Identification of Three Distinct Receptor Binding Sites of Murine Interleukin-11*

Victoria A. BartonDagger , Keith R. Hudson§, and John K. Heath

From the Cancer Research Campaign Growth Factor Group, Department of Biochemistry, University of Birmingham, Edgbaston, Birmingham, B15 2TT, United Kingdom

    ABSTRACT
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Abstract
Introduction
References

Interleukin-11 (IL-11) is a member of the gp130 family of cytokines. These cytokines drive the assembly of multisubunit receptor complexes, all of which contain at least one molecule of the transmembrane signaling receptor gp130. A complex of IL-11 and the IL-11 receptor (IL-11R) has been shown to interact with gp130, with high affinity, and to induce gp130- dependent signaling. In this study, we have identified residues crucial for the binding of murine IL-11 (mIL-11) to both the IL-11R and gp130 by examining the activities of mIL-11 mutants in receptor binding and cell proliferation assays. The location of these residues, as predicted from structural studies and a model of IL-11, reveals that mIL-11 has three distinct receptor binding sites. These are structurally and functionally analogous to the previously defined receptor binding sites I, II, and III of interleukin-6 (IL-6). This supports the hypothesis that IL-11 signals via the formation of a hexameric receptor complex and indicates that site III is a generic feature of cytokines that signal via association with gp130.

    INTRODUCTION
Top
Abstract
Introduction
References

Interleukin-11 (IL-11)1 is a secreted polypeptide cytokine. It was identified originally from its ability to stimulate the proliferation of a murine plasmacytoma cell line T1165 (1), although it has now been shown that IL-11 is widely expressed and has biological effects on a diverse range of cell types, including hematopoietic cells, hepatocytes, adipocytes, neurones, and osteoblasts (for review, see Ref. 2). In vivo administration of IL-11 results in the stimulation of megakaryopoesis and increased platelet counts (3), and, in fact, IL-11 has clinical potential for the treatment of thrombocytopenia (4) and oral mucositis (5), both of which can be induced by chemotherapy.

IL-11 mediates its effects by association with the transmembrane signal transducer gp130 (6), explaining why many of the in vitro functions of IL-11 overlap with those of other members of the gp130 family of cytokines, which includes interleukin-6 (IL-6), oncostatin M, leukemia inhibitory factor (LIF), cardiotrophin-1, ciliary neurotrophic factor (CNTF), and an IL-6-like protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV-IL-6). All of these cytokines elicit either hetero- or homodimerization of gp130 which activates intracellular signal transduction pathways via protein kinases belonging to the Janus kinase, mitogen-activated protein kinase, and Src families (7-11). Each cytokine drives the assembly of a multiprotein receptor complex that mediates the oligomerization of gp130. In the case of IL-6, association of the ligand with a ligand-specific IL-6 receptor (IL-6R) (12) mediates gp130 homodimerization. It has been shown that a hexameric complex is formed, consisting of two molecules each of IL-6, IL-6R, and gp130 (13, 14). IL-11 has been shown to function in a similar manner, and, as for IL-6, a ligand-specific IL-11 receptor (IL-11R) (15, 16) functions to promote the formation of a high affinity complex between IL-11 and gp130 (17). It has been shown that neither the IL-6R nor the IL-11R requires the cytoplasmic domain to induce gp130-dependent signaling, because soluble forms of each receptor are active (17, 18). There is structural homology between both the ligands and the receptors of the gp130 cytokine family. The extracellular region of all receptors within this superfamily of cytokine receptors contains a cytokine binding homology domain, which is characterized by a WSXWS motif, 4 positionally conserved cysteines, and a proline-rich hinge region (19). The extracellular domains of the IL-6R and the IL-11R share approximately 32% identity in their amino acid sequences.

