Identification of a Cys Motif in the Common beta  Chain of the Interleukin 3, Granulocyte-Macrophage Colony-stimulating Factor, and Interleukin 5 Receptors Essential for Disulfide-linked Receptor Heterodimerization and Activation of All Three Receptors*

Frank C. Stomski, Joanna M. Woodcock, Betty Zacharakis, Christopher J. BagleyDagger , Qiyu Sun, and Angel F. Lopez§

From the Cytokine Receptor Laboratory, The Hanson Centre for Cancer Research, The Institute of Medical and Veterinary Science, Adelaide 5000, South Australia, Australia

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors undergo covalent dimerization of the respective specific alpha  chains with the common beta  subunit (beta c) in the presence of the cognate ligand. We have now performed alanine substitutions of individual Cys residues in beta c to identify the Cys residues involved and their contribution to activation of the IL-3, GM-CSF, and IL-5 receptors. We found that substitution of Cys-86, Cys-91, and Cys-96 in beta c but not of Cys-100 or Cys-234 abrogated disulfide-linked IL-3 receptor dimerization. However, although Cys-86 and Cys-91 beta c mutants retained their ability to form non-disulfide-linked dimers with IL-3Ralpha , substitution of Cys-96 eliminated this interaction. Binding studies demonstrated that all beta c mutants with the exception of C96A supported high affinity binding of IL-3 and GM-CSF. In receptor activation experiments, we found that beta c mutants C86A, C91A, and C96A but not C100A or C234A abolished phosphorylation of beta c in response to IL-3, GM-CSF, or IL-5. These data show that although Cys-96 is important for the structural integrity of beta c, Cys-86 and Cys-91 participate in disulfide-linked receptor heterodimerization and that this linkage is essential for tyrosine phosphorylation of beta c. Sequence alignment of beta c with other cytokine receptor signaling subunits in light of these data shows that Cys-86 and Cys-91 represent a motif restricted to human and mouse beta  chains, suggesting a unique mechanism of activation utilized by the IL-3, GM-CSF, and IL-5 receptors.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cytokine receptor dimerization is a common theme in receptor activation (1). Following the binding of the cognate ligand to cytokine receptors, a sequential process takes place whereby receptor subunits associate and recruit cytoplasmic signaling molecules leading to receptor activation and cellular signaling (2). The general process of receptor dimerization exhibits variations among the cytokine receptor superfamily and may involve homodimerization or heterodimerization events depending on receptor subunit composition (3, 4). In the case of the growth hormone receptor, growth hormone binds initially to one receptor subunit and induces its homodimerization with a second, identical subunit (5). A similar process probably takes place with erythropoietin and granulocyte colony-stimulating factor, leading, in both cases, to receptor homodimerization and activation (6, 7).

With cytokine receptors that comprise multiple subunits, receptor activation is accompanied by homodimerization or heterodimerization of the signaling subunits. For example, in the IL-61 receptor system, IL-6 induces dimerization of IL-6Ralpha with gp130 (8), homodimerization of gp130, and receptor activation (9). On the other hand, the binding of CNTF to CNTFRalpha induces its association with gp130 and the LIF receptor, and the heterodimerization of gp130 and the LIF receptor is accompanied by receptor activation (10). Similarly, heterodimerization of IL-2Rbeta and IL-2Rgamma subunits is necessary for IL-2 receptor activation (11, 12). Interestingly, in these cases, each receptor alpha  chain constitutes the major binding subunit but does not seem to form part of the signaling receptor complex.

The mechanism of activation of the GM-CSF/IL-3/IL-5 receptor system exhibits features similar to the mechanism employed by the above receptors, although some unique features are becoming evident. One of the most important differences is the contribution that each receptor alpha  chain makes to signaling. This is manifested in two ways: first, unlike IL-6Ralpha , CNTFRalpha , and the IL-2Ralpha , the cytoplasmic domains of GM-CSFRalpha , IL-3Ralpha , and IL-5Ralpha are all required for full receptor activation and signaling (13-16). Second, IL-3Ralpha and GM-CSFRalpha form disulfide-linked dimers with the common beta  chain (beta c) of their receptor (4, 17). The disulfide-mediated dimerization of IL-3Ralpha with beta c and of GM-CSFRalpha with beta c is accompanied by tyrosine phosphorylation of beta c (4). In all of these cases, however, tyrosine phosphorylation is observed in the disulfide-linked dimers as well as in the monomeric molecules, and hence it is not clear which is the critical species for receptor activation. Furthermore, the location of the cysteines involved in disulfide linkage is not known, nor is it apparent whether they constitute a functionally conserved motif in the cytokine receptor superfamily. We have now performed single alanine substitutions of candidate cysteine residues in the N-terminal cytokine receptor module (CRM) of the IL-3, GM-CSF, and IL-5 receptor beta c and examined their contribution to disulfide-linked receptor dimerization, high affinity ligand binding, and receptor activation.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Mutagenesis of Human beta c and Expression Plasmid Constructs-- Cysteine residues were substituted with alanines in the human beta  chain cDNA using oligonucleotide-directed mutagenesis (Altered-sites, Promega, Sydney, New South Wales, Australia) as described previously (18). The mutations were confirmed by nucleotide sequencing, and the mutant beta c cDNAs were subcloned into the eukaryotic expression vector pcDNA1 (Invitrogen, San Diego, CA). The IL-3R, GM-CSFR, and IL-5R alpha  chain cDNAs were cloned into the eukaryotic expression vector pCDM8 (Invitrogen) for transfection (18).

