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Originally published In Press as doi:10.1074/jbc.M710230200 on March 7, 2008

J. Biol. Chem., Vol. 283, Issue 19, 13398-13406, May 9, 2008
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Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging*Formula

Meirav Zaks-Zilberman, Adrian E. Harrington, Tetsuya Ishino, and Irwin M. Chaiken1

From the Department of Biochemistry and Molecular Biology, Drexel University College of Medicine, Philadelphia, Pennsylvania 19102

Received for publication, December 14, 2007 , and in revised form, February 22, 2008.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-5 exerts hematopoietic functions through binding to the IL-5 receptor subunits, {alpha} and βc. Specific assembly steps of full-length subunits as they occur in cell membranes, ultimately leading to receptor activation, are not well understood. We tracked the oligomerization of IL-5 receptor subunits using fluorescence resonance energy transfer (FRET) imaging. Full-length IL-5R{alpha} and βc were expressed in Phoenix cells as chimeric proteins fused to enhanced cyan or yellow fluorescent protein (CFP or YFP, respectively). A time- and dose-dependent increase in FRET signal between IL-5R{alpha}-CFP and βc-YFP was observed in response to IL-5, indicative of heteromeric receptor {alpha}-βc subunit interaction. This response was inhibited by AF17121, a peptide antagonist of IL-5R{alpha}. Substantial FRET signals with βc-CFP and βc-YFP co-expressed in the absence of IL-5R{alpha} demonstrated that βc subunits exist as preformed homo-oligomers. IL-5 had no effect on this βc-alone FRET signal. Interestingly, the addition of IL-5 to cells co-expressing βc-CFP, βc-YFP, and nontagged IL-5R{alpha} led to further increase in FRET efficiency. Observation of preformed βc oligomers fits with the view that this form can lead to rapid cellular responses upon IL-5 stimulation. The IL-5-induced effects on βc assembly in the presence of nontagged IL-5R{alpha} provide direct evidence that IL-5 can cause higher order rearrangements of βc homo-oligomers. These results suggest that IL-5 and perhaps other βc cytokines (IL-3 and granulocyte/macrophage colony-stimulating factor) trigger cellular responses by the sequential binding of cytokine ligand to the specificity receptor (subunit {alpha}), followed by binding of the ligand-subunit {alpha} complex to, and consequent rearrangement of, a ground state form of βc oligomers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
It is generally thought that most cytokines trigger cellular signal transduction by inducing the association of receptor subunits that leads to increased proximity of associated intracellular kinases and initiation of phosphorylation cascades. Recent studies of human growth hormone and erythropoietin have shown that the assembly of their receptor subunits leads to conformational rearrangement and that this process plays a role in receptor activation (1-3). However, evidence for such cytokine-induced receptor rearrangement for other cytokine-receptor complexes remains limited. In the case of interleukin (IL)2-5 and the other βc cytokines IL-3 and granulocyte/macrophage colony-stimulating factor (GM-CSF), the states of receptor subunit assembly in membranes that lead to activation have been proposed, but experimental evidence for these has remained largely indirect.

Human IL-5 is a TH2 cell-derived cytokine that regulates hematopoiesis and inflammation. It is implicated in the pathogenesis of allergic disorders, asthma, and other forms of hypereosinophilic syndromes, through its influence on eosinophil maturation, proliferation, activation, expansion, and tissue distribution (4-7). IL-5 exerts its biological functions by binding to a heteromeric cell surface receptor complex that contains two types of subunits, {alpha} and βc (8). IL-5R{alpha} confers specificity and is essential for activation of the βc subunit. The IL-5R{alpha} subunit has been shown to bind IL-5 directly, even in the absence of βc, albeit at a lower affinity (9). IL-5R{alpha} is structurally related to the receptor {alpha} subunits of IL-3 and GM-CSF receptors. The βc receptor subunit is common to the IL-3, IL-5, and GM-CSF receptors and is considered the main signaling component of the activated receptor-ligand complexes (10, 11). Based on biophysical and high resolution crystallographic data, the βc receptor ectodomain exists as a stable homodimer (12, 13). On its own, βc does not measurably bind any of the cytokine ligands (11, 14, 15), but it cannot be ruled out that βc may have direct contact with IL-5 (or the other cytokines) in ternary cytokine-R{alpha}-βc complexes.

Previous binding studies support the view that the IL-5 ligand first binds to IL-5R{alpha} with an apparent Kd value of 0.3-0.6 nM (11, 16, 17), followed by binding of βc into the IL-5·IL-5R{alpha} complex. The affinity of IL-5 when {alpha}- and βc-chains are coexpressed in the ternary complex (IL-5·IL-5R{alpha}·βc) is 2-5-fold greater than in the IL-5·IL-5R{alpha} complex (11, 16, 18). In the GM-CSF and IL-3 ligand-receptor systems, βc binding increases ligand affinity even further. The initial binding of IL-5 to IL-5R{alpha} has a 1:1 stoichiometry, as shown by gel filtration, titration calorimetry, analytical ultracentrifugation, and surface plasmon resonance using soluble receptor molecules or truncated receptor chains (19-21). Cross-linking and immunoprecipitation studies in the presence of IL-5 demonstrated a close physical association of IL-5R{alpha} and βc (16, 17, 22, 23). Furthermore, the observation of cytokine-dependent formation of a disulfide linkage between IL-3R{alpha} and βc subunits (24-26) is consistent with a need for conformational changes in the extracellular domains of the receptor subunits (21). The reorganization of the intracellular domains upon cytokine binding has been inferred but has not been shown directly.

