the gel, compared to the
hexamer formed by IL-11, IL-11R and wild type gp130. This gp130(Ig-) receptor
complex co-migrates with the receptor complex formed by IL-11 W147A, IL-11R
and gp130 (results not shown) and is therefore predicted to be a trimeric receptor
complex.
17
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Discussion
Cytokines mediate their biological effects through the formation of oligomeric
receptor complexes, which initiates intracellular signalling. The nature of this signal,
and the resulting biological response, depends on the specificity and affinity of the
interactions between cytokines and their receptors. Understanding how cytokines
recognise and engage receptors is therefore an important issue in understanding
biological function. Information regarding the mechanism by which a cytokine
exerts a response can also aid the manipulation of cytokine signalling; for example,
in the generation of agonists and/or antagonists which could have therapeutic value.
Both the cytokines and the receptors of the gp130 family share common structural
features, and general patterns of receptor engagement are now emerging. This study
contributes to our understanding of how this family of cytokines form multi-subunit
receptor complexes.
Current evidence suggests that the gp130 family of cytokines initiate intracellular
signalling through either homo- or hetero-dimerization of gp130. IL-11 has
previously been shown to specifically interact with the IL-11R and gp130 (39).
Furthermore, cells expressing these two receptors are responsive to IL-11 (32). It
has also been shown that IL-11 can mediate a biological response through interaction
with a soluble form of the IL-11R and full length gp130, a response which does not
require LIFR or OSMR (16, 39). These results suggest that IL-11 mediates signal
transduction through gp130 homodimerization, but published data regarding the
composition and stoichiometry of the ternary IL-11 receptor complex has as yet not
been conclusive. From immunoprecipitation experiments, similar to those reported
here, Neddermann et al described a pentameric receptor complex consisting of two
IL-11, two IL-11R and one gp130 (40). IL-6, on the other hand, has been shown to
form a hexameric receptor complex, consisting of two molecules each of IL-6, IL-6R
and gp130 (37, 38).
18
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
The results described here from immunoprecipitation experiments show that multiple
copies of gp130 are present in the IL-11 receptor complex. Homo-dimerization of
gp130 was demonstrated to require both IL-11 and IL-11R. Furthermore, it was
shown that multiple copies of both IL-11 and IL-11R are also present in the IL-11
receptor complex. The high affinity IL-11 receptor complex therefore contains at
least two copies each of IL-11, IL-11R and gp130 and since ligand mediated homo-
dimerization of gp130 is clearly sufficient for signal transduction to be initiated, as
demonstrated for IL-6, we propose that the IL-11 receptor complex is a hexamer
(depicted in Figure 8), although we can not formally rule out the possibility of larger
complexes. The fact that wild type IL-11 was shown to induce gp130
homodimerization, in the presence of IL-11R, contradicts previous published results
(40). The formation of a ternary receptor complex containing at least two molecules
of gp130, observed here, excludes the requirement for a novel signalling receptor
component as proposed by Neddermann et al (40).
Further evidence supporting the hexameric model was obtained using two mutant
forms of IL-11, R111A/L115A and W147A, the activities of which have previously
been reported (32). Results from immunoprecipitation experiments revealed that
neither the site II mutant, R111A/L115A, nor the site III mutant, W147A, were able
to dimerize gp130. The fact that both of these mutants were able to bind a single
molecule of gp130 in the presence of soluble IL-11R shows that sites II and III of IL-
11 are independent gp130 binding sites. IL-11 has been shown to interact with the
IL-11R through site I (32), and assuming IL-11 binds to the IL-11R with a 1:1
stoichiometry, this suggests that the mutants formed trimeric complexes consisting of
one molecule each of IL-11, IL-11R and gp130.
