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Originally published In Press as doi:10.1074/jbc.M209560200 on September 26, 2002

J. Biol. Chem., Vol. 277, Issue 48, 46073-46078, November 29, 2002
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Kinetic Analysis of the Interleukin-13 Receptor Complex*

Allison-Lynn Andrews, John W. HollowayDagger, Sarah M. Puddicombe, Stephen T. Holgate, and Donna E. Davies§

From the Infection, Inflammation and Repair Division, School of Medicine, University of Southampton, 97 Tremona Rd., Southampton General Hospital, Southampton SO16 6YD, United Kingdom

Received for publication, September 18, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interleukin (IL)-13 is a key cytokine associated with the asthmatic phenotype. It signals via its cognate receptor, a complex of IL-13 receptor alpha 1 chain (IL-13Ralpha 1) with IL-4Ralpha ; however, a second protein, IL-13Ralpha 2, also binds IL-13. To determine the binding contributions of the individual components of the IL-13 receptor to IL-13, we have employed surface plasmon resonance and equilibrium binding assays to investigate the ligand binding characteristics of shIL-13Ralpha 1, shIL-13Ralpha 2, and IL-4Ralpha . shIL-13Ralpha 1 bound IL-13 with moderate affinity (KD = 37.8 ± 1.8 nM, n = 10), whereas no binding was observed for hIL-4Ralpha . In contrast, shIL-13Ralpha 2 produced a high affinity interaction with IL-13 (KD = 2.49 ± 0.94 nM n = 10). IL-13Ralpha 2 exhibited the binding characteristics of a negative regulator with a fast association rate and an exceptional slow dissociation rate. Although IL-13 interacted weakly with IL-4Ralpha on its own (KD > 50 µM), the presence of hIL-4Ralpha significantly increased the affinity of shIL-13Ralpha 1 for IL-13 but had no effect on the binding affinity of IL-13Ralpha 2. Detailed kinetic analyses of the binding properties of the heteromeric complexes suggested a sequential mechanism for the binding of IL-13 to its signaling receptor, in which IL-13 first binds to IL-13Ralpha 1 and this then recruits IL-4Ralpha to stabilize a high affinity interaction.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IL-131 is a pleiotropic cytokine with roles in asthma and allergy (1, 2). It is produced by activated T-cells to promote B-cell proliferation and, IgE synthesis. It also down-regulates the production of tumor necrosis factor alpha , increases expression of vascular cell adhesion molecule-1 on endothelial cells, and enhances the induction of major histocompatibility complex Class II and CD23 antigens on monocytes. IL-13 is a key cytokine in asthma not only because of its pro-allergic role but also its wide ranging effects on epithelial cells and fibroblasts linked to airway wall remodeling. Overexpression of IL-13 in the bronchial epithelium of transgenic mice causes lymphocytic and eosinophilic infiltration, goblet cell metaplasia, subepithelial fibrosis, and smooth muscle proliferation associated with marked bronchial hyper-responsiveness (3-9).

IL-13 mediates these functions through interacting with its cognate receptor on hematopoietic and other cell types, but no functional receptors have been identified on human or mouse T-cells (7). The human IL-13 receptor (IL-13R) is a heterodimer composed of the interleukin-4 receptor alpha  chain (IL-4Ralpha ) and an IL-13 binding protein, IL-13Ralpha 1 (10). Although they only share 25% homology, IL-13 shares many functional properties with IL-4 as a result of the common IL-4Ralpha component in their receptors. The IL-13Ralpha 1·IL-4Ralpha complex can act as an alternative IL-4 receptor especially in cells that lack the common gamma chain (gamma c) that usually forms a heterodimer with IL-4Ralpha to bind IL-4. However, IL-13 appears to have a distinct role in allergic inflammation. It has been shown, in a mouse model, that IL-13 acts as a key regulator of allergen-induced airway inflammation and remodeling, which were independent of IL-4 (1, 11).

In a cellular context, IL-13 binds to IL-13Ralpha 1 with moderate affinity (KD = 2-10 nM) in the absence of IL-4Ralpha ; however, this affinity increases when IL-4Ralpha is present (12). IL-13 does not bind IL-4alpha in the absence of IL-13Ralpha 1. A second IL-13 binding protein, IL-13Ralpha 2, has also been identified. IL-13Ralpha 2 shares a 37% homology with IL-13Ralpha 1 and binds IL-13 with high affinity (50 pM) (10, 13-15). Despite this increased binding affinity, IL-13Ralpha 2 is believed to be non-signaling and therefore may act as a "decoy" receptor. The presence of IL-13Ralpha 1 and IL-4Ralpha on the cell surface renders cells responsive to IL-13; thus IL-13Ralpha 2 is not required for receptor-ligand interactions. Similarly, the expression of IL-13Ralpha 2 in vitro does not make cells responsive to IL-13. The discovery of a soluble receptor, homologous to IL-13Ralpha 2, in the serum and urine of mice has lent support to this regulatory role (15, 16). However, this soluble protein has yet to be identified in humans.

