Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 19, 2019
`
`THE JOURNAL OF BIOLOGICAL CHEMISTRY
`© 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
`
`Vol. 275, No. 46, Issue of November 17, pp. 36197–36203, 2000
`Printed in U.S.A.
`
`Interleukin-11 Signals through the Formation of a Hexameric
`Receptor Complex*
`
`Received for publication, May 30, 2000, and in revised form, August 15, 2000
`Published, JBC Papers in Press, August 17, 2000, DOI 10.1074/jbc.M004648200
`
`Victoria A. Barton‡, Mark A. Hall‡§, Keith R. Hudson¶, and John K. Heathi
`From the Cancer Research Campaign Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston,
`Birmingham B15 2TT, United Kingdom
`
`Interleukin-11 (IL-11) is a member of the gp130 family
`of cytokines. These cytokines drive the assembly of mul-
`tisubunit receptor complexes, all of which contain at
`least one molecule of the transmembrane signaling re-
`ceptor gp130. IL-11 has been shown to induce gp130-de-
`pendent signaling through the formation of a high affin-
`ity 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 nondenaturing
`polyacrylamide gel electrophoresis, and its composition
`verified by second dimension denaturing polyacryl-
`amide gel electrophoresis. Results indicate that the
`three receptor binding sites of IL-11 and the Ig-like do-
`main 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.
`
`Interleukin-11 (IL-11)1 is a secreted polypeptide cytokine,
`which has been shown to exhibit in vitro biological effects on a
`diverse range of cell types including hemopoietic cells, hepato-
`cytes, adipocytes, neurons, and osteoblasts (reviewed in Ref. 1).
`
`In vivo administration of IL-11 results in the stimulation of
`megakaryopoeisis and increased platelet counts (2). Recombi-
`nant human IL-11 is now used for the treatment of chemother-
`apy-induced thrombocytopenia (3). IL-11 also has clinical po-
`tential
`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 multisubunit receptor com-
`plexes, which initiates intracellular signal transduction path-
`ways. 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),
`leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary
`neurotrophic factor (CNTF), cardiotrophin-1, and a viral homo-
`logue of IL-6 encoded by the Kaposi’s sarcoma-associated her-
`pesvirus. Each of these cytokines exerts its action by either
`homo- or heterodimerization of gp130, which leads to the stim-
`ulation of signaling 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 the biological
`response depends on the exact composition of the signaling
`receptor complex. Signaling specificity of the gp130 cytokines is
`conferred by the use of ligand-specific receptors. Specific recep-
`tors for IL-6 (15), IL-11 (16, 17), and CNTF (18) have been
`identified. These receptors are not directly involved in cytoplas-
`mic signaling, 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 mem-
`bers of the hemopoietic family of receptors (reviewed in Ref.
`19), characterized by the presence of a cytokine binding homol-
`ogy domain (CHD).
`The CHD, of approximately 200 amino acids, comprises 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 that are important for ligand binding (22–24).
`In addition to the CHD, gp130 and all known receptors that
`bind to the gp130 family of cytokines also contain an amino-
`terminal domain predicted to adopt a seven-b-stranded immu-
`noglobulin-like conformation in their extracellular region (Ig-
`like domain).
`36197
`
`* This work was supported by Cancer Research Campaign grants and
`a Cancer Research Campaign studentship from 1996 to 1999 (to
`V. A. B.). The costs of publication of this article were defrayed in part by
`the payment of page charges. This article must therefore be hereby
`marked “advertisement” in accordance with 18 U.S.C. Section 1734
`solely to indicate this fact.
`‡ These authors contributed equally to this work and should both be
`considered first authors.
`§ Current address: Walter and Elisa Hall Inst. for Medical Research
`and the Rotary Bone Marrow Research Laboratories, Royal Melbourne
`Hospital, Parkville, Victoria, Australia 3050.
`¶ Current address: Genesis Research and Development Corp.,
`Parnell, Auckland, New Zealand.
`i To whom correspondence should be addressed. Tel.: 44-0-121-414-
`7533; Fax: 44-0-121-414-3983; E-mail: j.k.heath@bham.ac.uk.