It has been proposed that IL-11 consists of 4 alpha -helices (A-D) in an up-up-down-down topology (20). This structure is common to all members of the hematopoietin family of cytokines (21), of which human growth hormone (hGH) is the best characterized (22). hGH homodimerizes two identical receptor subunits, forming a trimeric signaling complex (22, 23). The co-crystal structure of hGH and its receptors (22) together with mutagenesis studies (24-27) have allowed the interaction sites between the units of the signaling complex to be examined. hGH uses two topologically distinct sites (sites I and II) to bind to the two hGH receptors (GHRs). Site I is composed of residues in the carboxyl ends of both the A and D helices and the AB loop, whereas site II is composed of residues in the A and C helices (23-25). It has been shown that IL-6 has two distinct regions that are functionally equivalent to sites I and II on hGH (28). These sites, also termed sites I and II, allow IL-6 to form a trimer with the IL-6R (via site I) and gp130 (via site II), just as hGH forms a trimer with two receptors. The hexameric IL-6 receptor complex described earlier is composed of two of these IL-6·IL-6R·gp130 trimers (13, 14). In addition to sites I and II, a third topologically distinct site has been identified for IL-6 which allows it to bind to a second gp130 molecule (14, 29). This site (site III) contributes to stabilizing the hexameric receptor complex.

In this study we identify residues critical for the binding of mIL-11 to the IL-11R and gp130, which provides evidence that IL-11 has three topologically distinct receptor binding sites structurally and functionally equivalent to sites I, II, and III of IL-6.

    MATERIALS AND METHODS

IL-11 Constructs-- Polymerase chain reaction was used to amplify the cDNA sequence (Genetics Institute) encoding the mature form of mIL-11 (amino acids 1-178). The fragment was then cloned into pGEX-2T (Amersham Pharmacia Biotech) using restriction enzymes BamHI and EcoRI. A recognition site for human rhinovirus protease 3C (30) was introduced by the 5'-primer (the sequence of the primers used is available on request). Cleavage of the glutathione S-transferase-mIL-11 fusion protein with 3C protease produces a protein consisting of amino acids 1-178 of mIL-11 with an extra glycine at the NH2 terminus. This was confirmed by NH2-terminal sequencing. All mutant mIL-11 DNA sequences were created by polymerase chain reaction (31) overlap using pGEX-mIL-11 as a template and specific oligonucleotide primers encoding each mutation. The mutant mIL-11 sequences were cloned into pGEX, and the nucleotide sequences of all constructs were confirmed by DNA sequencing using a sequencing reaction kit (Perkin-Elmer).

Expression and Purification of IL-11-- mIL-11 and all mutants were expressed as glutathione S-transferase fusion proteins in the Escherichia coli strain JM109. The expression, purification, and cleavage of the fusion proteins were carried out as described previously for human leukemia inhibitory factor (32). The protein concentrations were determined using the Coomassie Plus Protein assay (Pierce). Mutant proteins were also examined by SDS-polyacrylamide gel electrophoretic analysis.

Monoclonal Antibody Binding Experiments-- The structural integrity of the mIL-11 mutant proteins was assessed by their reactivity with two monoclonal antibodies raised against rhIL-11 (kindly donated by Genetics Institute) in an enzyme-linked immunosorbent assay. A neutralizing monoclonal antibody (11 h3/19.6.1) served as the capture reagent, and a second biotinylated monoclonal antibody (11 h3/15.6.13) was used as the detector. Dot-blot analysis showed that both monoclonal antibodies preferentially recognized native mIL-11 and not denatured mIL-11, indicating that their reactivity with mIL-11 is conformation-dependent.

IL-11R-Fc and gp130-Fc Expression Constructs-- The construction of the eukaryotic expression plasmids pIG/IL-11R-Fc and pIG/gp130-Fc has been described previously (17).