Fluorescence resonance energy transfer (FRET) has been used to directly visualize protein-protein interactions in cells by confocal microscopy. Because energy transfer depends on the inverse 6th power of the distance between donor and acceptor fluorophores, this method allows the detection of energy exchange between these two fluorophores fused to proteins that are in a close spatial proximity (2-10 nm), a distance that correlates with the size of the molecules on the cell surface (27, 28). Using cell FRET and relatively small fluorescent tags that are available, the cellular environment is maintained, conserving transmembrane interactions with minimal perturbation of signaling systems (29-31). Recent studies have applied FRET to different cytokine receptor systems, including those of IL-2 (32), IL-10 (33), IL-13 (34), IL-17 (35), leptin (36), and growth hormone (1).

In the current study, we used cell FRET to assess assembled states of IL-5R{alpha} and βc and the impact of IL-5 on the assembly process. In order to test whether IL-5 receptor subunits multimerize at the plasma membrane and to measure their dependence on IL-5, we expressed the receptor subunits as full-length fusion proteins linked at their carboxyl termini to either cyan or yellow fluorescent proteins. We assessed the assembly of IL-5 receptors and the effect of IL-5 by FRET after acceptor photobleaching. Our data demonstrate that the IL-5R{alpha} subunit is present as a monomer on the cell surface, whereas βc exists as a dimer or a higher order homo-oligomer. We found that IL-5 binding induces IL-5R{alpha}·βc assembly. Interestingly, we also found that IL-5 binding leads to organizational changes within the intracellular domains of βc·βc multimers in the presence of IL-5·IL-5R{alpha}. The results demonstrate a hitherto unknown structural rearrangement in the intracellular domain of βc subunit that could be important in switching on cytokine signal transduction.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—The human IL-5 protein was expressed and purified as previously described (18, 37). AF17121 peptide was synthesized and purified as before (33, 42).

Reagents—All enzymes were purchased from New England Biolabs Inc. (Beverly, MA). The Phoenix cell line was purchased from ATCC (Manassas, VA). DNA oligonucleotide primers were purchased from Invitrogen. Dulbecco's modified Eagle's medium was from Invitrogen. pEYFP-N1 and pECFP-N1 were from Clontech (Palo Alto, CA). Opti-MEM was from Invitrogen. Fetal bovine serum was from Sigma. FuGENE 6 was from Roche Applied Science. Vectashield mounting medium was from Vector Laboratories (Burlingame, CA). Poly-D-lysine coverslips were from BD Biosciences. The CD4-YFP cDNA was a gift from Dr. Tian Jin (Laboratory of Immunogenetics, Twin-brook II Facility, NIAID, National Institutes of Health, Rockville, MD).

cDNA, Mutagenesis, Cell Lines, and Transfections—A 1300-base pair fragment encoding the full-length human IL-5R{alpha} was amplified by PCR using forward (5'-TATAGAATTCATGATCATCGTGGCGCATGTATTAC-3'; EcoRI site underlined) and reverse (5'-TATAACCGGTCCAAACACAGAATCCTCCAGGG-3'; AgeI site underlined) oligonucleotides. A 2700-base pair fragment encoding the full-length human βc receptor was amplified by PCR using forward (5'-TATAGAATTCATGGTGCTGGCCCAGGGGCTGC-3'; EcoRI site underlined) and reverse (5'-TATAACCGGTCCACACACCTCCCCAGGC-3'; AgeI site underlined) oligonucleotides. These fragments were digested by restriction enzyme, purified from agarose gel, ligated into pECFP-N1 (CFP) and pEYFP-N1 (YFP), and then transformed using DH5{alpha}-competent cells.

The βc-pEYFP-N1 mutant constructs (Y15S, F79A, Y347Q or Y347S, and Y390S) were generated from the corresponding wild type vector by site-directed mutagenesis using QuikChange (Stratagene). Nontagged IL-5R{alpha} was generated by introducing an additional AgeI restriction enzyme site at the end of the receptor coding sequence using site-directed mutagenesis, followed by removal of the Y/CFP DNA and religation into the original vectors.

Phoenix cells, a derivative of human embryonic kidney 293 cells, were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin (Sigma), and 2 mML-glutamine at 37 °C in 5% CO2. Exponentially growing cells were dispersed with trypsin (Sigma). For immunoblotting analysis, cells were seeded at a density of 2 x 105/well in 6-well plates and transiently transfected with a combination of tagged IL-5R{alpha} and βc chains using FuGENE 6 (Roche Applied Science) according to the manufacturer's recommendations and challenged with IL-5 or IL-5/AF17121 mixture for the times and doses indicated. For fixed cell imaging and acceptor photobleaching assays, cells were seeded at a density of 1 x 105/well on poly-D-lysine-treated coverslips in 6-well plates and transiently co-transfected with combinations of IL-5R{alpha}-CFP and βc-YFP chains, IL-5R{alpha}-CFP and IL-5R{alpha}-YFP, or βc-CFP and βc-YFP chains. Forty-eight hours post-transfection, cells were challenged with IL-5 or IL-5/AF17121 mixture for the times and doses indicated and then fixed with 4% paraformaldehyde and washed with phosphate-buffered saline containing Ca2+ and Mg2+, and the coverslips were mounted onto Superfrost microscope slides (Fisher) using Vectashield (Vector Laboratories).

