A Novel Functional Epitope Formed by Domains 1 and 4 of the Human Common beta -Subunit Is Involved in Receptor Activation by Granulocyte Macrophage Colony-stimulating Factor and Interleukin 5*

James M. MurphyDagger §, Sally C. FordDagger , Ursula M. WiedemannDagger , Paul D. Carr§, David L. Ollis§, and Ian G. YoungDagger

From the Dagger  Division of Molecular Bioscience, John Curtin School of Medical Research and the § Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia

Received for publication, November 15, 2002

    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The receptors for human interleukins 3 and 5 and granulocyte macrophage colony-stimulating factor are composed of ligand-specific alpha -subunits and a common beta -subunit (beta c), the major signaling entity. The way in which beta c interacts with ligands in the respective activation complexes has remained poorly understood. The recently determined crystal structure of the extracellular domain of beta c revealed a possible ligand-binding interface composed of domain 1 of one chain of the beta c dimer and the adjacent domain 4 of the symmetry-related chain. We have used site-directed mutagenesis, in conjunction with ligand binding and proliferation studies, to demonstrate the critical requirement of the domain 1 residues, Tyr15 (A-B loop) and Phe79 (E-F loop), in high affinity complex formation and receptor activation. The novel ligand-receptor interface formed between domains 1 and 4 represents the first example of a class I cytokine receptor interface to be composed of two noncontiguous fibronectin III domains.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Granulocyte-macrophage colony stimulating factor (GM-CSF),1 IL-5, and IL-3 are three cytokines produced by activated T-cells during immune responses that are important mediators of inducible hematopoiesis and inflammation. They signal through a shared receptor (the beta c receptor) and play an important role in the pathogenesis of allergic disorders and inflammatory diseases of the lung, such as asthma (1, 2). Eosinophilia is controlled primarily by IL-5 (3, 4) and to a lesser extent by IL-3 and GM-CSF. GM-CSF and IL-3 are believed to be centrally involved in other chronic inflammatory diseases, such as arthritis and multiple sclerosis (5, 6).

The receptors for IL-5, IL-3, and GM-CSF consist of cytokine-specific alpha  receptors essential to the activation of the shared common beta -receptor subunit (beta c), which is believed to be the main signaling entity (7-10). Human GM-CSF and IL-3 bind to their cognate alpha  receptors with low affinities, but in the presence of beta c, high affinity complexes are formed. For example, with GM-CSF, the Kd values for low and high affinity binding are 2-10 nM and 50-100 pM, respectively. Human IL-5 differs from IL-3 and GM-CSF in that it binds to its alpha  receptor (IL-5Ralpha ) with greater affinity, and there is only a small affinity conversion by the beta c subunit (10, 11). The formation of a complex involving ligand and the alpha  and beta c receptors is necessary for receptor activation and signaling. The cytoplasmic portions of the alpha  and beta c subunits possess no intrinsic tyrosine kinase activity (12) but in the activated receptor complexes formed with all three ligands interact with and activate Janus kinase 2 (13), leading to the phosphorylation of eight tyrosine residues located in the beta c cytoplasmic domain (14, 15). Subsequently, several signaling pathways are induced, including the Janus kinase/STAT, Ras/mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways (reviewed in Ref. 16). The structures of the activation complexes involving the beta c receptor are unknown.

Structurally, the alpha  and beta c subunits of the GM-CSF, IL-3, and IL-5 receptors belong to the cytokine class I receptor superfamily (or hemopoietin receptor family), which includes the growth hormone, erythropoietin, gp130, and IL-4alpha receptors. The characteristic feature of this family is the extracellular cytokine receptor homology module, composed of two fibronectin III domains that contain a number of conserved sequence elements (17, 18). The crystal structures of the receptor-ligand complexes of the growth hormone (19), erythropoietin (20, 21), and IL-4alpha (22) receptors have been solved, and through mutagenesis studies (23-27), the ligand-binding epitopes have been elucidated. The extracellular domains of these receptors contain two approximately orthogonal fibronectin III domains that each contribute key residues for ligand binding from loops at the receptor "elbow" region. To what extent these principles apply to the beta c subunit has not been clear, because beta c does not bind any of its three ligands directly, has four fibronectin III domains in its extracellular region rather than two, and heterodimerizes with the respective alpha  receptors. By analogy with the other class I receptors, several groups have predicted the existence of an elbow region formed by domains 3 and 4 of beta c that would serve as a ligand-binding interface. Mutagenesis studies have been successful in identifying residues in the B'-C' (28, 29) and F'-G' (30) loops of domain 4 that are critical for high affinity ligand binding, but no critical residues have been detected in domain 3.