Cell Culture and DNA Transfection-- COS cells were maintained in RPMI 1640 medium supplemented with 10% v/v fetal calf serum and transfected by electroporation. Routinely, 2 × 107 COS cells were co-transfected in 0.8 ml of PBS at 0 °C with 25 µg of wild type or mutated beta c cDNA together with 10 µg of GM-CSFR and 10 µg of IL-3R alpha  chain cDNA at 500 microfarads with 300 V. After electroporation, cells were centrifuged through a 1-ml cushion of fetal calf serum, and cells were plated in either 25 ml of medium/150-cm2 flask or 24-well plates for binding analysis. Transfectants were incubated for 2 days prior to cytokine treatment (18).

The HEK293T cell line, derived from the adenovirus type transformed human embryonic kidney 293 cell line and containing the simian virus 40 large tumor antigen (19), was maintained in RPMI 1640 medium supplemented with 10% v/v fetal calf serum. On the day before transfection, 1.4 × 106 cells were plated into 6-cm tissue culture dishes to adhere overnight. Four hours after a medium change, 6 µg of wild type or mutated beta c cDNA together with 4 µg of GM-CSFRalpha , 4 µg of IL-3Ralpha , or 4 µg of IL-5Ralpha cDNA and 0.5 µg of JAK-2 cDNA were added to cells in the form of a calcium phosphate precipitate (20), and the cells were placed in an incubator for 4 h to permit the uptake of the DNA-calcium phosphate precipitate. The cells were then washed, replated in 4 plates/150 cm2, and placed in the incubator for 48 h prior to cytokine treatment.

GM-CSF, IL-3, and IL-5-- Recombinant human IL-3, GM-CSF, and IL-5 were produced in Escherichia coli essentially as described before (21, 22). Cytokine purity and quantitation was determined by high performance liquid chromatography analysis. The unit activity of the cytokines based on the ED50 values in a proliferation assay (23) was 0.03 ng/ml GM-CSF, 0.1 ng/ml IL-3, and 0.3 ng/ml IL-5; each value represents 1 unit of that substance.

Radiolabeling Cytokines-- Recombinant IL-3 and GM-CSF were radioiodinated by the iodine monochloride method (24) to a specific activity of about 36 mCi/mg. Routinely, 4 µg of protein was iodinated and separated from iodide ions on a Sephadex G-25 column (Pharmacia Biotech Inc.), eluted with PBS containing 0.02% v/v Tween 20, and the iodinated proteins were stored at 4 °C for up to 4 weeks.

Saturation Binding Assays-- Binding assays were performed on confluent monolayers in 24-well plates over a concentration range of 10 pM to 10 nM 125I-labeled GM-CSF or IL-3 in binding medium (RPMI containing 0.5% (w/v) BSA/0.1% (w/v) sodium azide) essentially as described previously (25). After incubation at room temperature for 2 h, radioligand was removed, and the cells were briefly washed twice in binding medium. Specific counts were determined after lysis of the cell monolayer with subsequent transfer and counting on a gamma  counter (Cobra Auto Gamma; Packard Instruments Co., Meridien, CT). Dissociation constants were calculated using the EBDA and LIGAND programs (26) (Biosoft, Cambridge, United Kingdom).

Monoclonal Antibodies to the IL-3, GM-CSF, and IL-5 Receptors-- Monoclonal antibodies directed against GM-CSFRalpha , IL-3Ralpha , IL-5Ralpha , or beta c were generated as described previously (27) and purified and characterized as detailed elsewhere (17, 18, 27). The monoclonal antibodies 8E4, 4H1, 9F5, and A14 were selected for their ability to specifically immunoprecipitate beta c, GM-CSFRalpha , IL-3Ralpha , and IL-5Ralpha , respectively. The monoclonal antibody 1C1 conjugated to biotin was used for immunoblotting beta c, and 8E4, 4H1, 9F5, and A14 were used for cell surface expression staining for beta c, GM-CSFRalpha , IL3Ralpha , and IL5Ralpha , respectively. The monoclonal antibodies were purified from ascites as described (27). The monoclonal antibody (mAb) against phosphorylated tyrosine was the peroxidase-conjugated anti-phosphotyrosine 3-365-10 (Boehringer Mannheim).