19
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
The results presented here from non-denaturing PAGE analysis confirm that
interactions between IL-11 and the extracellular domains of IL-11R and gp130 are
sufficient to form a stable ternary complex. A complex of IL-11, IL-11R and gp130
was resolved by PAGE under non-denaturing conditions and its composition verified
by second dimension denaturing PAGE. A complex of IL-11 and IL-11R was also
detected, although it appeared to be less stable than the ternary IL-11 receptor
complex. Similarly, the predicted IL-11/IL-11R/gp130 trimers formed by both the
IL-11 site II and site III mutants appeared to be much less stable than the ternary IL-
11 receptor complex. This, together with the observation that IL-11 wild type did
not form a stable complex with gp130 in the absence of IL-11R and that fact that IL-
11 binds the IL-11R through site I (32), shows that all three binding sites of IL-11
are required to drive the formation of a stable ternary receptor complex.
It has recently been proposed that the Ig-like domain of gp130 is involved in the
interaction of gp130 with ligands that induce homo-dimerization of gp130, namely
IL-6 and IL-11 (47, 48). The evidence presented here clearly shows that the Ig-like
domain of gp130 is essential for IL-11 to form a high affinity hexameric complex
with IL-11R and gp130. It is predicted that the Ig-like domain of gp130 interacts
with site III of IL-11, as shown in Figure 8. Results from experiments examining a
series of human/murine LIFR chimeras show that the recognition site for OSM and
LIF lies in the Ig-like domain of the LIFR (49, 50). This suggests that the Ig-like
domain of signalling receptors, belonging to the gp130 family, represents a common
motif for binding to site III of the ligand. There is strong evidence, from
mutagenesis studies, to suggest that the gp130 recognition epitope for site II of the
ligands lies in the hinge region of the CHD (22, 48, 51). The existence of two ligand
binding epitopes of gp130, and the evidence presented here that shows IL-11 has two
independent gp130 binding sites, strongly supports the hexameric model depicted in
Figure 8.
20
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
The experimental data reported here clearly shows that IL-11, like IL-6 (52), forms a
stable hexameric receptor complex in vitro, via trimer intermediates. Recently,
Grotzinger et al have proposed that a tetrameric IL-6 receptor complex, not the
hexamer, is responsible for initiating signal transduction (41). They suggest, on the
basis of modelling studies, that the arrangement of one IL-6, one IL-6R and two
gp130 molecules in a tetramer is more favourable for dimerizing gp130 in such a
way that signal transduction can be initiated, and that at high concentrations of IL-6
there is a conformational shift from functional tetramers to inactive hexamers. The
data presented here do not provide any evidence for the existence of tetramers.
Non-denaturing PAGE analysis of IL-11 receptor complexes revealed only one
stable ternary species which, based on the results of immunoprecipitation
experiments, was predicted to be a hexamer. If IL-11 is able to form both stable
tetrameric and hexameric receptor complexes, why were they not both detected
following non-denaturing PAGE? Even with an excess of gp130, only one stable
receptor complex was observed following non-denaturing PAGE (results not shown).
The work presented here also favours the idea that a hexameric receptor complex is
formed by means of intermediate trimeric species. These results do not exclude the
possibility that active tetramers exist and it seems likely that analysis of the
signalling receptor complexes in live cells will be required for decisive testing of this
proposal.
From the evidence presented here it is clear that IL-11, like IL-6, forms a hexameric
receptor complex in vitro. It is predicted that IL-11 binds the IL-11R though site I,
the CHD of gp130 through site II and the Ig-like domain of gp130 through site III.
This is analogous to the way in which IL-6 is thought to bind the IL-6R and two
molecules of gp130, hence forming a hexameric receptor complex, which suggests
21
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
that the hexameric model is common to ligands that drive the homodimerization of
gp130.