Our recent determination of the crystal structure of the complete extracellular domain of the human beta c subunit (Fig. 1) showed it to exist as a stable, intertwined homodimer (32). A number of lines of evidence indicate that the dimer is not confined to the crystal and is likely to be the functional form of the receptor in vivo (32). A highly novel feature of the structure is that strand G of domain 1 forms strand G of domain 3 of the dimer-related molecule, and likewise strand G of domain 3 partners with the dimer-related domain 1. The unique interlocking dimeric structure of beta c results in the juxtapositioning of domain 1 of one chain adjacent to domain 4 of its dimer-related equivalent chain (Fig. 1). The interface between domains 1 and 4 is approximately orthogonal and resembles the ligand-binding interface of the growth hormone, erythropoietin, and IL-4alpha receptors (Fig. 1). Thus, by analogy with other class I cytokine receptors, domains 1 and 4 could form a ligand-binding interface involved in high affinity complex formation. However, it has not been clear to what extent the binding interactions in the beta c activation complex conform to the principles for ligand binding already established in the cytokine class I receptor family or whether novel types of interactions are involved.


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Fig. 1.   Structure of the beta c receptor. Top panel, structure of the beta c homodimer. Chain A is yellow, and chain B is red. Glycosylation is shown by sticks colored by atom type (green, carbon; red, oxygen; and blue, nitrogen). The domains of chain A are labeled in yellow text, and those of chain B are in red text. The loops of domains 1 and 4 potentially relevant to ligand-receptor interaction are labeled in white text. Bottom left panel, the IL-4·IL-4 alpha  receptor complex. IL-4 is green, and IL-4alpha receptor is orange. The loops of the IL-4 alpha  receptor involved in complex formation are labeled. Bottom right panel, the domain 1-domain 4 interface of beta c, which resembles the ligand-binding interface of the IL-4alpha receptor. The beta c loops equivalent to the IL-4alpha ligand-binding loops are labeled. The figures were drawn using RIBBONS (31). The structures of beta c and the IL-4:IL-4 alpha  receptor complex are taken from Protein Data Bank codes 1gh7 and 1iar, respectively.

In the present work, we have used site-directed mutagenesis, ligand binding, and proliferation assays to establish that Tyr15 and Phe79 of domain 1 of beta c are required for high affinity ligand binding and receptor activation. The novel ligand-receptor interface, which involves cooperation between domains 1 and 4 of two different chains of the beta c receptor dimer, is the first example in the class I cytokine receptor family of a functional epitope that involves noncontiguous fibronectin III domains.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Site-directed Mutagenesis of Human beta c cDNA-- A cDNA encoding the extracellular domain of the human beta c subunit (residues 1-443) was cloned from HL60 eosinophils (33). A full-length cDNA was constructed by substituting a BstXI-BssHII fragment of the extracellular domain of the cDNA for beta c isolated from TF1 cells (8). Site-directed mutagenesis was carried out using the QuikChange method (Stratagene, La Jolla, CA) with Pfu Turbo DNA polymerase. The complete sequences of mutant beta c cDNAs were verified by Big Dye Terminator cycle sequencing (Applied Biosystems, Gladesville, Australia).

Expression Constructs-- For expression in COS7 cells, the cDNAs encoding human GM-CSFRalpha (from T. Willson, Walter and Eliza Hall Institute, Melbourne, Australia) or IL-5Ralpha and wild type or mutant beta c subunits were subcloned into pCEX-V3-Xba, a vector derived from pCEX-V3 (34). For expression in CTLL-2 cells, cDNAs encoding human GM-CSFRalpha or IL-5Ralpha were subcloned into pEF-IRES-N, and the beta c subunits were subcloned into pEF-IRES-P (35) (from S. Hobbs, Institute of Cancer Research, London, UK).