`1 The abbreviations used are: IL-11, interleukin-11; R, receptor; IL-6,
`interleukin-6; LIF, leukemia inhibitory factor; OSM, oncostatin M;
`CNTF, ciliary neurotrophic factor; CHD, cytokine binding homology
`domain; Ig, immunoglobulin; m (prefix), murine; Fc, constant region of
`human IgG; PCR, polymerase chain reaction; PAGE, polyacrylamide
`gel electrophoresis; BSA, bovine serum albumin; PBS, phosphate-buff-
`ered saline; HRP, horseradish peroxidase; b (prefix), biotinylated; HA,
`hemagglutinin.
`
`This paper is available on line at http://www.jbc.org
`
`Lassen - Exhibit 1006, p. 1
`
`

`

`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 19, 2019
`
`36198
`
`IL-11 Forms a Hexameric Receptor Complex
`
`The gp130 cytokines share a common four-a-helix bundle
`fold. Crystal structures have been determined for LIF (25),
`CNTF (26), IL-6 (27), and OSM.2 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 hu-
`man growth hormone (35). In contrast, LIF and OSM have been
`shown to have two binding sites (sites II and III), which enable
`them to form trimeric receptor complexes (36).2 IL-6 is known
`to form a hexameric receptor complex consisting of two mole-
`cules 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, while sites II and III both
`mediate binding to gp130. Taken together with the finding that
`IL-11 signaling requires IL-11R and gp130, but not LIF recep-
`tor (LIFR) or OSM receptor (OSMR) (16, 39), it is predicted that
`IL-11 forms a signaling 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. (40) reported a pentameric
`IL-11 receptor complex consisting of two IL-11, two IL-11R, and
`one gp130. They suggested that gp130 homodimerization is not
`involved in IL-11-mediated signaling and that another, as yet
`unidentified, signaling receptor component is required. Fur-
`thermore, Grotzinger et al. (41) 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.
`
`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 described previously (32, 39). The
`pIG/mgp130(Ig2)Fc plasmid was derived from pIG/mgp130-Fc (39) by a
`PCR overlap technique. Two DNA fragments upstream and down-
`stream of the region encoding the NH2-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 am-
`plified 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 COOH-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
`NotI, was replaced with a 401-base pair Eco4711/NotI fragment from
`pIG/mLIFR-poly(Asn)-Myc3 that encodes six Asn residues followed by
`the Myc antibody epitope (EEQKLISEEDL).
`pGEX/HA-IL-11 was constructed as follows. The sequence encoding
`the hemagglutinin (HA) tag (YPYDVPDYA) was added to the 59 end of
`the IL-11 coding region by PCR, using a 59 primer encoding a BamHI
`restriction site and the HA tag, and a 39 primer encoding an EcoRI
`restriction site. The PCR product was then subcloned as a 571-base pair
`BamHI/EcoRI fragment into pGEX-3C (42).
`pCDNA3/IL-11R-HA(3C)Fc was generated by the addition of the HA
`tag coding sequence at the 39 end of IL-11R by PCR. The IL-11R-
`ectodomain coding region was amplified using a 59 primer encoding an
`
`EcoRI restriction site, and a 39 primer encoding the HA tag and a
`BamHI restriction site. The PCR fragment was then subcloned as a
`1137-base pair EcoRI/BamHI fragment into pcDNA3–3C-Fc.4
`Expression and Purification of Proteins—Murine IL-11R-Fc, gp130-
`Fc, gp130(Ig2)Fc, IL-11R-HA-Fc, IL-11R-Myc, and gp130-Myc were ex-
`pressed in human embryonic kidney 293T cells (43) by transient trans-
`fection, as described previously (39). Conditioned media containing Fc
`fusion receptors were then subjected to Protein A affinity chromatog-
`raphy and receptor ectodomains were released by on-column cleavage
`with the human rhinovirus protease 3C (44). Conditioned media con-
`taining Myc-tagged proteins were stored at 220 °C until required.
`IL-11, wild type and mutants, and HA-tagged IL-11 were expressed as
`glutathione S-transferase fusion proteins in Escherichia coli (strain
`JM109). Details of induction, purification using glutathione-Sepharose
`(Amersham Pharmacia Biotech) and cleavage using human rhinovirus
`protease 3C, are as described previously for leukemia inhibitory factor
`(36).
`Proteins were analyzed 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 polyvi-
`nylidene difluoride (Millipore) using a standard protocol (45). Mem-
`branes 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).