Analysis of Receptor Cell Surface Expression by Flow Cytometry-- Cell surface expression of transfected receptor subunits was confirmed by indirect immunofluorescence staining using anti-receptor alpha  and beta  chain specific monoclonal antibodies. Staining was performed as described previously (17) and analyzed with a EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL).

Surface Labeling of Cells and Immunoprecipitation-- COS cells were cell surface-labeled with 125I by the lactoperoxidase method as described previously (28). Approximately 108 cells were washed twice in PBS and then labeled with 1 mCi of 125I (NEN Life Science Products and AMRAD Pharmacia) in PBS. Cell were lysed in lysis buffer consisting of 137 mM NaCl, 10 mM Tris-HCl (pH 7.4), 10% glycerol, 1% Nonidet P-40 with protease inhibitors (10 µg/ml leupeptin, 2 mM phenylmethlysulfonyl fluoride, 10 µg/ml aprotinin), and 2 mM sodium vanadate for 30 min at 4 °C followed by centrifugation of the lysate for 15 min at 12,000 g 4 °C. Following a 1-h preclearance with protein A-Sepharose (Pierce) at 4 °C, the supernatant was incubated for 18 h with 5 µg/ml antibody. Protein-immunoglobulin complexes were captured by incubation for 1 h with protein A-Sepharose followed by six subsequent washes in lysis buffer. Samples were boiled for 10 min in SDS sample load buffer either in the presence or absence of 2-mercaptoethanol (i.e. reducing or nonreducing) before separating immunoprecipitated proteins by SDS-PAGE. Immunoprecipitation from HEK293T cells was carried out similary except the cells were not surface labeled.

SDS-Polyacrylamide Gel Electrophoresis-- Immunoprecipitated proteins were analyzed by SDS-PAGE on polyacrylamide gels. Samples were boiled in SDS loading buffer for 5 min prior to loading. Molecular weights were estimated using SeeBlueTM prestained standards (Novex French's Forest, New South Wales, Australia). Radiolabeled proteins were visualized using an ImageQuant PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Immunoblot and ECL-- Immunoprecipitated proteins separated by SDS-PAGE were transferred to nitrocellulose membrane by electroblotting. Routinely, nitrocellulose membranes were blocked in a solution of PBS/0.05% (v/v) Tween 20 containing 1% (w/v) blocking reagent 1096 176 (Boehringer Mannheim) and probed with either peroxidase-conjugated anti-phosphotyrosine 3-365-10 (Boehringer Mannheim) or anti-beta c (1C1-biotin) followed by Streptavidin-POD (Boehringer Mannheim). Immunoreactive proteins were detected by chemiluminescence using the ECL kit (Amersham Corp.) following the manufacturer's instructions.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Rationale for Mutagenesis of N-terminal Cysteine Residues of beta c-- To study the molecular events involved in the activation of the IL-3, GM-CSF, and IL-5 receptors, we replaced several extracellular cysteine residues of beta c by alanine residues. So as to target Cys available for intermolecular interactions, we sought to avoid Cys involved in structurally important intramolecular disulfide bonds. By homology with other cytokine receptors, domains one and three are expected to possess two disulfide bonds each. This is clearly the case with domain three, which contains only four Cys residues. However, domain one beta c possesses seven Cys, of which only Cys-34, Cys-45, and Cys-75 could be aligned readily with equivalent Cys residues in other receptors (Fig. 1). Of the remaining Cys residues, Cys-86, Cys-91, and Cys-96 are conserved in murine beta c. Of these, Cys-96 is followed by an Ile at position 98, which aligns with conserved hydrophobic residues in other cytokine receptors, suggesting that this Cys is part of the second conserved disulfide bond. Cys-96 and Cys-91 are proposed to lie in an extended loop between the D and E beta strands of the first domain. Although Cys-86 and Cys-91 were favored candidates for intermolecular disulfide bond formation, we chose to also mutate the nearby Cys-96 and Cys-100 and the single Cys residue in domain 2 at position 234.