After binding to its receptor, both IL-4 and IL-13 signal through phosphorylation-dependent activation of Jak kinases and the transcription factor, STAT6 (17). The IL-13Ralpha 1·IL-4alpha complex is thought to initiate its signal via IL-4Ralpha chain, because binding to the complex by either IL-13 or IL-4 results in the phosphorylation of IL-4Ralpha , insulin-receptor substrate-2, JAK1, and Tyk2, which is characteristic of the IL-4 response (18, 19). IL-13Ralpha 2 is unable to initiate the downstream signaling pathway because it lacks box-1 and -2 signaling motifs in its cytoplasmic domain, but it does contain a putative consensus phosphorylation site that may interact with Src homology 2-containing proteins. Recently it has been shown that IL-13Ralpha 2 may play an important role in receptor internalization (20, 21).

Unlike the closely related IL-4 receptor, the receptors for IL-13 have not been characterized in detail. The composition and binding affinities of IL-13R have been investigated in various cell types by cross-linking and displacement of radiolabeled IL-13 and/or IL-4. However, the contribution by individual subunits to the overall affinity of the different types of IL-13 receptor complexes is still unclear. In our studies of the IL-13 receptor system, we have used surface plasmon resonance (SPR) and equilibrium binding assays to analyze the binding properties of soluble forms of IL-13R subunits comprising the extracellular ligand binding domain either alone or as complexes with each other. We present the first kinetic analysis of IL-13 receptor binding using a biosensor and confirm previous values reported from equilibrium binding studies.

By using several different experimental strategies to direct the assembly of receptor complexes, we have also been able to isolate and analyze homomeric and heteromeric complexes of the IL-13Ralpha 1 and IL-13Ralpha 2 subunits. From these experiments we were able to observe a sequential mechanism for the binding of IL-13 to its receptor in which IL-13 first binds to IL-13Ralpha 1, and then this complex recruits IL-4Ralpha to create a high affinity interaction. This study describes the first detailed analysis of the interaction of IL-13 with its binding chains and, given the importance of IL-13 in allergic inflammation, may provide insight into the modulation of IL-13 responses.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents-- Sensor chips (SA and CM5), HBS buffer (10 nM Hepes with 0.15 M NaCl, 3.4 mM EDTA, and 0.005% surfactant P20), amine coupling kit, and regeneration agents were supplied by BIAcore, unless otherwise stated. Protein A and human IgG were obtained from Sigma. Recombinant shIL-13Ralpha 1.Fc, shIL-13alpha 2.Fc, and biotinylated IL-13 were gifts from the Genetics Institute (Cambridge, MA). shIL-4Ralpha .Fc chimera and biotinylated IL-4 were obtained from R&D Systems. Recombinant IL-13 and IL-4 were purchased from Peprotech. The bindability of the receptor and ligand preparations was determined by serial incubation with corresponding ligand or receptor (100 ng/ml IL-13, IL-4, shIL-13Ralpha 2.Fc, or shIL-4Ralpha , as appropriate) immobilized in the wells of a Maxisorb enzyme-linked immunosorbent assay plate until the unbound receptor (or ligand) reached a constant value, enabling computation of the bindable fraction. This was 80% for biotinylated IL-13, 97% for IL-13Ralpha 2.Fc, 99% for IL-13Ralpha 1, and 88% for IL-4Ralpha . Analyte concentrations were corrected for bindability.

Sensor Chip Preparation-- The molecular interactions between shIL-13Ralpha 1.Fc, shIL-13Ralpha 2.Fc, and IL-13 were determined by surface plasmon resonance measurements using a BIAcore 2000TM biosensor (BIAcore AB, Uppsala, Sweden) as described in detail elsewhere (22, 23). Biotinylated IL-13 was diluted in HBS running buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3.4 mM EDTA, and 0.005% surfactant P20) and immobilized on a streptavidin-coated sensor chip (SA chip) that had been conditioned with (3×) 1-min injections of 1 M NaCl in 50 mM NaOH.