Cytokines and Radiolabeling-- Human IL-5, human GM-CSF, and murine IL-2 were produced using the baculovirus expression system (33). Purified IL-5 was radiolabeled as described previously (36). Radiolabeled IL-5 was stored at 4 °C and used for up to 7 days. Radiolabeled GM-CSF was purchased from PerkinElmer Life Sciences, stored at -70 °C, and used for up to 6 weeks.

Cells and DNA Transfections-- COS7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% bovine serum and CTLL-2 cells as described previously (33). DNA expression constructs were introduced into cells by electroporation (33). COS7 cells were harvested using trypsin and electroporated with 10 µg of GM-CSFRalpha or IL-5Ralpha and 25 µg of wild type or mutant beta c constructs at 200 V and 960 microfarads. Binding studies and antibody staining were performed on cells 64-68 h after transfection. CTLL-2 cell lines stably expressing GM-CSFRalpha or IL-5Ralpha and wild type or mutant beta c subunits were generated in two steps, as described previously (33).

Equilibrium Binding Analysis for GM-CSF and IL-5-- COS7 cells expressing the relevant receptors were used to study IL-5 and GM-CSF binding and were harvested as described previously (28). CTLL-2 cells expressing the IL-5Ralpha and beta c subunits were cultured in IL-5 and washed four times with factor-free basal medium before use in binding assays. 106 cells in resuspended binding medium (RPMI 1640 supplemented with 10 mM HEPES and 0.5% w/v bovine serum albumin) were incubated with increasing concentrations of radioligand for 2-3 h at 4 °C. Nonspecific binding was determined by the addition of a 100-fold excess of unlabeled ligand to samples in which high radioligand concentrations were added. The nonspecific binding was interpolated for lower radioligand concentration data using linear regression. The assays were terminated by centrifugation of each binding solution through 0.2 ml of 2:1 v/v dibutyl phthalate:dinonyl phthalate at 12,000 × g for 4 min (37). The tips of tubes, containing the visible cell pellet and associated radio-iodinated ligand, were counted using a Packard 5780 Auto-gamma counter. The dissociation constants (Kd) and Scatchard transformations were calculated from specific binding data using the EBDA (38) and LIGAND (39) programs (Biosoft, Cambridge, UK). Multiple data files were co-analyzed to obtain more accurate estimates of Kd values. One- and two-site models were evaluated in LIGAND and only when statistically significant (p < 0.05), a two-site model was used to determine Kd values.

Flow Cytometry-- COS7 cells transfected with cDNAs encoding wild type or mutant beta c subunits were incubated in the presence of rabbit antiserum raised against the beta c(HL60) extracellular domain for 30min at 4 °C. The cells were washed and incubated with a fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (Sigma) for 25 min at 4 °C and then washed before analysis of cell surface expression was performed on a FACScan flow cytometer (Becton Dickinson, North Ryde, Australia).

Proliferation Assays-- [3H]Thymidine incorporation assays were performed as described previously (33) to determine the capacity of the wild type or mutant beta c subunits to deliver a proliferative signal in CTLL-2 stable cell lines.

    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Involvement of Domain 1 of beta c in Ligand Binding-- In the well established class I cytokine receptor superfamily ligand-binding motif, critical ligand-binding residues are commonly contributed from the E-F and A-B loops of the membrane-distal fibronectin III domain and from the B'-C' and F'-G' loops of the membrane-proximal domain (Fig. 1). From the x-ray structure of beta c (Fig. 1), it is clear that if ligand-binding with beta c follows the previously established principles, the interface would involve residues from loops in domain 1 cooperating with those from domain 4 of the partner molecule of the beta c dimer. The loops of domain 1 that could potentially be involved in ligand binding are the A-B, E-F, and C-D loops. Site-directed mutagenesis of the human beta c cDNA was therefore performed to generate alanine mutants of the residues in these loops. In studies on cytokine class I receptors, alanine substitution is commonly used for the determination of residues whose side chains are involved in ligand binding. Alanine has a hydrophobic side chain but is often found in solvent-exposed regions of proteins, and its introduction does not disrupt the geometry of the main-chain, nor does the methyl side chain cause electrostatic or steric interference. Because GM-CSF has readily distinguishable low and high affinity binding, we used this ligand as a model to test the binding of the mutants. We also examined the binding of IL-5, because this cytokine, unlike GM-CSF, is an interlocking dimer and could potentially interact with beta c in a different way.