Expression and Purification of IL-11R and gp130-- Both the murine IL-11R and murine gp130 were expressed as fusion proteins with the Fc region of human IgG1. The human epithelial kidney 293T cell line (33) was used for the transient expression of the pIG constructs, as described previously (17). Cell medium containing the Fc fusion proteins was harvested after 6 days. Filtered supernatant was then used either directly in ligand binding assays or for the purification of IL-11R and gp130. Murine IL-11R and gp130-Fc fusion proteins were purified by affinity chromatography using protein A-Sepharose, as described previously (17). Soluble IL-11R and gp130 were cleaved from the Fc portion while bound to the protein A-Sepharose by human rhinovirus 3C protease, as described previously (17). The purified soluble proteins were examined using SDS-polyacrylamide gel electrophoresis.

Biotinylation of IL-11-- The method of mIL-11 biotinylation was adapted from a published procedure (34). After purification of mIL-11, buffer exchange to 100 mM borate (pH 8.2) was carried out using NAP-5 Sephadex columns (Amersham Pharmacia Biotech). Biotin amidocaproate N-hydroxysuccinimide ester (Sigma) was added to give a 10:1 (biotin:mIL-11) molar ratio. After a 4-h incubation at room temperature NH4Cl (1 mg/250 mg of biotin ester) was added and incubated for 10 min. Biotinylated IL-11 (bIL-11) was dialyzed against 50 mM Tris (pH 8.5), 150 mM NaCl, and 10 mM EDTA for 20 h at 4 °C to remove unbound biotin.

Ligand Binding Assays-- Maxisorp 96-well plates (Nunc) were coated with protein A (2 µg/ml) overnight at room temperature and then blocked with PBS and 1% bovine serum albumin for 1 h. Wells were washed with PBS and incubated with 100 µl of IL-11R-Fc-containing supernatant for a minimum of 2 h. After washing the wells with PBS the plates were used for binding assays. The binding of biotinylated ligands to IL-11R-Fc was measured by adding varying concentrations of the biotinylated ligand to each well. Competition assays between bIL-11 and varying concentrations of nonbiotinylated ligand were also carried out. In all cases binding was left for 4 h at room temperature. The wells were washed in PBS and incubated with streptavidin-horseradish peroxidase conjugate (Amersham Pharmacia Biotech) for 1 h. After washing with PBS, bound horseradish peroxidase was visualized using orthophenylenediamine as substrate (Dako), and A490 was determined.

Complex formation studies were carried out in a similar manner. As for IL-11R-Fc, gp130-Fc was immobilized on protein A-coated plates. Varying concentrations of biotinylated ligand were then added to each well in the presence of a constant 1 µg/ml soluble IL-11R. After a 4-h incubation at room temperature the amount of biotinylated ligand captured in complex with IL-11R and gp130-Fc was measured using streptavidin-horseradish peroxidase and orthophenylenediamine substrate, as described above. To assess nonspecific binding, the amounts of biotinylated ligand bound in the absence of either gp130-Fc or soluble IL-11R were also measured.

Cell Culture and Ligand Bioassays-- Ba/F3 cells cultured in RPMI (Life Technologies, Inc) supplemented with 10% fetal calf serum, 1 mM glutamine, 1 mM streptomycin, and 1 mM penicillin were transfected with BCneo/mgp130 (35), which encodes the entire open reading frame for mgp130 (35). The cells were then selected on 600 µg/ml G418. Stable Ba/F3-mgp130 transfectants were transfected with pCDNA3/mIL-11R, which encodes the entire open reading frame for mIL-11R (donated by M. Hall) and selected on 100 ng/ml mIL-11. All mutant mIL-11 proteins were tested for biological activity on the Ba/F3-mgp130/mIL-11R cells, using the 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) proliferation assay as described previously (17).

IL-11 Structure Modeling-- The amino acid sequences of IL-11 and IL-6 were aligned using the progressive pairwise algorithm of Feng and Doolittle (36) implemented in the Pileup program of the GCG package. Predictions of secondary structure of mIL-11 were also made using the GCG package. A structural model of mIL-11 was created, from the IL-6 crystal structure coordinates (37) and the IL-6/IL-11 sequence alignment (with minor manual editing), using the Modeller 4 computer program (38).