Western Blotting—Phoenix cells expressing either the wild type human IL-5R{alpha} and wild type human βc or wild type human IL-5R{alpha} and human βc mutants were washed with cold phosphate-buffered saline and then lysed on ice using radioimmune precipitation buffer (Sigma). After centrifugation at 10,000 x g for 15 min, soluble proteins were separated by 4-20% SDS-PAGE and electroblotted onto nitrocellulose membranes. The membranes were blocked with 5% milk in Tris-buffered saline with 0.05% Tween 20 (TBST) and probed with the specific primary antibodies against IL-5R{alpha} (SC-673; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), βc (SC-676; Santa Cruz Biotechnology), C/YFP (monoclonal antibody JL-8; Clontech), or actin (SC-1616; Santa Cruz Biotechnology) for 1 h at room temperature in 5% milk with TBST. Washed membranes were then incubated with a 1:5000 dilution of the appropriate secondary horseradish peroxidase-conjugated anti-rabbit or mouse-IgG (GE Healthcare) for 1 h at room temperature and developed using ECL+, an enhanced chemiluminescence detection kit (GE Healthcare).


Figure 1
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  		FIGURE 1.
Western blot analysis of expression of CFP- or YFP-tagged full-length IL-5R{alpha} and βc receptors in Phoenix cells. A, schematic representation of IL-5R{alpha}-C/YFP and βc-C/YFP fusion proteins used in this study. B, whole cell lysates of Phoenix cells transiently expressing IL-5R{alpha}-C/YFP or βc-C/YFP or co-expressing IL-5R{alpha}-CFP and βc-YFP (as denoted below) were immunoblotted with anti-IL-5R{alpha} (left) and anti-βc (right) antibodies. Left, lane 1, IL-5R{alpha}-CFP alone; lane 2, IL-5R{alpha}-YFP alone; lane 3, co-expressed IL-5R{alpha}-CFP and βc-YFP. Right, lane 1, βc-CFP alone; lane 2, βc-YFP alone; lane 3, co-expressed IL-5R{alpha}-CFP and βc-YFP.

 
Acceptor Photobleaching FRET Assay by Confocal Microscopy—Phoenix cells transiently transfected and co-expressing combinations of IL-5R{alpha}-CFP, βc-YFP, IL-5R{alpha}-YFP, and/or βc-CFP were examined with a x40 objective lens on a Leica TCS SP2 laser-scanning confocal microscope at the Imaging Facility, Drexel University College of Medicine. Cells transfected with IL-5R{alpha}-CFP or βc-YFP alone were used to calibrate laser intensity to prevent bleed-through. For each slide, cells were viewed using the differential interface contrast function, and the field of cells was scanned to find healthy cells. Following this, cells were digitally imaged using the acceptor photobleaching module of the Leica Confocal Software (version 2.61.1537), and a region including cells expressing both fused receptors was defined as the region of interest (ROI). Images of the donor were acquired by exciting with a 458-nm laser and reading emitted spectra between 467 and 505 nm. Images of the acceptor (YFP) were acquired by exciting with a 514-nm laser and reading emitted spectra between 520 and 575 nm. These provided reference images for the donor and acceptor expression levels before photobleaching. The Leica Confocal Software was configured to achieve 80% photobleaching of YFP in the selected ROI, using maximum power of the 514-nm line. This led to nearly complete loss of YFP fluorescence when visualized by microscopy. Images of the donor and acceptor fluorescence after photobleaching were obtained by exciting at 458 nm and at 514 nm, respectively. These were generated simultaneously as soon as 80% photobleaching was achieved. Data were collected from 15-50 different cells in different fields from the same slide, and 25 ROIs were measured per slide in each experiment. Each experiment was performed at least three times. After photobleaching, additional ROIs were chosen at cell membrane locations on the cells within the photobleached area (4-5 ROIs/cell), and the mean donor fluorescence before and after photobleaching was obtained using the acceptor photobleaching application of the Leica Confocal Software. The FRET efficiency between CFP (donor) and YFP (acceptor) was quantified with the acceptor photobleaching method (38) using the equation FRET efficiency (E) = ((Dpost - Dpre)/Dpost) and calculated with the Leica Confocal Software acceptor photobleaching application for each ROI. Here, Dpost is the fluorescence intensity of the CFP (D for "donor") after photobleaching, and Dpre is the fluorescence intensity of the CFP before photobleaching.

Statistical Analysis—Data are presented as mean ± S.E. The significance of differences between the means of various treatment conditions was determined by Student's t test assuming equal variance. For each treatment, at least 25 ROIs were chosen per experiment. Differences were considered to be significant at p < 0.05 (not corrected for multiple comparisons). Each experiment was performed at least three times (unless otherwise indicated), carried out on different days and with different cell preparations.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
IL-5R{alpha}-C/YFP and βc-C/YFP Expression in Transfected Phoenix Cells—To assess IL-5 receptor subunit assembly in cells by FRET, full-length human IL-5R{alpha} and βc were fused to either CFP or YFP at their carboxyl termini to prepare four distinct tagged receptor constructs (Fig. 1A). These were then transiently transfected into Phoenix cells. Cells were co-transfected with the following combination of vectors: 1) IL-5R{alpha}-CFP and βc-YFP, 2) IL-5R{alpha}-CFP and IL-5R{alpha}-YFP, and 3) βc-CFP and βc-YFP. CFP and YFP were chosen because they have excitation and emission wavelengths favorable for FRET, coupled with suitable extinction coefficients and quantum yields (39). Cells were initially analyzed by Western blotting to confirm protein expression. Fig. 1B (left) shows the expression of full-length IL-5R{alpha}-YFP ({alpha}Y) and IL-5R{alpha}-CFP ({alpha}C) in cells transfected with each or co-transfected with βc({alpha}C/βY). Fig. 1B (right) shows the expression of full length βc-YFP (βY) and βc-CFP (βC) in cells transfected with a single vector or co-transfected with both IL-5R{alpha}-CFP and βc-YFP ({alpha}C/βY). Transfected proteins were detected with anti-IL-5R{alpha} or anti-βc antibodies, respectively. The 154 kDa band corresponds to βc-C/YFP fusion protein, whereas the lower band at 82 kDa corresponds to the IL-5R{alpha}-C/YFP. Fig. S1 shows the expression and co-expression of these fusion proteins as detected with anti-C/YFP monoclonal antibody JL-8 raised against GFP and cross-reactive with both CFP and YFP.