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Fig. 1.   Alignment of domain 1 of the CRM present in the common beta  chain of the GM-CSF, IL-3, and IL-5 receptors and other signaling subunits of the cytokine receptor superfamily. Four conserved Cys residues form the basis of the alignment, with the second Cys followed by a conserved Trp and the fourth Cys followed by a hydrophobic residue at the i+2 position. The sequences of human and mouse beta c and beta IL-3 are shown; the numbering corresponds to the human sequence, with residue 1 being the initiation Met. Human and mouse beta  subunits are aligned with human gp130, the IL-2Rbeta and IL-2Rgamma chains, the erythropoietin receptor (EPOR), and the growth hormone (GHR). The dashes represent spaces introduced to optimize the alignment.

Mutation of Cys-86 and Cys-91 Selectively Disrupt Ligand-induced, Disulfide-linked Heterodimer Formation-- Expression plasmids encoding IL-3R alpha  and wild type (wt) or C86A, C91A, C96A, C100A, and C234A mutant beta c were co-transfected into COS cells. After 48 h, the cells were 125I surface-labeled and either left unstimulated or stimulated with IL-3. IL-3Ralpha and beta c were then immunoprecipitated with specific MAbs 9F5 and 8E4, respectively, and the proteins were resolved by 6% SDS-PAGE under either nonreducing or reducing conditions. We found that the beta c mutants C100A and C234A behaved very similarly to wt beta c. Both mutants allowed the formation of two high molecular weight complexes in response to IL-3 (Fig. 2A), which, as with wt beta c, contain IL-3Ralpha and beta c (Ref. 17 and data not shown). These two complexes were immunoprecipitated by both anti-IL-3Ralpha mAb 9F5 and anti-beta c mAb 8E4 (Fig. 2A). In the absence of IL-3, the anti-IL-3Ralpha mAb 9F5 immunoprecipitated only monomeric IL-3Ralpha , whereas the anti-beta c mAb 8E4 immunoprecipitated monomeric beta c as well as the high molecular weight complex corresponding to disulfide-linked beta c homodimers (4, 17) (Fig. 2A). As with the disulfide-linked dimers, the noncovalent IL-3Ralpha and beta c heterodimers were not affected by mutating Cys-100 or Cys-234 because both the anti-IL-3Ralpha mAb and the anti-beta c mAb co-immunoprecipitated both IL-3Ralpha and beta c, and they did so only in the presence of IL-3 (Fig. 2B).


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Fig. 2.   Substitutions of Cys-86 and Cys-91 in beta c abolish disulfide-linked IL-3Ralpha and beta c heterodimerization without affecting their noncovalent association. COS cells transfected with IL-3Ralpha and either wild type or mutant beta c C86A, C91A, C96A, C100A, and C234A were 125I-surface labeled and incubated in medium without (-) or with (+) 6.5 nM IL-3 for 5 min at 4 °C. After cell lysis, proteins were immunoprecipitated (IP) with mAb 9F5 (anti-IL-3Ralpha ) or mAb 8E4 (anti-beta c). The immunoprecipitated proteins were separated either under nonreducing conditions on an SDS-6% polyacrylamide gel (A) or under reducing conditions on an SDS-7.5% polyacrylamide gel (B) and visualized by phosphorimaging.

In contrast, the mutants C86A, C91A, and C96A had a profound effect on disulfide-linked receptor dimerization. In the presence of IL-3, anti-IL-3Ralpha mAb 9F5 and anti-beta c mAb 8E4 did not immunoprecipitate the high molecular weight complexes corresponding to IL-3Ralpha and beta c heterodimers (Fig. 2A). In fact, under nonreducing conditions, very little or no monomeric beta c was immunoprecipitated by either mAb; most of the label was observed in the high molecular weight region, probably representing aggregated beta c. With the anti-IL-3Ralpha mAb 9F5, a nonspecific band migrating slightly faster than beta c was seen (Fig. 2A). Under reducing conditions, however, monomeric beta c could be detected (Fig. 2B). An important difference was noted between the C86A and C91A mutants on one hand and the C96A mutant on the other hand. Anti-IL-3Ralpha mAb 9F5 co-immunoprecipitated C86A and C91A in the presence of IL-3 but did not co-immunoprecipitate C96A (Fig. 2). Reciprocally, in the presence of IL-3, the anti-beta c mAb co-immnunoprecipitated IL-3Ralpha with C86A and C91A but not with C96A (Fig. 2). This was more clearly seen under reducing conditions (Fig. 2B) than under nonreducing conditions (Fig. 2A), where an overall lower signal was observed.

To verify that the surface expression levels of the individual beta c mutants was similar to beta c wt, COS cells transfected with the various constructs were analyzed by flow cytometry. Flow cytometry analysis indicated that the surface expression of wt beta c and the Cys right-arrow Ala beta c mutants was very similar both in terms of percentage of transfected cells expressing the different receptor subunits and in absolute levels (Fig. 3), suggesting that the mutations did not affect subunit transport and expression at the cell surface.