GM-CSF Binding Properties of Wild Type and Mutant beta c Receptors-- COS7 cells were co-transfected with expression vectors encoding human GM-CSFRalpha and wild type or mutant beta c subunits. Saturation binding assays were then performed on the transiently transfected COS7 cells using 125I-labeled human GM-CSF, and the dissociation constants (Kd) were determined (Table I).


                              
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Table I
Dissociation constants for GM-CSF binding to COS7 cells expressing GM-CSFRalpha alone and with wild type or mutant beta c subunits

COS7 cells transfected with the cDNA encoding GM-CSFRalpha were found to bind GM-CSF with low affinity (Kd, 8.5 nM). Cells expressing both wild type beta c and GM-CSFRalpha exhibited two GM-CSF-binding sites, as illustrated by the curvilinear Scatchard plot (Fig. 2). The Kd values of 58 pM (high affinity) and 8.3 nM (low affinity) are consistent with those previously reported for the GM-CSF receptor system (8, 28-30). The wild type beta c receptor used in this work and in the x-ray structural studies has a six-amino acid insertion in the C-D loop of domain 3. This beta c variant is the major form in HL60 eosinophils and appears to be a splice variant with normal function (33). It has not previously been examined in binding studies. The Kd values obtained indicate that this variant has normal binding properties. We also included as a control a mutant (Y403A) affected in one of the key ligand-binding residues in domain 4. In agreement with previous studies (30, 40), mutation of Tyr403 was found to abolish high affinity binding (Table I).


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Fig. 2.   Y15A and F79A mutant beta c subunits are unable to bind GM-CSF with high affinity. Scatchard plots of 125I-labeled GM-CSF saturation binding data for COS7 cells expressing GM-CSFRalpha with wild type beta c derived from HL-60 cells (A), Y15A (B), or F79A mutant beta c (C). Data from a representative saturation binding experiment are shown in each plot with the line-of-best-fit determined by co-analysis of data from several binding experiments using LIGAND (39). The derived Kd values are shown in Table I.

Involvement of Loops in Domain 1 of the beta c Subunit in GM-CSF Binding-- The first loop of domain 1 to be examined was the A-B loop. This loop consists of residues Tyr15, Thr16, Ser17, and His18. Each residue was individually mutated to alanine, and the resultant mutants were examined for GM-CSF binding. No high affinity GM-CSF receptors were detected on cells co-transfected with cDNAs encoding GM-CSFRalpha and the mutant beta c subunit Y15A (Table I and Fig. 2). This result indicates that Tyr15 is a critical binding determinant in the high affinity binding of GM-CSF. The Scatchard plot of GM-CSF binding with Y15A beta c indicates the presence of only low affinity sites. No other residues located in the A-B loop were found to be essential for high affinity binding (Table I). It was also of interest to determine whether phenylalanine could substitute for tyrosine in GM-CSF binding, and therefore the mutant beta c, Y15F, was prepared. Y15F was found to give normal high affinity binding (Table I), indicating that the hydroxyl group of Tyr15 is not involved and that Phe is an effective substitution for Tyr at this position.

We next examined the involvement of the C-D and E-F loops of domain 1 in GM-CSF binding. The C-D loop consists of residues Asn42, Glu43, Asp44, Leu45, and Leu46. A mutant was generated in which all of the C-D loop residues were mutated to alanine. This mutant gave normal high affinity binding, indicating that the C-D loop is not involved (Table I). The E-F loop is made up of residues Cys76, Gln77, Ser78, Phe79, Val80, Val81, and Thr82. Mutation of the first three residues in the loop to alanine did not affect high affinity GM-CSF binding. The last four residues were individually mutated to alanine. Of these, only Phe79 was found to be required for high affinity GM-CSF binding. The F79A mutation of beta c completely abolished high affinity binding (Table I and Fig. 2), indicating that the E-F loop also plays an important role in forming the high affinity GM-CSF complex.