`Biotinylation of Proteins—IL-11 was modified by biotinylation on
`e-amino groups of lysine residues using biotin amidocaproate N-hy-
`droxysuccinimide (Sigma) following a published protocol (32). gp130
`was biotinylated on oxidized 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 min 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 h
`at 4 °C. Dialysis against PBS was carried out to remove unbound biotin.
`Biotinylated proteins were examined by SDS-PAGE followed by West-
`ern blotting and detection with streptavidin-HRP conjugate (Amer-
`sham Pharmacia Biotech) and ECL.
`Co-immunoprecipitation of Differentially Tagged Cytokine-Receptor
`Complexes—Slightly different strategies were adopted for co-immuno-
`precipitation of the different components of the IL-11 receptor complex.
`For immunoprecipitation of bIL-11zHA-IL-11 complexes, equimolar con-
`centrations (100 nM) of HA-IL-11, bIL-11, gp130, and IL-11R were
`mixed together in various combinations, in a total volume of 500 ml of
`binding buffer (PBS, 1% BSA, 0.05% Tween 20). Mixtures were incu-
`bated for 3 h atroom temperature at which point, NeutrAvidin-agarose
`(Pierce) (10 ml) was added and then agitated for 16 –18 h at 4 °C.
`For immunoprecipitation of complexes containing Myc-tagged com-
`ponents, 5 ml of anti-Myc monoclonal antibody (clone 9E10, BABCo) was
`first immobilized on 8 ml of Protein G-Sepharose (Amersham Pharma-
`cia Biotech), in a final volume of 500 ml of binding buffer. The resin was
`then used to immunoprecipitate gp130-Myc from 500 ml of 293T condi-
`tioned medium. This “loaded” resin was then added to 500 ml of binding
`buffer containing equimolar concentrations (100 nM) of IL-11, IL-11R,
`and bgp130, in various combinations, and incubated for 16 –18 h at 4 °C
`with agitation. Similarly, IL-11R-Myc was immunoprecpitated using
`resin coated with anti-Myc monoclonal antibody and then added to 500
`ml of binding buffer containing equimolar concentrations (100 nM) of
`IL-11, IL-11R-HA, and gp130 or bgp130, in various combinations, and
`incubated for 16 –18 h at 4 °C with agitation.
`Following all immunoprecipitation reactions, complexes were har-
`vested by centrifugation, extensively washed with PBS, 0.1% Tween 20,
`and resuspended in 20 ml of SDS-PAGE loading buffer (300 mM Tris, pH
`6.8, 600 mM dithiothreitol, 12% SDS, 0.6% bromphenol blue, 30% glyc-
`erol). The immunoprecipitated proteins were then resolved by SDS-
`PAGE, Western-blotted, and detected using a mouse anti-HA mono-
`clonal antibody (clone 12CA5, Roche Molecular Biochemicals) or
`streptavidin-HRP conjugate, followed by ECL.
`Nondenaturing 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 16 ml
`of PBS, 0.05% Tween 20. Complexes were allowed to form for a mini-
`mum of 4 h at 18 –22 °C. 4 ml of native gel loading buffer (120 mM Tris,
`pH 6.8, 745 mM glycine, 50% glycerol, 0.5% bromphenol blue) was then
`added and each sample loaded onto a 4 –20% Tris-glycine gel (Novex).
`
`2 Deller, M. C., Hudson, K. R., Ikemizu, S., Bravo, J., Jones, F. Y., and
`Heath, J. K. (2000) Structure 8, 863– 874.
`3 K. R. Hudson, unpublished data.
`
`4 M. A. Hall, unpublished data.
`
`Lassen - Exhibit 1006, p. 2
`
`

`

`IL-11 Forms a Hexameric Receptor Complex
`
`36199
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 19, 2019
`
`FIG. 1. Immunoprecipitation of gp130-Myc complexes. gp130-
`Myc was immobilized on resin and incubated with combinations of
`bgp130, IL-11, and IL-11R (100 nM amounts of each). After incubation
`and washing, bound components were analyzed by SDS-PAGE, followed
`by Western blotting. Detection was carried out using streptavidin-HRP
`conjugate (diluted 1 in 4,000) and ECL.
`
`Electrophoresis was then carried out at 15 mA for 2 h innative 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 and soaked in SDS loading buffer (62.5 mM Tris, pH
`6.8, 5% 2-mercaptoethanol, 2% SDS, 0.1% bromphenol blue, 10% glyc-
`erol) for 5 min. The proteins were then resolved by SDS-PAGE using a
`12% polyacrylamide gel and detected by silver staining (46).