Surface Plasmon Resonance Measurements-- All receptor proteins were diluted from stock to the desired concentration in HBS buffer. To determine kinetic constants, sensograms were collected at 25 °C, flow rate 10 µl/min, and data collection rate of 1 Hz. For binding and kinetic analysis, five serial dilutions (200-1000 nM) of each protein were injected separately over the IL-13 surface. 200 nM IL-13 was present in HBS buffer during the dissociation phase to minimize rebinding to the sensor chip surface. Sensograms were recorded and normalized to a base line of 0 resonance units (RU). Equivalent concentrations of each protein were injected over an untreated surface to serve as blank sensograms for subtraction of bulk refractive index background. The sensor chip surface was regenerated between runs with a 1-min injection of 10 mM HCl, at 10 µl/min. The resultant sensograms were then evaluated using the BIA evaluation 2.0 software to provide kinetic data.

Briefly, the association of an analyte to the immobilized ligand is described by dR/dt = konCRmax - (konC + koff)Rtn, where kon is the association rate constant, C is the concentration of the analyte, Rmax is the maximum binding capacity of the ligand, koff is the dissociation rate constant, and Rtn is the amount of analyte bound to the ligand at time tn. A plot of dR/dt versus Rtn gives a line with slope ks = -(konC + koff). A plot of ks versus C for various concentrations of analyte results in a slope of the fitted line, which is equal to kon.

The dissociation rate constant, koff, is determined by the first rate order equation dR/dt = koffRtn, measured when analyte passing over the ligand is replaced with buffer and under the conditions where R approaches Rmax to minimize the effect of rebinding. koff is obtained from the slope of the plot, ln(Rt1 - Rtn) versus t, during the initial phase of dissociation (first 60 s) where Rt1 is the amount of bound analyte at time t1, Rtn is the amount of bound analyte at time tn, and t is tn - t1. The equilibrium dissociation constant is KD = koff/kon.

shIL-13Ralpha 1·shIL-13Ralpha 2·shIL-4Ralpha Complex Interactions-- A CM5 sensor chip was activated with a 10-µl injection of N-hydroxysuccinimide/N-ethyl-N'-[(3-diethylamino)propyl]carbodiimide) followed by an injection of 10-µl Protein A in 10 mM sodium acetate (pH 4.0). The remaining activated esters were blocked by an injection of 35 µl of 1 M ethanolamine. shIL-13Ralpha 1, shIL-13Ralpha 2, or shIL-4Ralpha were injected either singly or in combination and allowed to bind non-covalently to the immobilized Protein A. This allows the preparation of homogeneous surfaces at controlled densities of either shIL-13Ralpha 1, shIL-13Ralpha 2, or IL-4Ralpha as well as mixed co-receptor surfaces at varying ratios of each subunit. A series of concentrations (50-200 nM) of IL-13 or IL-4 in HBS buffer were passed over the sensor chip surface, and the resulting sensograms were recorded and analyzed as previously described.

Equilibrium Binding Assays-- Recombinant receptors (0.13-81 µg/ml) were immobilized on Protein A (100 µg/well) coupled to Maxisorb enzyme-linked immunosorbent assay plates (Nalgene Nunc, Herefordshire UK); unoccupied sites were blocked with the addition of human IgG (50 µg/ml). The receptors were then allowed to bind to serial dilutions of biotinylated IL-13 or IL-4 overnight at 4 °C; nonspecific binding at each ligand concentration was determined by the addition of a 10-fold excess of unlabeled IL-13 or IL-4. Bound ligand was detected using streptavidin-horseradish peroxidase conjugate using tetramethylbenzidine as chromagen. The absorbance was read on microplate spectrophotometer at 450 nm with a 630-nm reference filter, and the amount of bound ligand was determined by reference to a standard curve generated using serial dilutions of biotinylated IL-13 or IL-4. After determination of free and bound ligand and correction for nonspecific binding, equilibrium binding constants were determined by Scatchard analysis.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Kinetic Binding Analysis of Receptor Subunits-- The binding kinetics of IL-13 to shIL-13Ralpha 1 and shIL-13Ralpha 2 were analyzed in real-time by SPR using a BIAcoreTM 2000 biosensor and biotinylated IL-13 immobilized on an SA sensor chip. This was found to be the most efficient use of reagents because the IL-13 surface could be regenerated with 10 mM HCl more than 50 times and still retained over 95% of its binding capacity. Fig. 1 shows an overlay of a series sensograms obtained after the interaction of various concentrations of shIL-13Ralpha 1 with IL-13 as described under "Experimental Procedures." Analysis of the rate of binding versus concentration of bound shIL-13Ralpha 1 at various time points enabled the derivation of both the association and dissociation rate constants (3.76 ± 2.6 × 104 M-1s-1 and 1.41 ± 0.13 × 10-3 s-1, respectively). The calculated dissociation constant (KD = koff/kon) was found to be 37.8 ± 1.8 nM. Similar experiments were also carried for shIL-13Ralpha 2, the results of which are shown in Fig. 1. In this case the association and dissociation rate constants were found to be 5.41 ± 1.42 × 104 M-1s-1 and 1.39 ± 0.15 × 10-4 s-1, and this gave a KD value of 2.49 ± 0.94 nM. shIL-4Ralpha was also injected over the IL-13-coated chip, but no binding interactions were observed. Fig. 2 illustrates the specificity of the interaction; i.e. incubation of the receptor solution with increasing concentrations of IL-13 reduced the interaction of both soluble receptors with the IL-13 sensor chip surface in a dose-dependent manner. Preincubation with 500 nM IL-4 had no effect on the binding of either receptor chain (data not shown).