Cell Surface Expression of Y15A and F79A Mutant beta c Receptors-- To verify that the loss of high affinity binding when the Y15A and F79A beta c mutants and GM-CSFRalpha were co-expressed did not result from a perturbation of the receptor translation, folding, or presence on the cell surface, flow cytometry was used to detect beta c cell surface expression. COS7 cells transiently expressing wild type or mutant beta c subunits were stained with rabbit antiserum raised against the human beta c(HL60) extracellular domain and a fluorescein isothiocyanate-conjugated secondary antibody and examined by indirect flow cytometry (Fig. 3). The antiserum used was shown to immunoprecipitate human beta c from CTLL-2 GM-CSFRalpha /beta c cell lysates, and the affinity of the antiserum for the human beta c extracellular domain was verified using an enzyme-linked immunosorbent assay.2 The level of cell surface expression of the Y15A and F79A mutant beta c subunits was observed to be equivalent to wild type beta c expression with only small variations observed because of differences in transfection efficiency.


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Fig. 3.   Cell surface expression of the mutant beta c subunits, Y15A and F79A, as detected by flow cytometry. COS7 cells were transfected with cDNAs encoding wild type beta c (B), Y15A beta c (C), or F79A beta c (D) and, after 64-68 h, stained with rabbit anti-beta c serum and fluorescein-isothiocyanate-conjugated goat anti-rabbit antibody (solid line). Cells stained with only the fluorescein isothiocyanate-conjugated antibody were used as a control (broken line). COS7 cells were used also used as a control (A).

Growth Responses of the Mutant beta c Receptors to GM-CSF-- Although the formation of a high affinity complex is believed to be a prerequisite for receptor activation and downstream signaling, we considered it important to correlate the binding data with an unequivocal signaling response, in this case proliferation. The GM-CSFRalpha cDNA was installed in the murine IL-2-dependent lymphoid cell line, CTLL-2, using the pEF-IRES-N vector, which encodes G418 resistance. G418-resistant cells were subsequently transfected with wild type or mutant beta c cDNAs in the vector pEF-IRES-P, which encodes puromycin resistance. Following selection with these antibiotics, resistant cells, which had been maintained in the presence of murine IL-2, were used in proliferation assays to assess their responsiveness to GM-CSF after murine IL-2 was removed. The dose of GM-CSF required for half-maximal stimulation of each cell line (ED50) was determined by GM-CSF titration. The amount of GM-CSF giving 50% of maximal stimulation of the CTLL-2 cell line co-expressing the wild type beta c and GM-CSFRalpha subunits was defined as 1 unit, and the relative amounts of GM-CSF required for 50% stimulation of cell lines expressing mutant receptors were determined. Duplicate CTLL-2 cell lines co-expressing the Y15A or F79A mutant beta c and GM-CSFRalpha subunits were generated from separate transfections. The transfected cells expressing the mutant receptors exhibited severely reduced responsiveness to GM-CSF compared with cells expressing the wild type receptor (Table II). The amount of GM-CSF for a 50% growth response was increased 55-85-fold for Y15A and 8-16-fold for F79A. The parent cell line, CTLL-2 GM-CSFRalpha , did not respond detectably to GM-CSF, even at doses in excess of 2000 ED50 units, verifying the absolute requirement of beta c for the growth response.


                              
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Table II
Proliferation of CTLL-2 cell lines expressing reconstituted wild type or mutant GM-CSF or IL-5 receptors