`
`RESULTS
`Multiple Copies of gp130, IL-11R, and IL-11 Are Present in
`the IL-11 Receptor Complex—IL-11-mediated signaling 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 us-
`ing differentially tagged receptor components, similar to the
`assays used by Paonessa et al. (38), were carried out. Each
`component of the receptor complex was labeled with two dif-
`ferent tags. Receptor complexes were then immunoprecipitated
`using one tag and examined by Western blot analysis to deter-
`mine 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, immu-
`noprecipitated bgp130 was detected using streptavidin-HRP
`conjugate. The results, as shown in Fig. 1, indicate that bgp130
`(which migrates with an approximate molecular mass of 97
`kDa) 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.
`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 immobilized IL-11R-Myc and var-
`ious combinations of IL-11, gp130, and IL-11R-HA, the pres-
`ence of the latter was detected using anti-HA antiserum. The
`
`FIG. 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 (100 nM amounts of each). After incuba-
`tion and washing, bound components were analyzed by SDS-PAGE
`followed by Western blotting. Detection was carried out using a bioti-
`nylated anti-HA monoclonal antibody (diluted 1 in 5,000) followed by
`streptavidin-HRP conjugate (diluted 1 in 5,000) and ECL.
`
`FIG. 3. Immunoprecipitation of bIL-11 complexes. Combina-
`tions of bIL-11, HA-IL-11, IL-11R, and gp130 were mixed together and
`incubated (100 nM amounts of each). Complexes were then immunopre-
`cipitated using NeutrAvidin-agarose. After washing, bound components
`were analyzed 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 HRP conjugate (diluted 1 in 5,000)
`and ECL.
`
`results, as shown in Fig. 2, show that IL-11R-HA (which mi-
`grates with an approximate molecular mass of 45 kDa) 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 immo-
`bilized 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 immu-
`noprecipitates was detected using anti-HA antiserum. The re-
`sults, as shown in Fig. 3, show that HA-IL-11 (which migrates
`with an approximate molecular mass of 22 kDa) was co-precip-
`itated 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 de-
`
`Lassen - Exhibit 1006, p. 3
`
`

`

`36200
`
`IL-11 Forms a Hexameric Receptor Complex
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 19, 2019
`
`FIG. 4. Immunoprecipitation of gp130-Myc and IL-11R-Myc
`complexes formed by IL-11 mutants. A, gp130-Myc was immobi-
`lized on resin and incubated with combinations of bgp130, IL-11R, and
`bIL-11 (100 nM amounts of each). B, IL-11R-Myc was immobilized on
`resin and incubated with combinations of bgp130 and bIL-11 (100 nM
`amounts of each). WT represents wild type, while D2 and D3 represent
`the site II mutant R111A/L115A and the site III mutant W147A, re-
`spectively. After incubation and washing, bound components were an-
`alyzed by SDS-PAGE followed by Western blotting. Detection was car-
`ried out using streptavidin-HRP conjugate (diluted 1 in 4,000) and ECL.
`
`scribed above, were also used to examine the ability of IL-11
`mutants to form high affinity receptor complexes. It has previ-
`ously 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, while maintaining
`normal affinity for IL-11R (32). Immunoprecipitation assays
`were performed using immobilized gp130-Myc and various
`combinations of 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 Fig.
`4A, confirm those described earlier, i.e. 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 (Fig. 4A, see lanes 5 and 6). 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
`
`FIG. 5. Nondenaturing PAGE and second dimension SDS-
`PAGE of IL-11 receptor complex. A, native PAGE. Equimolar con-
`centrations (1 mM) of IL-11, IL-11R, and gp130 were mixed together in
`various combinations. After incubation, complexes were subjected to
`nondenaturing PAGE. Proteins were detected using Coomassie stain.
`B, SDS-PAGE. Bands 1* and 2* (see Fig. 5A) were excised from the
`Coomassie-stained gel, soaked in SDS loading buffer, and subjected to
`SDS-PAGE. Proteins were detected by silver staining.