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Fig. 3.   Surface expression of IL-3Ralpha and wild type or mutant beta c transfected into COS cells as measured by flow cytometry. COS cells expressing both IL-3Ralpha and GM-CSFRalpha chains together with wild type (A) or mutant beta c C86A (B), C91A (C), C96A (D), C100A (E), and C234A (F) were stained with negative control antibody mouse IgG1 (control), mAb 9F5 (anti-IL-3Ralpha ), and mAb 8E4 (anti-beta c).

The beta c Mutants C86A and C91A Do Not Disrupt IL-3 and GM-CSF High Affinity Binding-- We next examined the ability of Cys mutants of beta c to support high affinity IL-3 or GM-CSF binding. COS cells were transfected with IL-3Ralpha and GM-CSFRalpha and either wt beta c or the Cys right-arrow Ala beta c mutants and subjected to saturation binding studies with 125I-IL3 and 125I-GM-CSF. Scatchard transformation of the saturation binding curves were performed and the Kd and receptor numbers determined using the Ligand program. We found that beta c bearing Cys right-arrow Ala substitutions at positions 86, 91, 100, and 234 were able to form high affinity binding sites (Fig. 4 and Table I). The range of affinities for IL-3 high affinity binding of these Cys right-arrow Ala beta c substitution mutants varied from 31 to 280 pM, compared with 330 pM for wt beta c, whereas GM-CSF high affinity binding ranged from 27 to 230 pM, compared with 120 pM for wild type beta c. In contrast, COS cell transfectants expressing the C96A beta c showed no detectable high affinity binding (Fig. 4 and Table I).


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Fig. 4.   High affinity 125I-IL-3 and 125I-GM-CSF binding using Cys right-arrow Ala beta c mutants. COS cells expressing both IL-3Ralpha and GM-CSFRalpha chains together with wild type beta c (open circles) or beta c mutants (filled circles) containing different Cys right-arrow Ala substitutions were subjected to Scatchard transformation of saturation binding curves. The derived values are shown in Table I.

                              
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Table I
Effect of alanine substitutions of Cys-86, Cys-91, Cys-96, Cys-100, and Cys-234 of beta c on IL-3 and GM-CSF high affinity binding
COS cells were transfected with IL-3Ralpha , GM-CSFRalpha , and either wild type beta c or mutated beta c carrying alanine substitutions of Cys at positions 86, 91, 96, 100, and 234 and subjected to saturation binding studies with 125I-labeled IL-3 and 125I-labeled GM-CSF. The radioiodinated ligand concentration for both IL-3 and GM-CSF ranged from 10 pM to 10 µM. Nonspecific binding was determined in the presence of 1 µM unlabeled ligand. Scatchard transformation of the saturation binding curves were performed, and the Kd and receptor numbers determined using the LIGAND program. In the case of IL-3, due to the extremely low affinity of IL-3Ralpha , the affinity was estimated to be 50 nM based on our own previous studies (25). In the case of GM-CSF binding, a two-site fit was statistically preferred (P < 0.05) with all the beta c constructs except for C96A, in which no high affinity sites were detected. Values from two representative experiments are shown. Experiment 2 is the same as the experiment shown in Fig 4.

Although beta c mutants C86A and C91A were able to support high affinity binding of IL-3 and GM-CSF, a reduction in the number of high affinity receptors was observed compared with wild type beta c and the C100A and C234A analogues (Fig. 4 and Table I). This is probably a reflection of the tendency of C86A and C91A beta c analogues to oligomerize as observed in the immunoprecipitations under nonreducing conditions (Fig. 2A), thereby reducing the amount of free beta c available for interaction with alpha  chain.