22
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
References
1. Du, X., and Williams, D. A. (1997) Blood 89, 3897-908
2. Gordon, M. S., McCaskill-Stevens, W. J., Battiato, L. A., Loewy, J., Loesch, D.,
Breeden, E., Hoffman, R., Beach, K. J., Kuca, B., Kaye, J., and Sledge, G. W.,
Jr. (1996) Blood 87, 3615-24
3. Schwertschlag, U. S., Trepicchio, W. L., Dykstra, K. H., Keith, J. C., Turner, K.
J., and Dorner, A. J. (1999) Leukemia 13, 13075-1315
4. Sonis, S., Muska, A., O'Brien, J., Van Vugt, A., Langer-Safer, P., and Keith, J.
(1995) European Journal of Cancer. Part B, Oral Oncology 31B, 261-6
5. Bank, S., Sninsky, C., Robinson, M., Katz, S., Singleton, J., Miner, P., Safdi,
M., Galandiuk, S., Hanauer, S., Varilek, G., Sands, B., Buchman, A., Rogers,
V., Salzberg, B., Cai, B., Rogge, H., and Schwertschlag, U. (1997) Shock 7, 520
6. Hermann, J. A., Hall, M. A., Maini, R. N., Feldmann, M., and Brennan, F. M.
(1998) Arthritis & Rheumatism 41, 1388-97
7. Bilinski, P., Roopenian, D., and Gossler, A. (1998) Genes & Development 12,
2234-43
8. Robb, L., Li, R., Hartley, L., Nandurkar, H. H., Koentgen, F., and Begley, C. G.
(1998) Nature Medicine 4, 303-8
9. Hibi, M., Murakami, M., Saito, M., Hirano, T., Taga, T., and Kishimoto, T.
(1990) Cell 63, 1149-57
10. Yin, T., and Yang, Y. C. (1994) Journal of Biological Chemistry 269, 3731-8
11. Wang, X. Y., Fuhrer, D. K., Marshall, M. S., and Yang, Y. C. (1995) Journal of
Biological Chemistry 270, 27999-8002
12. Fuhrer, D. K., and Yang, Y. C. (1996) Experimental Hematology 24, 195-203
13. Heinrich, P. C., Behrmann, I., Muller-Newen, G., Schaper, F., and Graeve, L.
(1998) Biochemical Journal 334, 297-314
14. Taga, T., and Kishimoto, T. (1997) Annual Review of Immunology 15, 797-819
23
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
15. Yamasaki, K., Taga, T., Hirata, Y., Yawata, H., Kawanishi, Y., Seed, B.,
Taniguchi, T., Hirano, T., and Kishimoto, T. (1988) Science 241, 825-8
16. Hilton, D. J., Hilton, A. A., Raicevic, A., Rakar, S., Harrison, S. M., Gough, N.
M., Begley, C. G., Metcalf, D., Nicola, N. A., and Willson, T. A. (1994) EMBO
journal 13, 4765-4775
17. Nandurkar, H. H., Hilton, D. J., Nathan, P., Willson, T., Nicola, N., and Begley,
C. G. (1996) Oncogene 12, 585-93
18. Davis, S., Aldrich, T. H., Valenzuela, D. M., Wong, V. V., Furth, M. E.,
Squinto, S. P., and Yancopoulos, G. D. (1991) Science 253, 59-63
19. Cosman, D. (1993) Cytokine 5, 95-106
20. Bazan, J. F. (1990) Proceedings of the National Academy of Sciences of the
United States of America 87, 6934-8
21. Bravo, J., Staunton, D., Heath, J. K., and Jones, E. Y. (1998) EMBO Journal
17, 1665-74
22. Horsten, U., Muller-Newen, G., Gerhartz, C., Wollmer, A., Wijdenes, J.,
Heinrich, P. C., and Grotzinger, J. (1997) Journal of Biological Chemistry 272,
23748-57
23. Olivier, C., Auguste, P., Chabbert, M., Lelievre, E., Chevalier, S., and Gascan,
H. (2000) Journal of Biological Chemistry 275, 5648-5656
24. Timmermann, A., Pflanz, S., Grotzinger, J., Kuster, A., Kurth, I., Pitard, V.,
Heinrich, P. C., and Muller-Newen, G. (2000) FEBS Letters 468, 120-124
25. Robinson, R. C., Grey, L. M., Staunton, D., Vankelecom, H., Vernallis, A. B.,
Moreau, J. F., Stuart, D. I., Heath, J. K., and Jones, E. Y. (1994) Cell 77, 1101-