    RESULTS

Murine IL-11 Mutagenesis-- The strategy for mIL-11 mutagenesis was influenced by mutagenesis studies for IL-6 (for review, see Ref. 39) and LIF (32). A structural model of IL-11, created using the IL-6 crystal structure coordinates (37) and a sequence alignment of IL-6 and IL-11 combined with predictions of secondary structure, solvent accessibility, and helical wheel projections, was used to identify candidate mIL-11 amino acids for mutagenesis. Multiple alanine substitutions were made to identify those residues important for the binding of mIL-11 to mIL-11R and mgp130.

Amino acids located at the carboxyl terminus (Trp-166, Arg-169, Leu-172, Leu-173, and Thr-176) and within the predicted AB loop (Leu-64 and Leu-67) were selected as potential site I residues. Amino acids predicted to be on the exposed surfaces of helices A (Arg-9, Asp-13, Arg-15, Asp-19, and Val-22) and C (Arg-111, Leu-115, Arg-118, and Leu-121) were selected as potential site II residues, and amino acids predicted to lie within the CD loop or the NH2-terminal end of the D helix (Trp-147 and Arg-151) were selected as potential site III residues. Many of the selected residues within each predicted binding site were found to be in close proximity to one another in the structural model of mIL-11.

Structural Integrity of the Mutant Proteins-- The abilities of the site I mutants to bind to two conformation-dependent monoclonal antibodies were examined, as described under "Materials and Methods." All of the site I mutants exhibited binding similar to that of the wild type protein (results not shown), indicating that the mutations had not significantly disturbed the native conformation of the protein. In the case of the sites II and III mutants, the ability of the mutants to bind to IL-11R-Fc served as a control for protein folding and the structural integrity of the proteins. If a mutant has reduced affinity for one receptor and not the other, it suggests that the mutation has caused a local conformational change rather than effecting the global structure of the protein.

Activity of Site I Mutants-- The activities of the mIL-11 mutants were assessed using two different assays. First, the ability of each mutant to inhibit the binding of bIL-11 to IL-11R-Fc was measured (Table I and Fig. 1A), and second the ability of each mutant to stimulate the proliferation of Ba/F3-mgp130/mIL-11R cells was measured (Table I and Fig. 2A).

                              
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Table I
Summary of receptor binding and BAF assay data for mIL-11 mutants


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Fig. 1.   Binding of biotinylated IL-11 (60 ng/ml) to IL-11R-Fc in the presence of competing mIL-11 wild type or mutant. Results are expressed as the A490 value after detection of bound bIL-11 with streptavidin-horseradish peroxidase and orthophenylenediamine substrate. Values are the mean of triplicate samples, and error bars represent the S.E. Panel A, site I mutants: , mIL-11; open circle , L64A/L67A; ×, L173A; black-square, R169A; , L172A. Panel B, site II mutants: , mIL-11; open circle , R111A/L115A; ×, D13A. Panel C, site III mutants: , mIL-11; open circle , W147A; ×, R151A.


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Fig. 2.   Response of Ba/F3-mgp130/mIL-11R cells to mIL-11 wild type and mutants. Results are expressed as the A570 value of cells assayed for proliferation by MTT. Values are the mean of triplicate samples, and error bars represent the S.E. Panel A, site I mutants: , mIL-11; open circle , L64A/L67A; ×, L173A; black-square, R169A; , L172A. Panel B, site II mutants: , mIL-11; open circle , R111A/L115A; ×, D13A. Panel C, site III mutants: , mIL-11; open circle , W147A; ×, R151A.

Several of the site I mutants were shown to have reduced binding to IL-11R-Fc compared with that of the wild type protein. The mutant R169A showed the most dramatic (in excess of 1,000-fold) reduction in binding to IL-11R-Fc (Table I and Fig. 1A). Two of the mutants, L64A/L67A and L172A, showed a 50-fold reduction in IL-11R-Fc binding, and others, including L173A and W166A, showed only a weak (less than 10-fold) reduction in binding to the IL-11R-Fc (Table I and Fig. 1A). The mutation T176A had no significant effect on the activity of the protein, suggesting that this residue does not contribute directly to the protein-protein interactions that drive the formation of the signaling complex.