IL-5-induced Oligomerization of IL-5R{alpha} and βc Subunits—A variety of studies have indicated that IL-5R{alpha} associates with βc in the presence of ligand (16, 17, 22, 40-42). We examined whether the addition of IL-5 ligand affected receptor subunit hetero-oligomerization. This was done by analyzing the effect of IL-5 on FRET between IL-5R{alpha} and βc subunits (Fig. 2A). We first assessed IL-5R{alpha}-CFP and βc-YFP expression and localization by capturing images of cells using the acceptor photobleaching mode of the confocal microscope (see "Experimental Procedures"), recording signals simultaneously in the CFP and YFP channels. CFP or YFP alone was expressed mainly in the cytoplasm (data not shown). In contrast, the tagged receptors were expressed predominantly at the cell membrane (Figs. 2B and S2), along with some accumulation of the tagged receptors in the cytoplasm. This type of distribution has been seen with other cytokine receptors expressed ectopically (35). These data showed that co-transfected cells expressed both IL-5 receptor subunits appropriately on the cell surface.


Figure 2
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  		FIGURE 2.
FRET tracking of IL-5-dependent oligomerization of IL-5R{alpha} and βc subunits in Phoenix cells. A, schematic diagram of FRET between full-length human IL-5R{alpha}-CFP and βc-YFP on cell membrane. Following donor (CFP) excitation, fluorescence emission can be absorbed by a nearby acceptor (YFP). The effect of FRET was measured from the increased signal of the donor after acceptor photobleaching. B, representative confocal images of Phoenix cells co-expressing IL-5R{alpha} (cyan) andβc (yellow) before and after treatment with 30 nM IL-5 for 30 min, with and without 300 nM AF17121 for 30 min. From left to right, images of the CFP channel emission before photobleaching (CFP pre), images of the YFP channel emission before photobleaching (YFP pre), images of the CFP channel emission after photobleaching (CFP post), and images of the YFP channel emission after photobleaching (YFP post). Scale bars, 10µm. C, FRET efficiency plotted as a function of IL-5, between βc-CFP and βc-YFP subunits (white histograms), between IL-5R{alpha}-CFP and IL-5R{alpha}-YFP subunits (black histograms) and between IL-5R{alpha}-CFP and βc-YFP subunits (striped histograms). FRET signals after acceptor photobleaching were measured for Phoenix cells co-expressing the indicated receptor fusions and exposed to IL-5 for 30 min before fixation. FRET efficiency was analyzed in plasma membrane ROIs from multiple cells (n ≥ 25). Data were pooled from three independent experiments. FRET data are shown ± S.E. D, AF17121 dose-dependent inhibition of FRET with IL-5R{alpha}-CFP and βc-YFP. Phoenix cells co-expressing IL-5R{alpha}-CFP and βc-YFP were used to evaluate FRET before and after IL-5 treatment (30 nM) with the indicated concentrations of AF17121 for 30 min. Data are representative of three experiments.

 
We next assessed the possible presence of FRET between IL-5R{alpha} and βc. Full-length receptors IL-5R{alpha}-CFP and βc-YFP were co-expressed in Phoenix cells, which were then treated with IL-5 or with a combination of IL-5 and AF17121 and subjected to FRET analysis after photobleaching YFP to 20% of basal levels. The addition of IL-5 for 30 min to cells coexpressing IL-5R{alpha}-CFP and βc-YFP had no effect on basal surface expression levels of either IL-5R{alpha} or βc (Fig. 2B). When IL-5 was added to cells co-expressing IL-5R{alpha}-CFP and βc-YFP, an increase in the fluorescence of the donor was observed after photobleaching (Fig. 2B, compare data in the first and third panels of the second row). This visible increase in fluorescence of donor after photobleaching suggested that FRET was occurring between the tagged IL-5R{alpha} and βc subunits in the presence of IL-5. This response was inhibited by AF17121, a peptide antagonist of IL-5R{alpha} (37, 43) (Fig. 2B, third row).