16
26. McDonald, N. Q., Panayotatos, N., and Hendrickson, W. A. (1995) EMBO
Journal 14, 2689-99
27. Somers, W., Stahl, M., and Seehra, J. S. (1997) EMBO Journal 16, 989-97
24
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
28. Deller, M. C., Hudson, K. R., Ikemizu, S., Bravo, J., Jones, E. Y., and Heath, J.
K. (2000) manuscript submitted
29. Bravo, J., and Heath, J. K. (2000) EMBO Journal 19, 1-13
30. Brakenhoff, J. P., Hart, M., De Groot, E. R., Di Padova, F., and Aarden, L. A.
(1990) Journal of Immunology 145, 561-8
31. Ciapponi, L., Graziani, R., Paonessa, G., Lahm, A., Ciliberto, G., and Savino, R.
(1995) Journal of Biological Chemistry 270, 31249-54
32. Barton, V. A., Hudson, K. R., and Heath, J. K. (1999) Journal of Biological
Chemistry 274, 5755-61
33. Panayotatos, N., Radziejewska, E., Acheson, A., Somogyi, R., Thadani, A.,
Hendrickson, W. A., and McDonald, N. Q. (1995) Journal of Biological
Chemistry 270, 14007-14
34. Di Marco, A., Gloaguen, I., Graziani, R., Paonessa, G., Saggio, I., Hudson, K.
R., and Laufer, R. (1996) Proceedings of the National Academy of Sciences of
the United States of America 93, 9247-52
35. de Vos, A. M., Ultsch, M., and Kossiakoff, A. A. (1992) Science 255, 306-12
36. Hudson, K. R., Vernallis, A. B., and Heath, J. K. (1996) Journal of Biological
Chemistry 271, 11971-8
37. Ward, L. D., Howlett, G. J., Discolo, G., Yasukawa, K., Hammacher, A.,
Moritz, R. L., and Simpson, R. J. (1994) Journal of Biological Chemistry 269,
23286-9
38. Paonessa, G., Graziani, R., De Serio, A., Savino, R., Ciapponi, L., Lahm, A.,
Salvati, A. L., Toniatti, C., and Ciliberto, G. (1995) EMBO Journal 14, 1942-51
39. Karow, J., Hudson, K. R., Hall, M. A., Vernallis, A. B., Taylor, J. A., Gossler,
A., and Heath, J. K. (1996) Biochemical Journal 318, 489-95
40. Neddermann, P., Graziani, R., Ciliberto, G., and Paonessa, G. (1996) Journal of
Biological Chemistry 271, 30986-91
25
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
41. Grotzinger, J., Kernebeck, T., Kallen, K. J., and Rose-John, S. (1999) Biological
Chemistry 380, 803-813
42. Staunton, D., Hudson, K. R., and Heath, J. K. (1998) Protein Engineering 11,
1093-102
43. DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., and Calos,
M. P. (1987) Molecular & Cellular Biology 7, 379-87
44. Walker, P. A., Leong, L. E., Ng, P. W., Tan, S. H., Waller, S., Murphy, D., and
Porter, A. G. (1994) Bio/Technology 12, 601-5
45. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, First Ed.,
Cold Spring Harbor Press, New York
46. Ansorge, W. (1985) Journal of Biochemical and Biophysical Methods 11, 13-
20
47. Hammacher, A., Richardson, R. T., Layton, J. E., Smith, D. K., Angus, L. J.,
Hilton, D. J., Nicola, N. A., Wijdenes, J., and Simpson, R. J. (1998) Journal of
Biological Chemistry 273, 22701-7
48. Kurth, I., Horsten, U., Pflanz, S., Dahmen, H., Kuster, A., Grotzinger, J.,
Heinrich, P. C., and Muller-Newen, G. (1999) Journal of Immunology 162,
1480-7
49. Owczarek, C. M., Zhang, Y., Layton, M. J., Metcalf, D., Roberts, B., and
Nicola, N. (1997) Journal of Biological Chemistry 272, 23976-23985
50. Chobotova, K., Hudson, K. R., and Heath, J. K. (2000) manuscript submitted
51. Dahmen, H., Horsten, U., Kuster, A., Jacques, Y., Minvielle, S., Kerr, I. M.,
Ciliberto, G., Paonessa, G., Heinrich, P. C., and Muller-Newen, G. (1998)
Biochemical Journal 331, 695-702
52. Simpson, R. J., Hammacher, A., Smith, D. K., Matthews, J. M., and Ward, L. D.
(1997) Protein Science 6, 929-55
26
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Acknowledgements
We are grateful to the Cancer Research Campaign for funding this work and
providing V.A. Barton with a studentship (1996-1999). We would like to thank Dr
Lisa McGovern for discussion of the manuscript.