C86A and C91A Abolish IL-3-, GM-CSF-, and IL-5-dependent Tyrosine Phosphorylation of beta c-- It has been previously established that stimulation of cells with IL-3 leads to the formation of disulfide-linked heterodimers of IL-3 receptor alpha  and beta c chain, which is associated with phosphorylation of beta c (17). Similarly, in the case of the GM-CSF receptor, receptor heterodimerization occurs upon stimulation with GM-CSF, and this is accompanied by beta c phosphorylation (4). Here we show that in addition to the IL-3 and GM-CSF receptors, the IL-5 receptor forms disulfide-linked complexes that are similarly accompanied by beta c phosphorylation (Fig. 5). The relative proportion of phosphorylated beta c in the disulfide-linked heterodimer and in monomeric beta c varied between the three receptors. This may be due to kinetic differences in receptor assembly or in the stability of each receptor heterodimer. We have now taken advantage of the inability of beta c mutants to form disulfide-linked heterodimers to determine whether the formation of these is necessary for receptor activation or whether noncovalent dimerization is sufficient for activation as measured by beta c phosphorylation. We transfected wt beta c and the different beta c mutants in HEK293T cells together with JAK-2 and either IL-3Ralpha , GM-CSFRalpha , or IL-5Ralpha chain cDNA. After 48 h, the cells were either not treated or treated with IL-3, GM-CSF, or IL-5, lysed, and immunoprecipitated with mAb 8E4 anti-beta c. The immunoprecipitates were separated on SDS-PAGE gels under reducing conditions and Western blotted with antiphosphotyrosine antibody. Mutants C100A and C234A and wt beta c, which heterodimerize with the receptor alpha  chain in a disulfide-linked manner in response to ligand, showed phosphorylation of beta c. In contrast, mutants C86A, C91A, and C96A, which have lost the ability to heterodimerize in a disulfide-linked manner in response to ligand, have lost the potential to be phosphorylated in response to IL-3, GM-CSF, or IL-5 (Fig. 6).


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Fig. 5.   Ligand-induced disulfide-linked IL-3, GM-CSF, and IL-5 receptor dimerization results in phosphorylation of beta c. TF1.8 cells were either incubated with medium alone (-) or stimulated with medium containing 6.5 nM IL-3 (A), 6.5 nM GM-CSF (B), or 6.5 nM IL-5 (C) for 5 min at 37 °C. After cell lysis, proteins were immunoprecipitated with anti-beta c mAb 8E4, and the immunoprecipitates were separated under nonreducing conditions on an SDS-7.5% polyacrylamide gel and transferred onto nitrocellulose filters. The filters were then probed either with mAb 1C1 anti-beta c or anti-phosphotyrosine antibody 3-365-10.


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Fig. 6.   Alanine substitutions Cys-91, Cys-96, and Cys-100 of beta c abolish IL-3-, GM-CSF-, and IL-5-induced tyrosine phosphorylation of beta c. HEK293T cells transfected with either IL-3Ralpha (A), GM-CSFRalpha (B), or IL-5Ralpha (C) together with wild type or mutant beta c were incubated either with medium alone (-) or with medium containing 6.5 nM IL-3 (+IL-3), 6.5 nM GM-CSF (+GM-CSF), or 6.5 nM IL-5 (+IL-5) for 5 min at 4 °C. After cell lysis, proteins were immunoprecipitated with anti-beta c mAb 8E4, and the immunoprecipitates were separated under reducing conditions on an SDS-7.5% polyacrylamide gel transferred to nitrocellulose and probed with anti-phosphotyrosine antibody 3-365-10 (A-C). To control for the amount of beta c present, the filters were also probed with anti-beta c, mAb, 1C1 (D).

The expression of all mutants compared with wt was monitored by flow cytometry and by Western blot analysis with antibodies to beta c and indicated that the levels were very similar between all the mutants.(Figs. 3 and 5D). Because the number of high affinity sites for mutants C86A and C91A was decreased, the lack of phosphorylation with these two mutants could have been the result of a decrease in sensitivity due to less mutant beta c being heterodimerized compared with the total amount of mutant beta c expressed. To address this possibility, we have examined only beta c heterodimerized to the IL-3Ralpha by immunoprecipitating with IL-3Ralpha antibody and Western blotting with antiphosphotyrosine antibody. The results were identical to those seen when the beta c was directly immunoprecipitated, indicating that cysteines in position 86 and 91 are essential for receptor tyrosine phosphorylation (Fig. 7).


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Fig. 7.   Ligand-induced heterodimers of IL-3Ralpha and beta c with C86A and C91A substitution lack tyrosine phosphorylation of beta c. HEK293T cells transfected with IL-3Ralpha together with either wild type or mutant beta c were incubated with medium alone (-) or with medium containing 6.5 nM IL-3 (+IL-3) for 5 min at 4 °C. After cell lysis, proteins were immunoprecipitated with mAb 9F5 (IL-3Ralpha ), and the immunoprecipitates were separated under reducing conditions on a SDS-7.5% polyacrylamide gel, transferred onto nitrocellulose, and probed with antiphosphotyrosine antibody 3-365-10 (A). To control for similar amounts of immunoprecipitated beta c, the filters were also probed with mAb 1C1 anti-beta c (B).

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

We show here that disulfide-linked heterodimerization of the GM-CSF, IL-3, and IL-5 receptors is essential for receptor activation by the cognate ligand. Furthermore, we have identified Cys-86 and Cys-91 in the N-terminal domain of beta c as the key Cys residues involved in heterodimerization with the alpha  chain of each receptor. Comparison with other cytokine receptors indicates that these Cys residues constitute a conserved motif present only in human and mouse beta c and in beta IL-3, suggesting that it subserves a specialized function restricted to the GM-CSF, IL-3, and IL-5 receptor family.