In general, those mutants that showed a reduction in IL-11R-Fc binding also exhibited reduced activity in the cell proliferation assay (Table I and Fig. 2A). The mutant R169A, which showed a dramatic reduction in IL-11R-Fc binding, showed at least a 1,000-fold reduction in biological activity (Table I and Fig. 2A). The two mutants L64A/L67A and L172A, which showed a 50-fold reduction in IL-11R-Fc binding, exhibited a 2-3-fold reduction in the proliferation assay. In general, the reduction observed for IL-11R-Fc binding was greater than the reduction observed in the proliferation assay. A similar difference in the sensitivity between binding assays and biological assays was also observed for mutants of LIF (32). The ability of each mutant to bind to gp130-Fc in the presence of soluble IL-11R was also analyzed (results not shown). It was found that the reduction observed in the IL-11R-Fc binding assay was very similar to those observed in this complex formation assay. In addition, the mutants exhibited greater reductions in activity when assessed in a Ba/F3-mgp130 proliferation assay in the presence of soluble receptor (results not shown) compared with their activities in the Ba/F3-mgp130/mIL-11R proliferation assay, even though the Ba/F3-mgp130/mIL-11R assay was able to detect significantly lower concentrations of ligand. This observation that membrane anchoring of the IL-11R renders the IL-11 receptor complex less sensitive to changes in the affinity of the ligand for the IL-11R has also been reported for the CNTF receptor complex (40).

Activity of Site II Mutants-- The activities of the site II mutants were assessed in both the IL-11R-Fc binding assay and the Ba/F3-mgp130/mIL-11R proliferation assay (Table I and Figs. 1B and 2B). Most of the site II mutants, including R9A, D13A, R118A, and L121A, showed no significant difference in IL-11R-Fc binding compared with that of the wild type protein (Table I and Fig. 1B). The mutant R111A/L115A showed a 4-fold increase in affinity for the IL-11R-Fc (Table I and Fig. 1B), suggesting that changing these 2 residues in helix C has somehow altered site I such that the ligand binds to IL-11R with a higher affinity.

One of the site II mutants, D13A, which exhibited normal binding to IL-11R-Fc, showed a 4-fold reduction in activity in the cell proliferation assay (Table I and Fig. 1B). The mutant R111A/L115A, which showed an increased binding affinity for the IL-11R, showed more than a 10-fold reduction in biological activity (Table I and Fig. 1B). These results suggest that the residues Asp-13 and either or both of Arg-111 and Leu-115 are involved in the interaction between mIL-11 and gp130, as substitution of these residues reduces the ability of mIL-11 to form a signaling complex with gp130 without reducing the affinity of the ligand for the IL-11R. Other mutants, such as R9A, R118A, and L121A, had activities equivalent to wild type mIL-11 in both assays.

The mutant R15A/D19A/V22A showed a 10-fold reduction in the IL-11R-Fc binding assay (Table I and Fig. 1B) which was accompanied by a greater than 10-fold reduction in the cell proliferation assay (Table I and Fig. 2B). These data suggest that the alanine substitutions in this mutant have altered the global structure of the protein.