`
`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-pre-
`cipitation of bgp130 with IL-11R-Myc by the two mutants, as
`shown in Fig. 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 tri-
`meric 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 Re-
`solved by Nondenaturing PAGE—The results described above,
`together with the fact that a complex of IL-11 and soluble
`IL-11R can mediate signaling by association with gp130 (39),
`suggest that 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 be-
`tween IL-11 and soluble forms of IL-11R and gp130 can be
`resolved by nondenaturing PAGE. Equimolar quantities (1 mM)
`of IL-11, soluble IL-11R, and soluble gp130 were mixed to-
`gether in various combinations, incubated to allow complexes
`to form, and subjected to nondenaturing PAGE. The results, as
`shown in Fig. 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 follow-
`ing nondenaturing PAGE, as indicated in Fig. 5A. Free IL-11
`does not migrate into the gel, because of its high isoelectric
`
`Lassen - Exhibit 1006, p. 4
`
`

`

`IL-11 Forms a Hexameric Receptor Complex
`
`36201
`
`Downloaded from
`
`http://www.jbc.org/
`
` by guest on July 19, 2019
`
`FIG. 6. Nondenaturing PAGE of receptor complexes formed by
`IL-11 mutants. Equimolar concentrations (300 nM) of IL-11, IL-11R,
`and gp130 were mixed together in various combinations. WT represents
`wild type, while D2 and D3 represent the site II mutant R111A/L115A
`and the site III mutant W147A, respectively. After incubation, com-
`plexes were subjected to nondenaturing PAGE and detection was car-
`ried out using silver staining (46).
`
`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 with the IL-11zIL-11Rzgp130 complex (see
`Fig. 5A, lane 2). However, this IL-11zIL-11R complex was only
`observed if high concentrations (1 mM) of recombinant protein
`were used. If lower concentrations (in the nanomolar range) of
`recombinant protein were used, only the high affinity IL-11zIL-
`11Rzgp130 complex could be detected (see Fig. 6). This is prob-
`ably because complexes of IL-11 and IL-11R dissociate more
`easily compared with 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 with
`binding IL-11R alone (16, 39).
`The high affinity complex resolved by nondenaturing PAGE
`(labeled as band 1* in Fig. 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 Fig. 5B, indicate that IL-11, IL-11R, and
`gp130 were all present in the ternary complex, which resolved
`as a discrete band during nondenaturing PAGE. Similarly, the
`composition of the IL-11zIL-11R complex (labeled as band 2* in
`Fig. 5A) was confirmed using this method (see Fig. 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 nondenaturing 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 ex-
`amined using nondenaturing PAGE. Equimolar concentrations
`(300 nM) of IL-11, IL-11R, and gp130 were mixed together,
`incubated, and subjected to nondenaturing PAGE. Receptor
`complexes were then visualized using silver staining (46). The
`results, as shown in Fig. 6, were consistent with those de-
`scribed above, i.e. 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 (300 nM).
`The results in Fig. 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-migrat-
`
`FIG. 7. Nondenaturing PAGE of IL-11 receptor complexes
`formed by an Ig deletion mutant of gp130. Equimolar concentra-
`tions (300 nM) of IL-11, IL-11R, and gp130 or gp130(Ig2) were mixed
`together in various combinations. After incubation, complexes were
`subjected to nondenaturing PAGE and detection was carried out using
`silver staining (46).
`
`ing 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 with the earlier re-
`sults 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, while 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
`with IL-11 wild type, as described previously (32). The results
`in Fig. 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 nondenaturing PAGE. Instead, a single
`band that 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 com-
`plex, as opposed to a dimer of IL-11 W147A and IL-11R.
`The results described here from nondenaturing PAGE, and
`the immunoprecipitation experiments described earlier, sug-
`gest that the single discrete band formed by IL-11 wild type,
`IL-11R, and gp130 (as seen in lanes 1 of Figs. 5A and 6)
`represents a hexamer. Trimeric receptor complexes can also be
`detected as single bands although they co-migrate with dimers
`of IL-11zIL-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 hex-
`amer, compared with IL-11 wild type (i.e small amounts of
`hexamer were detected), while the site III mutant was unable
`to form a hexamer correlates with the observed biological ac-
`tivities of these two mutants. That is, R111A/L115A shows
`more than a 10-fold reduction in biological activity, compared
`with IL-11 wild type, while 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—The abil-
`ity of a gp130 mutant, lacking the Ig-like domain, to form a
`stable receptor complex was also examined using nondenatur-
`ing 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 nondenaturing
`PAGE. Receptor complexes were then visualized using silver
`staining (46). The results, as shown in Fig. 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