We have previously shown that the human IL-3 and GM-CSF receptors undergo both noncovalent and disulfide-linked dimerization upon ligand binding (4). We have now extended these observations to the IL-5 receptor, demonstrating that disulfide-linked dimerization is a common theme in this receptor subfamily. To identify the Cys residues in beta c responsible for disulfide linkage with the IL-3, GM-CSF, and IL-5 receptor alpha  chains, we mutagenized five of the eight Cys residues in the N-terminal CRM of beta c that, from alignment with other cytokine receptors, represented the best candidates for intermolecular interactions. We found a range of sensitivities to mutation of these Cys residues that could be correlated with their interspecies and interreceptor conservation.

The first class of Cys residue is exemplified by Cys-100 and Cys-234. These residues are not conserved with even the closely related mouse beta  chains and we found no phenotype on replacing them with alanine residues. The second class is represented by Cys-96, which is apparently a conserved residue in both the mouse beta  chains and the cytokine receptor family at large (29) and is inferred to be involved in a structurally conserved disulfide bond. This residue is apparently required for the structural integrity of the first domain of hbeta c if not the entire extracellular portion of the molecule. Although the C96A mutation permitted cell-surface expression of hbeta c, it did not support high affinity binding of GM-CSF or IL-3 despite the substitution being well removed in sequence, and presumably spatially distant, from the fourth domain of the receptor that encompasses the majority of the ligand-recognition determinants (18, 25). The exact molecular basis for this observation is uncertain, but it may be related to sequestration of hbeta c into very large aggregates (Fig. 2A) that obscure the ligand-contact site.

The third and most interesting class of cysteine mutation is that of C86A and C91A, analogues that have lost their ability to form disulfide-linked heterodimers but still retain the ability to associate noncovalently with the alpha  subunit upon stimulation with ligand (Fig. 2). Although these mutants exhibited some propensity to aggregate in the absence of stimulation, they retain the ability to interact with ligand as judged by their ability to support high affinity binding. Importantly, these analogues are deficient in phosphorylation of tyrosine residues in beta c; however, receptor-mediated functions and downstream signaling remains to be ascertained.

The observation of identical phenotypes with either mutation C86A or mutation C91A suggests that these residues may cooperate functionally in the native receptor such as via formation of an additional intramolecular disulfide. This is consistent with our molecular modelling of beta c, which suggests that Cys-86 and Cys-91 are sufficiently close to allow the formation of such a disulfide bond.2 In the presence of ligand, this bond is proposed to undergo disulfide exchange with a free sulfhydryl group from the alpha  chain that is brought into proximity via ligand-dependent noncovalent association.

Previous experiments have noted a correlation between disulfide-linked receptor dimerization and receptor activation. IL-6 induces covalent dimerization of two molecules of gp130 (9), and CNTF induces covalent dimerization of gp130 with the LIF receptor (10). Similarly, IL-3 (Fig. 5A), GM-CSF (Fig. 5B), and IL-5 (Fig. 5C) induce covalent dimerization of beta c with the corresponding alpha  chain. In all of these cases, concomitant phosphorylation of the receptor has been observed; however, a causal relationship has not been established. The use of the C86A and C91A mutants allowed us to demonstrate that noncovalent receptor associations are not sufficient for receptor tyrosine phosphorylation and that this requires disulfide linkage of receptor subunits. The role of covalent dimerization seems to be to ensure concomitant dimerization of the cytoplasmic portions of beta c, facilitating transphosphorylation of associated kinases of the JAK family. Indeed, experiments using chimeric receptors that cause artificial dimerization of the cytoplasmic portions of beta c lead to their activation (30, 31), and direct dimerization of JAK has been shown to transduce a growth signal (32). Although they are essential for normal activation (14-16), the role of the cytoplasmic domains of the receptor alpha  chains remains unclear; however, they may serve to orientate to beta  chains so as to juxtapose correctly the JAK molecules or to participate directly in certain functions (33, 34).

The beta  chain forms intermolecular disulfides specifically with the alpha  chains of the GM-CSF, IL-3, or IL-5 receptors. A common feature of these alpha  chains is the presence of an N-terminal FnIII-like domain of a type restricted to this subfamily of cytokine receptors. Within this N-terminal domain, all three receptor alpha  chains possess an uneven number of Cys residues in the N-terminal domain, suggesting that these Cys residues are the most likely candidates to act as partners for beta c. The IL-5Ralpha has only a single Cys residue in the N-terminal domain, at position 86, and this residue has been shown to be important for IL-5 binding to the receptor (35).