Role of Tyr15 and Phe79 in High Affinity Binding and Signaling with IL-5-- Because IL-5, unlike GM-CSF, is a dimer, it was of interest to determine whether the same residues that were critical for high affinity GM-CSF binding were also critical for IL-5 binding. Human IL-5 binds to its cognate alpha  receptor with unusually high affinity. Reported increases in binding in the presence of the beta c subunit range from 2- to 3-fold (10, 11, 29). We compared the binding of IL-5 to IL-5Ralpha in the presence and absence of beta c in COS7 cells and found an increase in binding of less than 1.5-fold in the presence of beta c (Table III). CTLL-2 cells co-expressing the IL-5Ralpha and beta c subunits that gave a normal growth response to IL-5 exhibited the same Kd as COS7 cells co-expressing the IL-5Ralpha and beta c subunits (Table III). The similarity of the dissociation constants observed for high and low affinity IL-5 binding made it difficult to reliably distinguish IL-5 binding to the IL-5Ralpha :beta c complex from binding to IL-5Ralpha alone. However, we were able to establish that even though only a small affinity conversion was detectable, IL-5 binding induced receptor activation, because CTLL-2 cells co-expressing beta c with the IL-5Ralpha showed a growth response not seen with cells expressing IL-5Ralpha alone (Table II). We therefore examined the roles of Tyr15 and Phe79 of the beta c subunit in the proliferative response to IL-5 in CTLL-2 cells. Separate transfection experiments were performed to generate duplicate CTLL-2 cell lines co-expressing IL-5Ralpha and wild type, Y15A, or F79A beta c subunits. The duplicate CTLL-2 cell lines co-expressing the wild type beta c and IL-5Ralpha subunits gave similar growth responses to IL-5, giving 50% of maximal stimulation with 1 and 1.93 units of IL-5, respectively. In contrast, cells expressing the mutant beta c subunit Y15A together with IL-5Ralpha gave no detectable growth response to IL-5, whereas cell lines expressing F79A together with IL-5Ralpha required 7-15-fold more IL-5 for a 50% growth response than CTLL-2 cells co-expressing IL-5Ralpha and wild type beta c (Table II). The parent cell line, CTLL-2 IL-5Ralpha , did not detectably respond to IL-5 doses in excess of 250 units. It is interesting that the effect of the two mutations on the growth responses to both GM-CSF and IL-5 was very similar, with the Y15A mutant being more severely affected in growth signaling than the F79A mutant, even though the loss of high affinity GM-CSF binding seemed comparable in both cases.


                              
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Table III
Dissociation constants for IL-5 binding to cells expressing IL-5Ralpha in the presence or absence of the beta c subunit


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To date, the definition of the ligand-binding interfaces in the cytokine class I receptor family has been accomplished primarily for two-domain receptors, like the growth hormone receptor, that bind their ligands directly. In these receptors, a conserved motif consisting of an approximately orthogonal elbow between the two fibronectin III domains serves as the ligand-binding interface. X-ray crystallography of ligand-receptor complexes has demonstrated that the structural epitopes for these receptors comprise a relatively large number of residues at the protein-protein interfaces. However, mutagenesis has shown that only a small subset of these residues (forming the functional epitope) play key roles in ligand binding. The functional epitopes determined so far involve at least one residue in each of the two fibronectin III domains comprising the interface, with the residues being located in the A-B or E-F loops of the membrane-distal domain and the B'-C' or F'-G' loops of the membrane-proximal domain. The key residues are clustered in a binding "hot spot."

In contrast to the two-domain receptors, much less is known about the mechanism of ligand binding for a receptor like the beta c receptor that consists of four fibronectin III domains and forms a high affinity complex only with the ligands bound to their specific alpha  receptors. Previous work established the involvement of residues in the B'-C' (28, 29) or F'-G' (30) loops of the membrane-proximal domain (domain 4) but unexpectedly no residues from the predicted membrane-distal domain (domain 3). Our recent determination of the crystal structure of the beta c extracellular domain revealed that beta c exists as a homodimer with domain 1 of one chain positioned adjacent to domain 4 of the other chain (32), suggesting the possibility that the functional epitope for high affinity ligand binding may involve domains 1 and 4. Indeed, we show here that the domain 1 residues, Tyr15 (A-B loop) and Phe79 (E-F loop), are critical for the formation of the high affinity GM-CSF complex and subsequent activation of human GM-CSF receptor, as measured by a proliferative response. They are also critical for the growth response to IL-5, another of the ligands that shares the beta c receptor subunit, even though IL-5 differs from GM-CSF in that it is a dimer.

CTLL-2 cells co-expressing the Y15A or F79A mutant beta c subunits and the appropriate alpha  receptors showed substantially diminished growth responses to GM-CSF and IL-5. The Y15A mutant showed no detectable growth response to IL-5 and a severely reduced response to GM-CSF. The F79A mutant was less severely affected, but the data clearly indicate the involvement of Phe79 in the growth response to the two ligands. It is interesting that the Y15A and F79A beta c mutants can still signal detectably with high concentrations of GM-CSF despite their inability to support high affinity binding of GM-CSF. This finding is similar to the observation that the murine GM-CSF E21A mutant is capable of stimulating the murine GM-CSF receptor at elevated ligand concentrations, despite its inability to form a high affinity complex (41). The detectable growth response of the mutant beta c receptors confirms their cell surface expression, in agreement with the fluorescence-activated cell sorter analysis, indicating that single amino acid substitutions did not disrupt their translation, folding, or transport to the membrane.