1 The abbreviations used are: IL-11, interleukin-11; R, receptor; IL-6, interleukin-6;
LIF, leukaemia inhibitory factor; OSM, oncostatin M; CNTF, ciliary neurotrophic
factor; CT-1, cardiotrophin-1; KSHV, Kaposi's sarcoma associated herpes virus;
CHD, cytokine binding homology domain; Ig, immunoglobulin; m (prefix), murine;
Fc, constant region of human IgG; PCR, polymerase chain reaction; PAGE,
polyacrylamide gel electrophoresis; PVDF, polyvinlyidene di-flouride; BSA, bovine
serum albumin; PBS, phosphate buffered saline; HRPO, horse-radish peroxidase;
ECL, enhanced chemiluminescence; b (prefix), biotinylated; kDa, kilodaltons.
27
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Figure 1. Immunoprecipitation of gp130-myc complexes. gp130-myc was
immobilized on resin and incubated with combinations of bgp130, IL-11 and IL-11R
(100nM of each). After incubation and washing, bound components were analysed
by SDS-PAGE followed by Western blotting. Detection was carried out using
streptavidin HRPO conjugate (diluted 1 in 4,000) and ECL.
Figure 2. Immunoprecipitation of IL-11R-myc complexes. IL-11R-myc was
immobilized on resin and incubated with combinations of IL-11R-HA, gp130 and
IL-11 (100nM of each). After incubation and washing, bound components were
analysed by SDS-PAGE followed by Western blotting. Detection was carried out
using a biotinylated anti-HA monoclonal antibody (diluted 1 in 5,000) followed by
streptavidin HRPO conjugate (diluted 1 in 5,000) and ECL.
Figure 3. Immunoprecipitation of bIL-11 complexes. Combinations of bIL-11,
HA-IL-11, IL-11R and gp130 were mixed together and incubated (100nM of each).
Complexes were then immunoprecipitated using Neutravidin-agarose. After
washing, bound components were analysed by SDS-PAGE followed by Western
blotting. Detection was carried out using an anti-HA monoclonal antibody (diluted 1
in 5,000) followed by sheep anti-mouse HRPO conjugate (diluted 1 in 5,000) and
ECL.
Figure 4. Immunoprecipitation of gp130-myc and IL-11R-myc complexes
formed by IL-11 mutants. A: gp130-myc was immobilized on resin and incubated
with combinations of bgp130, IL-11R and bIL-11 (100nM of each). B: IL-11R-myc
was immobilized on resin and incubated with combinations of bgp130 and bIL-11
28
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
(100nM of each). WT represents wild type, whilst ∆2 and ∆3 represent the site II
mutant R111A/L115A and the site III mutant W147A, respectively. After incubation
and washing, bound components were analysed by SDS-PAGE followed by Western
blotting. Detection was carried out using streptavidin HRPO conjugate (diluted 1 in
4,000) and ECL.
Figure 5. Non-denaturing PAGE and second dimension SDS-PAGE of IL-11
receptor complex. A: Native PAGE. Equimolar concentrations (1µM) of IL-11, IL-
11R and gp130 were mixed together in various combinations. After incubation,
complexes were subjected to non-denaturing PAGE. Proteins were detected using
Coomassie stain. B: SDS-PAGE. Bands 1* and 2* (see Figure 5A) were excised
from the Coomassie stained gel, soaked in SDS loading buffer, and subjected to
SDS-PAGE. Proteins were detected by silver staining.