The extent of FRET efficiency between IL-5 receptor subunits was further quantified in Fig. 2C. Full-length receptors IL-5R{alpha}-CFP and βc-YFP, IL-5R{alpha}-CFP and IL-5R{alpha}-YFP, or βc-CFP and βc-YFP were co-expressed in Phoenix cells, which were then treated with different concentrations of IL-5 for 30 min and analyzed for FRET after photobleaching YFP to 20% of basal levels. In the absence of IL-5 or at low ligand concentrations (0-10 nM), only a low base-line level of FRET efficiency was measured between the IL-5R{alpha} and βc chains (~10%) (Fig. 2C, striped histograms; other histograms are discussed below). As IL-5 levels were increased, significant increases in FRET efficiency between IL-5R{alpha} and βc chains were observed, to values of 38 ± 2.2% at 30 nM IL-5 and 45 ± 2.3% at 100 nM IL-5 (Fig. 2C, striped histograms). Whereas the low base-line FRET levels could be due to spontaneous or random receptor subunit interactions at the plasma membrane or the tendency of GFP mutants (C/YFP) to dimerize (39), the sharp dose-dependent increase in FRET signal over a narrow range of IL-5 concentration (10-30 nM) implies a ligand-dependent increase in hetero-oligomeric complex formation. Further increasing IL-5 levels to even higher levels (300 nM) caused a small decline from the peak of the hetero-oligomerization FRET signal (from peak levels of 45 ± 2.3% to a level of 34 ±1%), although the signal remained clearly above base line. The reason for this small but statistically significant decrease is not known at present. Overall, the IL-5-dependent increase in the donor (IL-5R{alpha}-CFP) fluorescence after acceptor (βc-YFP) photobleaching is consistent with IL-5 stimulation of heteromeric receptor subunit assembly. In contrast to IL-5R{alpha}-βc hetero-oligomerization, low baseline FRET efficiency values of 10 ± 2% were seen with IL-5R{alpha} chains alone; these did not change with either increasing concentrations of IL-5 (Fig. 2C, black histograms; Fig. S3, empty circles) or with the addition of AF17121, an IL-5R{alpha} antagonist (37, 43) (data not shown). The low magnitude of FRET coupled with the lack of IL-5 or AF17121 effects argues that the FRET signals observed with IL-R{alpha} alone are nonspecific. In contrast, a high base line of FRET efficiency was seen between βc-CFP and βc-YFP chains (>40%). Although this signal was relatively insensitive to IL-5 (Fig. 2C, white histograms), the large magnitude of FRET efficiency suggested that it could reflect higher order organization of βc beyond monomers. This βc-alone FRET behavior is described in detail below with respect to follow-up studies of βc homo-oligomerization.

To confirm that the observed effect of IL-5 was specific, we quantified the ability of the IL-5R{alpha} antagonist AF17121 to prevent IL-5-induced IL-5R{alpha}-CFP and βc-YFP hetero-oligomerization. AF17121 inhibited the IL-5-dependent FRET signal between IL-5R{alpha}-CFP and βc-YFP co-transfectants in a dose-dependent manner (Fig. 2D). AF17121 decreased the IL-5-induced FRET signal to base-line levels at both IL-5 concentrations tested (30 and 100 nM). This supports the conclusion that the IL-5-dependent FRET signal observed between IL-5R{alpha} and βc is specific and is dependent on IL-5 interactions with IL-5R{alpha}. It further suggests that an IL-5·IL-5R{alpha} complex is required to bind βc with high affinity. The lack of an AF17121 effect on the base-line signal argues that the basal FRET observed for the IL-5R{alpha} combinations is not specific.

Time Dependence of IL-5 Induction of Hetero-oligomerization of IL-5R{alpha} and βc Subunits—To ensure that the effect of IL-5 on the interactions between IL-5R{alpha} and βc is measured at an optimal time point, co-transfected cells were treated with 30 nM IL-5 for between 0 and 60 min and analyzed as above. The FRET signal between IL-5R{alpha}-CFP and βc-YFP in response to IL-5 behaved in a time-dependent manner, peaking at 30 min to FRET efficiency values of 58 ± 1.5% (Fig. S3, filled squares). Different expression levels of {alpha} and βc chains did not cause a nonspecific FRET signal (i.e. not triggered by IL-5), since switching CFP to the C-terminal of βc and YFP to the C terminus of IL-5R{alpha} yielded similar results. This preserved the ratio between IL-5R{alpha} and βc but reversed the directionality of the FRET effect by reversing the fluorescent tags (Fig. S3, open squares). Finally, IL-5 caused no significant difference in the base-line FRET signal when tested with a combination of IL-5R{alpha}-CFP and IL-5R{alpha}-YFP (Fig. S3).

Effects of Mutations in Subunit Interface Residues on Hetero-oligomerization—A series of mutations were made in βc residues that would be expected to participate in the receptor-ligand hetero-oligomer interface. The crystal structure of the extracellular region of βc has been solved (44, 45). In that structure, βc exists as a domain-swapped dimer composed of two identical polypeptide chains (Fig. 3A). Previous binding and biological growth response studies using extracellular domains showed that βc receptor mutants of Tyr15, Phe79, or Tyr347 co-expressed with the {alpha}-subunit of GM-CSF, IL-3, or IL-5 were unable to bind the appropriate ligand with high affinity (12, 46-48). These residues are located in domains 1 (D1) and 4 (D4) of βc, in a putative binding region formed by two chains in a domain-swapped βc homodimer (Fig. 3B). Thus, point mutations at Tyr15 (Y15S), Phe79 (F79A), and Tyr347 (Y347Q and Y347S) were made, and Phoenix cells were co-transfected with IL-5R{alpha}-CFP and a wild type or mutant YFP-tagged βc. Forty-eight hours post-transfection, cells were treated with 30 nM IL-5 for 30 min, and FRET acceptor photobleaching analyses were performed. The mutations did not affect the βc cell surface expression (as analyzed by fluorescence images and Western blot; data not shown) but did reduce the ligand-dependent IL-5R{alpha}-βc FRET signal (Fig. 3C). The addition of IL-5 ligand to cells co-expressing IL-5R{alpha} and βc mutant Y15S, F79A, Y347Q, or Y347S decreased FRET efficiency to values of 18 ± 3.2%, 15 ± 2.5%, 13 ± 2.8%, and 10 ± 2.78%, respectively, compared with a ligand-induced FRET efficiency between IL-5R{alpha} and wild type β (42 ± 5%). As a control experiment, we examined the mutational effect of a residue in βc, Y390S, that is not expected to be in the interface of ligand-receptor complex and confirmed that this mutation does not affect the FRET efficiency found in wild type βc (Fig. 3C). Disruption or reduction of the FRET signal between IL-5R{alpha}-CFP and βc-YFP in the presence of ligand by specific residue mutation is consistent with the involvement of these residues in the ligand-dependent hetero-oligomerization of IL-5R{alpha}-βconthe cell surface and further supports the specificity of the observed FRET signal.