The activities of the site II mutants D13A, L121A, and R111A/L115A were also assessed by direct binding of biotinylated ligands to the receptor. The abilities of these biotinylated ligands to bind to both IL-11R-Fc and gp130-Fc (in the presence of soluble IL-11R) were measured (Table II and Fig. 3) as well as their activities in the Ba/F3-mgp130/mIL-11R proliferation assay. This allowed us to examine the binding of these site II mutants to gp130-Fc (in the presence of soluble IL-11R), which we were unable to do by measuring competitive binding between bIL-11 and the mutants, as the mutant proteins were still able to compete for soluble IL-11R. The biological activities and IL-11R-Fc binding affinities of the biotinylated mutants confirmed the results observed for the nonbiotinylated site II mutants. This served as a control to show that biotinylation had not altered the activity of the mutant proteins. The only biotinylated ligand with activity that differed significantly from that of the wild type was R111A/L115A. This biotinylated mutant bound to IL-11R-Fc with a 5-fold increase in affinity compared with the wild type (Table II), as observed for nonbiotinylated R111A/L115A, whereas binding to gp130-Fc (in the presence of IL-11R) was barely detectable (Table II and Fig. 3). These data support the suggestion that either one or both of the residues Arg-111 and Leu-115 are crucial for the binding of mIL-11 to gp130.

                              
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Table II
Summary of complex formation and BAF data for biotinylated ligands


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Fig. 3.   Binding of biotinylated ligands to gp130-Fc in the presence of 1 µg/ml IL-11R. Results are expressed as the A490 value after detection of bound biotinylated ligand with streptavidin-horseradish peroxidase and orthophenylenediamine substrate. Values are the mean of triplicate samples, and error bars represent the S.E. , biotinylated IL-11; open circle , biotinylated R111A/L115A (site II mutant); ×, biotinylated W147A (site III mutant).

Activity of Site III Mutants-- The activities of the site III mutants W147A and R151A were assessed in the IL-11R-Fc binding assay and the Ba/F3-mgp130-mIL-11R proliferation assay (Table I and Figs. 1C and 2C). Both of these mutants exhibited normal binding to IL-11R-Fc (Table I and Fig. 1C). The mutant R151A showed a 5-fold reduction in the cell proliferation assay, and W147A produced undetectable stimulation of the Ba/F3-mgp130-mIL-11R cells (Table I and Fig. 2C). These data suggest that these substitutions reduce the affinity of the ligand for gp130 without affecting the affinity for the IL-11R, and as discussed earlier, this itself provides a control for the structural integrity of the mutant proteins.

The two site III mutants W147A and R151A were also biotinylated, and the binding affinities and biological activities of the biotinylated ligands were examined (Table II and Figs. 3 and 4). The biotinylated mutant R151A was found to behave in a manner similar to that of the biotinylated wild type IL-11. The biotinylated mutant W147A exhibited normal binding to IL-11R-Fc (Table II), as observed for nonbiotinylated W147A, whereas binding to gp130-Fc (in the presence of soluble IL-11R) was barely detectable (Table II and Fig. 3). This was accompanied by undetectable stimulation of the Ba/F3-mgp130/mIL-11R cells (Table II and Fig. 4). These data suggest that the residue Trp-147 is critical for the binding of mIL-11 to gp130 and hence the formation of a signaling complex.


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Fig. 4.   Response of Ba/F3-mgp130/mIL-11R cells to biotinylated IL-11 wild type and mutants. Results are expressed as the A570 value of cells assayed for proliferation by MTT. Values are the mean of triplicate samples, and error bars represent the S.E. , biotinylated IL-11; open circle , biotinylated R111A/L115A (site II mutant); ×, biotinylated W147A (site III mutant).


    DISCUSSION

Cytokines mediate biological functions through the formation of multichain receptor signaling complexes. Each cytokine drives the assembly of a receptor complex that is stabilized by multiple protein-protein interactions between the various components. The simplest and best characterized example is hGH, which homodimerizes two identical receptor subunits (22). The gp130 family of cytokines, of which IL-11 is a member, share the common signal transducer, gp130. In the case of IL-11, gp130-dependent signaling is activated by the homodimerization of gp130. A complex of IL-11 and the IL-11R interacts with gp130 to induce this homodimerization.