The two critical residues identified in this study, Tyr15 and Phe79, are in the A-B and E-F loops of domain 1 of beta c, respectively. Together with the previously identified residues in domain 4 (Tyr347, His349, and Ile350 in the B'-C' loop and Tyr403 in the F'-G' loop), they form a functional epitope that resembles those of the simpler two-domain cytokine class I receptors, such as the growth hormone receptor. However, the beta c receptor functional epitope is novel because it is composed of domains contributed from two different protein chains.

The beta c functional epitope shows a superficial similarity to cluster I of the IL-4alpha receptor, which also features three tyrosines contributed by the A-B, B'-C', and F'-G' loops that are critical for ligand-binding (22, 27). Additionally, like IL-4, GM-CSF, IL-3, and IL-5, each possesses a conserved glutamic acid residue on the A-helix that is critical for the interaction with beta c (42-45). Despite Tyr15 of beta c being in a position analogous to the critical Tyr13 of the IL-4alpha receptor, this study has illustrated that the phenolic hydroxyl group of Tyr15 in beta c is dispensable for high affinity GM-CSF binding, whereas the phenolic hydroxyl of Tyr13 is essential for IL-4alpha ligand binding (27). This suggests that these two receptors engage their ligands by different mechanisms.

The residues in the receptor hot spot of beta c form a less compact cluster than those of the two-domain cytokine receptors, raising the interesting possibility that beta c functional epitope residues (for example Tyr403) could be involved in contacts with the alpha  subunits. Of the beta c functional epitope residues, Tyr15, Phe79, and Ile350, in contrast to Tyr347, His349, and Tyr403, are not highly exposed in the beta c structure. The low solvent accessibility of these residues, of itself, does not preclude a role in direct ligand interaction because it is known that Phe205, a residue critical for ligand binding by the erythropoietin receptor, is also not highly exposed (26). In addition, ligand-induced receptor plasticity has been well documented (46). However, it is also possible that the role of Phe79 in beta c could be to maintain the conformation of Tyr15, because the two residues make interatomic contacts in the beta c structure.

There is relatively little information available regarding the key residues involved in beta c recognition by the ligands, GM-CSF, IL-3, and IL-5. To date only a single residue (Glu21 of GM-CSF, Glu22 of IL-3, or Glu13 of IL-5) has been identified as critical for interaction with the beta c subunits (42-45). In comparison, IL-5 has been shown by mutagenesis to interact with IL-5Ralpha via several residue side chains (45, 47). Similarly, clusters of several residues on other cytokines have been shown to be critical for receptor binding (48, 49). Thus, we would expect that additional residues in the ligands that are involved in beta c recognition remain to be discovered.

Although a detailed understanding of receptor activation must await the determination of the structures of the respective activation complexes, the present work provides compelling evidence for the existence of a novel ligand-receptor interface in beta c formed by domains 1 and 4 and identifies Tyr15 and Phe79 in domain 1 as residues essential for beta c function.

    ACKNOWLEDGEMENTS

We thank T. Willson for providing the GM-CSFRalpha cDNA, S. Hobbs for the pEF-IRES vectors, A. Church for the purification of IL-5, I. Walker for the purification of GM-CSF, and S. Gustin for preparation of the anti-beta c serum.

    FOOTNOTES

* 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.

To whom correspondence should be addressed: Div. of Molecular Bioscience, John Curtin School of Medical Research, Canberra, ACT 0200, Australia. Tel.: 61-261252439; Fax: 61-261250415; E-mail: Ian.Young@anu.edu.au.

Published, JBC Papers in Press, January 12, 2003, DOI 10.1074/jbc.M211664200

1 The abbrieviations used are: GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; GM-CSFRalpha , alpha  subunit of GM-CSF receptor; IL-5Ralpha , alpha  subunit of IL-5 receptor; beta c, common beta -subunit of GM-CSF, IL-3 and IL-5 receptors; STAT, signal transducers and activators of transcription.

2 S. E. Gustin, J. M. Murphy, and I. G. Young, unpublished results.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1. Foster, P. S., Hogan, S. P., Ramsay, A. J., Matthaei, K. I., and Young, I. G. (1996) J. Exp. Med. 183, 195-201[Abstract]
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