IL-5-independent Preassembly of βc as Homo-oligomers on the Cell Surface—Numerous lines of evidence suggest that βc chains can form dimers in the absence of IL-5, including the crystal structure of βc extracellular domains (12, 13), biochemical cross-linking studies (49, 50), and gel filtration and ultracentrifugation analyses (13, 44, 45). In addition, FRET allows the unique chance to probe the endogenous state of the βc chains in the membrane. In this study, the extent of interaction of full-length βc receptors tagged with both CFP and YFP was measured by FRET. Images of Phoenix cells co-transfected with βc-CFP and βc-YFP fusions showed a ring-shaped staining distribution and co-localization of βc chains on the cell membrane (Fig. S2). The addition of IL-5 for 30 min had no significant effect on surface expression levels of either βc-CFP (CFPpre) or βc-YFP (YFPpre) (data not shown).


Figure 3
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  		FIGURE 3.
Crystal structure of the extracellular region of βc and effects of βc mutations on IL-5-induced FRET. A, overall topology of βc dimer. One monomer is shown in magenta, and the other is shown in cyan. B, domains 1 and 4 (D1 and D4) showing the residues that we mutated in this study. Tyr15, Phe79, and Tyr347 are involved in the ligand-receptor complex and are located in the elbow region between domains 1 and 4. Tyr390 is not involved in the ligand-receptor complex and is located at the proximal-membrane site. C, quantitative analysis of FRET between IL-5R{alpha}-CFP and either βc-YFP wild type (wt) or βc mutated at residue Tyr15, Phe79, Tyr347, or Tyr390. FRET acceptor photobleaching signals from Phoenix cells co-expressing the indicated receptor fusions were measured for cells with or without the addition of 30 nM IL-5 for 30 min. FRET efficiency was analyzed in plasma membrane ROIs from multiple cells (n ≥ 25). FRET data are shown ± S.E. The data are representative of three experiments.

 


Figure 4
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  		FIGURE 4.
FRET analysis of βc-βc interactions. The interactions of βc chains in the cell membrane were studied by measuring FRET acceptor photobleaching. FRET efficiencies were quantitated between βc-CFP and βc-YFP with (black histograms) and without nontagged IL-5R{alpha} (striped histograms) or between βc-CFP and CD4-YFP (white histograms). FRET acceptor photobleaching signals from Phoenix cells co-expressing the indicated receptor fusions were measured with and without the addition of 30 nM IL-5 for 30 min and with the addition of 300 nM AF17121. FRET efficiency was analyzed in plasma membrane ROIs from multiple cells (n ≥ 25). FRET data are shown ± S.E. The data are representatives of three experiments.

 
When FRET acceptor photobleaching was used to test for βc chain interaction, photobleaching of the acceptor YFP led to an increase of donor CFP emission independent of the presence of IL-5. Phoenix cells were treated with IL-5 at concentrations of 0, 0.1, 1, 10, 30, 100, and 300 nM for 30 min, fixed, and analyzed for FRET using the acceptor photobleaching method. High FRET efficiency values of 46.9 ± 1.7% (Fig. 2C, white histograms) between the two βc chains were measured in the absence of IL-5. This demonstrated a close interaction between βc-CFP and βc-YFP in the plasma membrane in the absence of IL-5. The addition of 10 and 30 nM IL-5 caused small increases in FRET efficiency values (50 ± 2.78 and 54.8 ± 2.3%, respectively). Increasing the concentrations of IL-5 even higher (100 and 300 nM) appeared to cause a small decrease in FRET efficiencies (45.5 ± 1.5 and 39 ± 1.6%, respectively). This decrease reached statistical significance at the highest concentration tested of 300 nM IL-5 (p = 0.0012 for pairwise comparison between 0 and 300 nM). The reasons for the small variations in βc FRET are not well understood at present. In any case, the substantial FRET efficiency between βc-CFP and βc-YFP before IL-5 treatment is clearly evident (Fig. 2C, white histograms).

To confirm that the observed FRET between βc chains is due to specific interaction and not nonspecific crowding, we co-expressed CD4-YFP (51) and βc-CFP by transiently transfecting these constructs into Phoenix cells. Since CD4 and βc receptor subunits are not functionally related, they should not interact with each other. Strong CFP and YFP fluorescence was observed when cells co-expressing this mismatched receptor pair were excited at the appropriate wavelength, confirming expression of the CD4-YFP and βc-CFP fusion proteins (data not shown). Fig. 4 (white histograms) shows that the FRET efficiency values were <13%, similar to base-line levels observed in previous experiments. This result indicates that there is little interaction between CD4 and βc on the cell surface. When IL-5 was added, there was no significant change in FRET efficiency between CD4 and βc (Fig. 4, white histograms). Together, these results argue that βc molecules are preassembled specifically and independently of cytokine in the membrane.