Here we have examined the activities of mIL-11 mutants in receptor binding assays and a cell proliferation assay. We have identified residues crucial for the binding of mIL-11 to both IL-11R and gp130. The location of these residues, as predicted from structural studies and a model of IL-11, provides evidence that mIL-11 has three topologically distinct receptor binding sites, which are both structurally and functionally equivalent to the receptor binding sites I, II, and III of IL-6. This supports the suggestion that, in a manner similar to that of IL-6, IL-11 forms hexameric signaling complexes.

IL-6 has been shown to interact with the IL-6R through a region known as site I, which is formed by residues in the COOH-terminal end of helix D and the AB loop (41-43). Our data indicate that the region of mIL-11 responsible for binding to the IL-11R is topologically very similar to that of IL-6 (Fig. 5). The residues Arg-169, Leu-172, and Leu-173, which are predicted to lie within the COOH-terminal end of helix D, are very important for the binding of mIL-11 to the IL-11R. In fact, Arg-169 was found to be crucial for the binding of mIL-11 to the IL-11R and hence its biological activity in the cell proliferation assay. Sequence alignments reveal that the equivalent residues in all known IL-6 sequences and KSHV-IL-6 are also arginines. Mutagenesis studies have shown that this arginine of hIL-6 (Arg-179) is an important site I residue, and in fact, a positive charge in position 179 is an absolute requirement for the interaction between hIL-6 and the IL-6R (41). The mIL-11 mutant L64A/L67A also showed reduced binding to the IL-11R, indicating that either one or both of these residues, predicted to lie within the AB loop, also contribute to the binding of mIL-11 to the IL-11R. On examining the location of these five site I residues, within our structural model of IL-11, it was found that they collectively form a distinct region at one end of the four-helix bundle structure, as observed for other members of this cytokine family such as hGH (23, 26), LIF (32), and IL-6 (42). The residues appear to be clustered around a central Arg-169, which proved to be the most important residue for binding to the IL-11R. The configuration of a binding site as a few critical residues surrounded by ones of lesser importance has also been reported for both hGH (26) and LIF (32).


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Fig. 5.   Diagrammatic representation of the IL-11 structural model showing the location of an essential residue in each of the predicted binding sites. The diagram shows the four predicted alpha  helices, A (blue), B (purple), C (green), and D (yellow). The long interconnecting AB and CD loops are not shown. The residues shown in red are crucial for mIL-11 to form a signaling complex with the IL-11R and gp130. Note: residue Arg-111 was mutated in conjunction with Leu-115 (not shown). This diagram was created using Molscript software (46).

Site II of IL-6 may be defined as the region that interacts with gp130 in a manner similar to that of site II in hGH. It is formed by exposed residues on helices A and C (44). Residues of mIL-11 predicted to be found within this region of the protein were selected for alanine substitution. Binding data indicate that either one or both of the residues Arg-111 and Leu-115 are extremely important for the binding of mIL-11 to gp130. The mutant R111A/L115A had an unexpectedly high activity in the cell proliferation assay, considering the importance of these residues for interaction with gp130 as shown by binding studies. However, the affinity of this mutant for the IL-11R was approximately 4-fold greater than that of the wild type, therefore reducing the impact of the mutation on the biological activity of the ligand; and, as described earlier for CNTF (40), membrane anchoring of the IL-11R is likely to render the IL-11 receptor complex less sensitive to changes in the affinity of the ligand for either of the receptor subunits. The reason for this is not clear. The residues Arg-111 and Leu-115 are found within the predicted C helix. The data also indicate that Asp-13, which is found within the predicted A helix, may play some role in the interaction between mIL-11 and gp130. These residues form a distinct patch on the surface of the IL-11 structural model, well separated from site I (Fig. 5). Further mutagenesis is required to examine this binding region in more detail, although often it is only a few key residues that contribute to the energy of binding. Site II of IL-6 is thought to be composed of residues Tyr-31 (A helix), Ser-118, and Val-121 (C helix) (37, 44, 45). It was noted for hGH that only a few of the residues involved in contacts between the ligand and receptor in the crystal structure actually contributed to the energy of binding (26); that is, the functional epitope is much smaller than the structural epitope.