Figure 6. Non-denaturing PAGE of receptor complexes formed by IL-11
mutants. Equimolar concentrations (300nM) of IL-11, IL-11R and gp130 were
mixed together in various combinations. WT represents wild type, whilst ∆2 and ∆3
represent the site II mutant R111A/L115A and the site III mutant W147A,
respectively. After incubation, complexes were subjected to non-denaturing PAGE
and detection was carried out using silver staining (46).
Figure 7. Non-denaturing PAGE of IL-11 receptor complexes formed by an Ig
deletion mutant of gp130. Equimolar concentrations (300nM) of IL-11, IL-11R
and gp130 or gp130(Ig-) were mixed together in various combinations. After
29
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
incubation, complexes were subjected to non-denaturing PAGE and detection was
carried out using silver staining (46).
Figure 8. Schematic representation of the compositions and stoichiometries of
IL-11 receptor complexes. Data reported here provide evidence for IL-11/IL-11R
dimers (A), IL-11/IL-11R/gp130 trimers formed by both site II and site III mutants
of IL-11 (B) and the hexameric receptor complex (C). The IL-11, IL-11R and gp130
components are shown in red, blue and green, respectively. This model illustrates
the interactions between the three receptor binding sites of IL-11, IL-11R and two
epitopes of gp130, the cytokine binding homology domain (CHD) and the Ig-like
domain (Ig).
30
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
1 2 3 4
++IL-11
IL-11R +++
+ +gp130myc ++
+
_+bgp130 + +_
_
220K _
30 _
46 _
66 _97.4 _ bgp130_
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
220K _
30 _
46 _
66 _97.4 _
21.5 _
IL-11R-HA_
321 4
gp130
IL-11R-myc
IL-11
IL-11R-HA
_
+
++
+
+
_+
+
+
++
+
+
+
_
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
21.5 _
66K _
46 _
30 _
14 _
21 43
HA-IL-11 ++ _+bIL-11 ++ ++
IL-11R _+ ++gp130 ++ +_
HA-IL-11_
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
220K97.4
66
46
30
21.5
_
_
_
_
_
_ bgp130_
bIL-11_
gp130-myc
IL-11R
bgp130
bIL-11
+
++
WT
+
++
D2
+
++
D3
+
_+
WT
+
+
_
WT
1 5 62 3 4
+
++
_
A
B
220K97.4
66
46
30
21.5
_
_
_
_
_
_ bgp130_
bgp130
IL-11
IL-11R-myc
5
+
D2
+
6
+
D3
+
2
+
WT
+
3
_
WT
+
4
+_
+
1
+
WT
_
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
gp130
IL-11IL-11R
2
_
++
4
+
_+
1
+
++
3
+
+_
gp130_
IL-11R_
band 2* _band 1* _
A
B
1* 2*
220K _
30 _
46 _
66 _97.4 _
_21.5
gp130_
IL-11_
IL-11R_
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
gp130
IL-11R
1
+
+
WT
3
_+
WT
2
+
_
WT IL-11
4 5
+
+
D2
+
+
D3_+
+
6
hexamer _gp130IL-11R
dimer/trimer _
+ +
_
D2
7 8
_
D3
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
4 5 76
IL-11R + + _ +
IL-11 _ + + _
gp130
2
+
+
_
1
+
+
WT WT
3
_+
WT Ig- Ig-Ig-
gp130
IL-11R
hexamer _trimer_
gp130 Ig-
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
III
III
III
III
Ig
CHD
I II
III
Ig
CHD
CHD
Ig
III
III
Ig
CHD
III
III
A B
C
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Victoria A. Barton, Mark A. Hall, Keith R. Hudson and John K. HeathInterleukin-11 signals through the formation of a hexameric receptor complex
published online August 17, 2000J. Biol. Chem.
10.1074/jbc.M004648200Access the most updated version of this article at doi:
Alerts:
When a correction for this article is posted•
When this article is cited•
to choose from all of JBC's e-mail alertsClick here
by guest on April 13, 2018
http://ww
w.jbc.org/
Dow
nloaded from
Top Related