IL-5-induced Rearrangement in βc Subunits in the Three-component IL-5·IL-5R{alpha}·βc Complex—We tested whether IL-5 can induce the reorganization of preformed βc oligomers in the presence of IL-5R{alpha}. Interactions between βc-CFP and βc-YFP subunits in the presence of nontagged IL-5R{alpha} subunit were analyzed by FRET acceptor photobleaching (Fig. 4). Imaging and Western blotting analyses of Phoenix cells co-transfected with βc-CFP, βc-YFP, and nontagged IL-5R{alpha} confirmed cell membrane expression of transfected receptors (data not shown). Co-expression of these βc receptor chains produced a high FRET signal, as was seen in the absence of IL-5R{alpha}. The average FRET efficiency for these receptor constructs was 38 ± 1.3% (Fig. 4, black histograms). A significant increase in FRET efficiency between the βc chains in the presence of IL-5R{alpha} was observed when the cells were treated with IL-5 for 30 min, to values of 59 ± 3.5% (Fig. 4, black histograms). These data show a 55% increase in the donor fluorescence after acceptor photobleaching, indicating that the two βc chains are moving closer to one another in response to the addition of ligand. To confirm that the observed effect of IL-5 was specific, we tested the ability of the IL-5R{alpha} antagonist AF17121 to prevent IL-5 induction of FRET in this nontagged IL-5R{alpha} experiment. AF17121 prevented the IL-5-dependent FRET signal increase between βc chains in the presence of nontagged IL-5R{alpha} (Fig. 4, black histogram).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Investigation of IL-5-induced receptor assembly in the cellular context, in particular by chemical cross-linking (8, 11, 17, 22, 40, 52-54) and co-immunoprecipitation (23), has shown that IL-5 induces IL-5R{alpha} and βc subunit interaction at the cell surface. However, these methodologies do not lend themselves to a delineation of the specific receptor subunit assembly steps in the cellular environment. The purpose of the current study was to visualize this cellular assembly process at the molecular level by using FRET. The FRET methodology uses noninvasive and stable markers that enable direct visualization of protein-protein associations within the environment of the cell surface through energy transfer between donor and acceptor fluorophore tags that are in sufficiently close proximity. In the current study, we tagged the receptor subunits with fluorescent tags by fusing their intracellular C termini with CFP donor and YFP acceptor fluorescent proteins.

We developed the FRET assay to study the heteromeric interaction of IL-5R{alpha} with βc on the surface of recombinant Phoenix cells (Fig. 2A). The addition of IL-5 increased the FRET efficiency between {alpha} and βc subunits on the cell surface (Fig. 2B). IL-5 has been shown to bind to the extracellular region of IL-5R{alpha} (both soluble and full-length forms) in a dose-dependent manner (18, 37, 52, 55). No evidence exists to support direct binding of IL-5 to the βc subunit on its own (13, 56). We found that IL-5 stimulated a dose-dependent increase in the FRET efficiency between {alpha} and β subunits (Fig. 2C) and that this increased FRET was abolished by the addition of AF17121 (Fig. 2D), an IL-5R{alpha} antagonist peptide (37, 43). AF17121 inhibition of the IL-5/IL-5R{alpha} interaction at the ligand-binding epitope in IL-5R{alpha} is consistent with the view that complex formation of IL-5 with the extracellular region of IL-5R{alpha} is necessary to enable closer proximity between {alpha}-βc subunits in the heteromeric complex.

The specificity of FRET-tracked {alpha}-βc assembly was further supported using the βc mutants Y15S, F79A, Y347Q, and Y347S (Fig. 3, B and C). We found that the FRET efficiencies between {alpha} and βc subunits induced by IL-5 were largely suppressed by these mutations. The residues Tyr15, Phe79, and Tyr347 have been proposed to be involved in direct interaction between βc and IL-5 in complex with IL-5R{alpha} (12, 45, 47, 57). Our data are consistent with a role for the putative βc ligand-binding epitope in the assembly of {alpha}-βc subunits although not necessarily in a direct IL-5-βc contact.

The FRET approach was used to assess the nature of βc oligomerization in cells. Previous high resolution structure analyses have shown that the extracellular region of βc can exist as a dimer (44, 48). In this structure, the βc is a homodimer stabilized by domain swapping between domain 1 (D1) of one chain and domain 3 (D3) of the symmetry-related chain (Fig. 3A). The homodimeric nature of the extracellular regions of βc has also been demonstrated by solution analyses, including ultracentrifugation and chemical cross-linking (44, 48). In the current work, we observed substantial FRET efficiencies between CFP- and YFP-tagged βc subunits either in the presence or in the absence of IL-5, whereas FRET efficiencies between CFP- and YFP-tagged {alpha} subunits were at a base-line level (Fig. 2C). These results provided direct evidence that full-length βc chain was spontaneously preassembled as a homo-oligomer at the cell surface. Preassembled receptor complexes in the absence of ligand are not unique to class I cytokine receptors and have been described for other cytokine receptors, such as interfer-on-{gamma}, tumor necrosis factor, and FAS receptors (58-60). The preassembled signaling receptor subunits might enable more kinetically efficient signal transduction upon hetero-oligomerization with ligand-specific "co-receptor" subunits, such as {alpha} subunits for the βc cytokine receptors.