The epitopes identified in this study which allow mIL-11 to bind to the IL-11R and one gp130 molecule are functionally equivalent to sites I and II, originally identified on hGH for binding to the two GHR molecules. This illustrates how members of the hematopoietin family of cytokines use topologically conserved epitopes to bind to cytokine receptors. More recently a third epitope has been identified for some members of this family. Site III enables IL-6 to bind to a second gp130 molecule and form hexameric complexes (29), and site III of LIF enables it to bind to the LIF receptor (32). Our data provide evidence that mIL-11 also has a third receptor binding site (Fig. 5), which is predicted to interact with a second molecule of gp130. Binding studies show that the residue Tyr-147, predicted to be located at the beginning of the D helix or within the CD loop, is essential for mIL-11 to form a complex with gp130. Alanine replacement of this residue renders mIL-11 biologically inactive, without affecting the ability of the protein to bind to the IL-11R.

Site III of IL-6 is also composed of residues in the CD loop/NH2-terminal end of the D helix (29). This is a region of significant similarity among several members of the gp130 family of cytokines, including IL-6, IL-11, LIF, oncostatin M, and CNTF. A tryptophan residue (Tyr-157) found within this region of IL-6, is conserved in nearly all IL-6 sequences of different species, and it has been reported to be one of the most important site III residues (29). When the amino acid sequences of different gp130 family members are compared, this residue aligns not only with Tyr-147 of IL-11 (identified in this study as a site III residue) and a tryptophan in KSHV-IL-6 (which is known to interact with gp130) but with a conserved phenylalanine seen in LIF and CNTF. The significance of this becomes apparent when examining mutagenesis data for hLIF (32), which indicates that this conserved phenylalanine, Phe-156, is essential for the interaction of hLIF with the LIF receptor, an interaction involving site III of the LIF molecule. This indicates that site III, comprising a solvent-exposed nonpolar and basic residue, is a generic feature of gp130 cytokines.

These findings strengthen the hypothesis that IL-11, like IL-6, forms a hexameric signaling complex, involving multiple protein-protein interactions. It is highly likely that there are additional stabilizing interactions among the IL-11, IL-11R, and gp130 proteins, which remain to be identified. This work also supports the idea that protein-protein interactions involve a few essential residues that provide the majority of energy for binding and that the functional epitopes of cytokines are quite small and highly conserved, which highlights the potential for drug development. The identification of Tyr-147 as a crucial site III residue confirms that site III is a characteristic feature of gp130 cytokines and that the binding epitope is highly conserved among several members of this family. Finally, this study reinforces a pattern of binding site usage, which has emerged with the progressive characterization of gp130 signaling complexes.

    ACKNOWLEDGEMENTS

We are grateful to M. Hall for the pCDNA3/mIL-11R construct, Will Somers for the IL-6 crystal coordinates, and Genetics Institute for monoclonal antibodies against rhIL-11.

    FOOTNOTES

* This work was supported in part by the Cancer Research Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Supported by a studentship from the Cancer Research Campaign.

§ Present address: Genesis Research and Development Corporation, 1 Fox St., Parnell, Auckland, New Zealand.

To whom correspondence should be addressed. Tel.: 44-0-121-414-7533; Fax: 44h0-121-414-3982; E. mail: j.k.heath{at}bham.ac.uk.

    ABBREVIATIONS

The abbreviations used are: IL-11, interleukin-11; IL-6, interleukin-6; LIF, leukemia inhibitory factor; CNTF, ciliary neurotrophic factor; KSHV, Kaposi's sarcoma-associated herpesvirus; IL-11R, IL-11 receptor; IL-6R, IL-6 receptor; m, murine; h, human; hGH, human growth hormone; GHR, growth hormone receptor; rhIL-11, recombinant human IL-11; Fc, constant region domain of human gamma -immunoglobulin; PBS, phosphate-buffered saline; MTT, 3-(4, 5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide.

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Abstract
Introduction
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