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: j.k.heath@bham.ac.uk

* equal first authors

Running title: IL-11 forms a hexameric receptor complex

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Copyright 2000 by The American Society for Biochemistry and Molecular Biology, Inc.

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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.

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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

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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

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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.

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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).

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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).

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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

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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

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resolved by SDS-PAGE using a 12% polyacrylamide gel and detected by silver

staining (46).

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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.

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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

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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

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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,

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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

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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

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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.

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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).

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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.

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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.

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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

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that the hexameric model is common to ligands that drive the homodimerization of

gp130.

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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.

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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

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(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

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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).

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1 2 3 4

++IL-11

IL-11R +++

+ +gp130myc ++

+

_+bgp130 + +_

_

220K _

30 _

46 _

66 _97.4 _ bgp130_

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220K _

30 _

46 _

66 _97.4 _

21.5 _

IL-11R-HA_

321 4

gp130

IL-11R-myc

IL-11

IL-11R-HA

_

+

++

+

+

_+

+

+

++

+

+

+

_

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21.5 _

66K _

46 _

30 _

14 _

21 43

HA-IL-11 ++ _+bIL-11 ++ ++

IL-11R _+ ++gp130 ++ +_

HA-IL-11_

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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

_

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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_

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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

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4 5 76

IL-11R + + _ +

IL-11 _ + + _

gp130

2

+

+

_

1

+

+

WT WT

3

_+

WT Ig- Ig-Ig-

gp130

IL-11R

hexamer _trimer_

gp130 Ig-

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III

III

III

III

Ig

CHD

I II

III

Ig

CHD

CHD

Ig

III

III

Ig

CHD

III

III

A B

C

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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:

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