The stoichiometry of the IL-3, GM-CSF, and IL-5 receptor complexes is not known. The formation of an intermolecular disulfide bond between Cys-86 or Cys-91 of beta c and a Cys in the N-terminal domain of a receptor alpha  chain could potentially occur with either the alpha  chain with which it shares ligand or a second alpha  chain, recruited as part of a hexameric complex, as seen with the IL-6 receptor (36). Because the individual FnIII-like domains of the receptor alpha  chains and beta c are likely to be fairly rigid units with a length of 3.5-4.5 nm, the ability of beta c to contact alpha  chain will depend on the interdomain angles that they can adopt. The receptor alpha and beta c chains are class 1 cytokine receptors, and the angles observed between the two domains of the CRM in the known structures of this family, growth hormone receptor (5) and erythropoietin receptor (37) are approximately 90°. It is therefore reasonable to infer that the angles between domains 1 and 2 and domains 3 and 4 of beta c and between domains 2 and 3 of the receptor alpha  chains will also be approximately 90°. The conformation of the linker peptides between domains 2 and 3 of beta c or between domains 1 and 2 of the receptor alpha  chains cannot be gauged by reference to homologous structures. Even if the linker peptides permitted the membrane-distal portions to fold back over the cytokine binding portions of the receptors, this would be unlikely to facilitate a sufficiently close approach of Cys residues to allow formation of the observed intermolecular disulfide bonds. Rather, we propose that the intermolecular disulfide bond forms between beta c and an alpha  chain from a second receptor heterodimer (Fig. 8) because this can be accommodated readily with respect to both the sizes of the domains and their interdomain angles.


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Fig. 8.   Proposed model for assembly of GM-CSF-, IL-3-, and IL-5-induced receptor complexes. The binding of GM-CSF, IL-3, and IL-5 to GM-CSFRalpha , IL-3Ralpha , and IL-5Ralpha chain, respectively, induces alpha :beta c heterodimerization and a conformational change in alpha  chain that allows its disulfide linkage to beta c. Modeling of beta c suggests that this bridging would only be possible if the unpaired cysteines in the alpha  chain N-terminal domain (Nt) of receptor 1 formed a disulfide bridging with cysteine at position 86 or 91 in domain 1 (D1) of beta c in receptor 2. The bringing together of two beta c chains with their associated JAK-2 molecules would then lead to receptor activation. A schematic model representing the side view is shown in A and a top view in B.

Based on the likely orientation of their N-terminal domains with respect to the CRM of the alpha  chains, we favor a receptor complex arranged clockwise when viewed from outside the cell in the order alpha  chain1/ligand1/beta chain1S-Salpha chain2/ligand2/beta chain2. The disulfide linkage of beta c in one receptor heterodimer to an alpha  chain in a second receptor heterodimer would facilitate juxtaposing two beta c molecules with their associated JAK kinases and induce receptor phosphorylation. This initial association may also facilitate the formation of a second disulfide between beta c in receptor 2 and an alpha  chain in receptor 1 (Fig. 8, A and B). The formation of a 2:2:2 complex is also consistent with the requirement of two alpha  chains in an active ligand-receptor complex (38). On the other hand, a 1:1:1 stoichiometry has been suggested from experiments using chimeras of alpha  and beta c with fos and jun leucine zippers, although the formation of higher order complexes was not excluded (34). The direct measurement of alpha -beta c interactions in solution may ultimately resolve this question.

    ACKNOWLEDGEMENTS

We thank Anna Nitschke for excellent secretarial assistance and Dr. Michael Berndt for critical reading of the manuscript.

    FOOTNOTES

* This work was supported by the National Health and Medical Research Council of Australia.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 Florey Fellow of the Royal Adelaide Hospital Research Fund.

§ To whom correspondence should be addressed: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, P. O. Box 14, Rundle Mall, Adelaide 5000, South Australia, Australia. Tel.: 61-8-8222-3471; Fax: 61-8-8222-3538; E-mail: alopez{at}immuno.imvs.sa.gov.au.

1 The abbreviations used are: IL, interleukin; GM-CSF, granulocyte-macrophage colony-stimulating factor; GM-CSFR, GM-CSF receptor; IL-2R, IL-2 receptor; beta c, common beta  chain; CRM, cytokine receptor module; mAb, monoclonal antibody; PAGE, polyacrylamide gel electrophoresis; wt, wild type; LIF, leukemia inhibitory factor; CNTF, ciliary neutrotrophic factor; CNTFR, CNTF receptor.

2 F. C. Stomski, J. M. Woodcock, B. Zacharakis, C. J. Bagley, Q. Sun, and A. F. Lopez, unpublished observations.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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