The substantial FRET signal observed for βc oligomers allowed us to ask if βc organization is altered upon assembly in the three-component complex containing βc, IL-5R{alpha}, and IL-5. Using CFP- or YFP-tagged βc subunits and nontagged {alpha} subunits co-expressed in cells, we observed an increase in FRET efficiency in the presence of IL-5R{alpha} when IL-5 was added (Fig. 4). This IL-5 effect was suppressed in the presence of AF17121, showing that it was dependent on interaction of IL-5 with IL-5R{alpha}. It is possible that this increased FRET is due to an equilibrium shift between monomers and oligomers of βc; however, we think that βc monomers are unlikely to participate in cytokine-triggered βc rearrangement based on the following observations. First, extensive biophysical evidence has shown that the ectodomain of the βc receptor subunit is a stable homodimer. For example, no βc monomer was detected previously using analytical ultracentrifugation and native gel electrophoresis studies of βc ectodomain expressed and purified from insect cells (48). Second, only dimers of βc have been observed in cell membranes by cross-linking studies of full-length βc receptor expressed in insect cells or of a truncated βc form expressed in Ba/F3 cells (44, 50). Thus, we conclude that IL-5 induces some form of rearrangement of preassembled βc oligomers upon binding to IL-5 and IL-5R{alpha}. Although our data cannot exclude the possibility that βc dimers might exist as higher order aggregates and that cytokine-receptor {alpha} complexes may shift the distribution among different dimer oligomerization states as a part of the βc rearrangement process, the data support the hypothesis derived in this work that the addition of IL-5 in the presence of both receptor subunits causes a structural change in βc dimers themselves. Further, since each of the βc cytokines appears able to sequester βc receptors on cells expressing all receptor {alpha} subunits (54), it can be surmised that each of them, when bound to their specific receptors {alpha}, can trigger the βc dimer rearrangement observed here with IL-5.


Figure 5
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  		FIGURE 5.
Proposed model for assembly of IL-5 and receptor subunits in cells. IL-5 ligand (red circle), IL-5R{alpha} (blue), and βc (green) are shown prior to ligand binding (IL-5R{alpha} monomer and βc dimer; left), as IL-5 binds IL-5R{alpha} (center), followed by hetero-oligomerization of {alpha} and β subunits that leads to further proximity of βc subunit intracellular domains (right).

 
Genetic and crystallographic studies have suggested that the growth hormone complex forms sequentially, by binding of site I of growth hormone to one receptor subunit followed by binding of the second receptor subunit through site II of growth hormone (61, 62). Moreover, recent analyses using FRET (1) have suggested that the growth hormone receptors can preexist as homodimers and that growth hormone binding to the growth hormone receptor dimer induces a reorientation of receptor ectodomains, leading to rearrangement of the transmembrane and cytoplasmic regions. These conformational rearrangements in the receptor dimer are believed to be important for signal transduction (1). Crystallographic studies on the erythropoietin receptor showed that it forms homodimers in the absence of ligand (63). Further biochemical studies established that the specific orientation of receptor subunits is important for activation and signal transduction and that the transmembrane-juxtamembrane segment in the receptor plays an important role in such receptor activation (1). The crystallographic structure of βc homodimers suggests that the distance between the two membrane-proximal domains in the intertwined unliganded dimer is ~100 Å (13, 45). This distance has been suggested to be too great for the interaction of signaling components, such as JAK2 (13, 45), which constitutively associates with the cytoplasmic domains of βc (13, 20, 23, 64, 65). One can speculate that IL-5 binding to the extracellular domain of βc initiates intracellular signaling by bringing the βc-binding kinase proteins into closer proximity, triggering receptor phosphorylation and initiation of intracellular signaling pathways.

Whether IL-5 receptor {alpha} and βc subunits preassemble has been controversial (10, 48-50). Our cell FRET data demonstrate the presence of preformed βc homo-oligomers but not {alpha}-βc hetero-oligomers. Moreover, our data show that IL-5 not only stabilizes the IL-5R{alpha}·βc complex but also induces βc rearrangement. These data fit with a sequential model of IL-5 receptor activation, in which IL-5 first binds to IL-5R{alpha} followed by binding of βc (Fig. 5). In this model, the structural rearrangements in the cytoplasmic regions of βc that occur upon formation of a three-component complex are envisioned to enhance kinase binding and phosphorylation, leading to consequent signal transduction (Fig. 5). The ability of the IL-5R{alpha} subunit to bind ligand even in the absence of βc, albeit at a lower affinity (9), supports the notion that ligand-linked conformational changes occur in one or more chains of the heteromeric receptor-ligand complex. Since βc is the shared signaling receptor subunit for IL-3, IL-5, and GM-CSF, the rearrangement of preformed βc subunits could be a common feature in the activation of IL-5, IL-3, and GM-CSF receptor systems, although there may be differences in the assembly processes among the three βc cytokines, as hinted by the observation of preassembled GM-CSFR{alpha}-βc (66, 67). We believe that the cell FRET approach developed here offers the potential to compare cell surface receptor assembly and activation processes among the βc cytokines. This method, combined with recombinant protein sequence variation, can help reveal more about structural features of the receptor subunits and cytokines that control receptor assembly and cell activation.


   FOOTNOTES
 
* This work was supported, in whole or in part, by National Institutes of Health Grants R01 GM55648 and AI 40462. This work was also supported by National Science Foundation Grant DMI-0422010. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S3. Back

1 To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Drexel University College of Medicine, 11102 New College Bldg., 245 N. 15th St., Philadelphia, PA 19102. Tel.: 215-762-4197; Fax: 215-762-4452; E-mail: ichaiken{at}drexelmed.edu.

2 The abbreviations used are: IL, interleukin; GM-CSF, granulocyte/macrophage colony-stimulating factor; βc, common receptor β subunit; FRET, fluorescence resonance energy transfer; CFP, cyan fluorescent protein; YFP, yellow fluorescent protein; ROI, region of interest. Back


   ACKNOWLEDGMENTS
 
We thank Louise Bertrand for help and advice performing FRET, Sabine C. Baxter for technical assistance purifying IL-5, and Dr. Michael Edidin for advice on FRET analysis in cells. We also thank Drs. Patrick Loll, Bradford Jameson, Mauricio Reginato, and William Williams for insightful suggestions and helpful discussions.



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