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  		Fig. 1.   Analysis of ligand binding to the IL-13 surface. Representative sensograms of shIL-13Ralpha 1 (A, lower to upper curve: 200, 400, 600, 800, and 1000 nM); shIL-13Ralpha 2 (B) and shIL-4Ralpha (C) binding to a biotinylated IL-13 surface at a flow rate of 10 µl/min. Resonance units (RU) are shown after background subtraction. D shows plots of ks versus concentration (M × 107) for shIL-13Ralpha 1 (black-triangle), shIL-13Ralpha 2 (black-square), and shIL-4Ralpha (black-diamond ). Data are mean ± S.D., n = 10.


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  		Fig. 2.   Specificity of ligand binding to the IL-13 surface. Sensograms of shIL-13Ralpha 1 (A) and shIL-13Ralpha 2 (B), which have been preincubated with IL-13 (lower to upper curve: 300, 200, 100, and 500 nM IL-4 acted as a negative control) prior to injection. The amount of receptor (expressed in RUs) binding to biotinylated IL-13 on the sensor chip decreased in a dose-dependent manner with increasing amounts of IL-13. The presence of 500 nM IL-4 had no effect on the amount of binding of either IL-13Ralpha 1 or IL-13Ralpha 2.

To provide confirmation of the data obtained in the kinetic analyses, equilibrium binding studies were also carried out. In the case of IL-13Ralpha 1, it was difficult to obtain a reliable KD, consistent with its low affinity as determined using SPR. In contrast, IL-13Ralpha 2 bound to biotinylated IL-13 with an affinity similar to that determined by SPR (KD = 0.76 ± 0.09 nM) (Fig. 3). Little or no interaction between IL-13 and IL-4Ralpha was detected in either SPR or equilibrium assays (data not shown).


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  		Fig. 3.   Equilibrium binding assays. A, representative curves for IL-13 binding to IL-13Ralpha 2: 0.13 µg/ml (), 0.65 µg/ml (black-square), 3.25 µg/ml (black-triangle), 16 µg/ml (black-square), and 81 µg/ml (black-diamond ). B, a Scatchard plot constructed from equilibrium binding data for shIL-13Ralpha 2. Data are mean ± S.D., n = 3.

Combination Assays-- We then used the SPR protocol to determine if the presence of a particular subunit or ligand influenced binding affinity. 250 nM shIL-13Ralpha 1 was preincubated with equimolar solutions of IL-13 or IL-4 in the presence or absence of IL-4Ralpha . Fig. 4 is a summary of results from sets of sensograms obtained for each experimental condition. The binding of shIL-13Ralpha 1 to the sensor surface was unaffected by the presence of IL-4 with or without IL-4Ralpha . The binding of shIL-13Ralpha 1 was reduced in the presence of IL-13 with or without IL-4Ralpha . However, in the presence of IL-4Ralpha , the dissociation rate was reduced when compared with shIL-13Ralpha 1 on its own, leading to an overall increase in affinity.


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  		Fig. 4.   Preincubation of soluble receptors with other components of the IL-13 receptor complex. A summary of sensograms collected from experiments when shIL-13Ralpha 1 (A) or shIL-13alpha 2 (B) was preincubated with hIL-4Ralpha , IL-4, IL-13 prior to injection (lower to upper curves: +IL-13, +IL-13 and IL-4Ralpha , +IL-4Ralpha , +IL-4, +IL-4 and IL-4Ralpha , control with no additions) of 1000 nM IL-13Ralpha 1 (A) or 1000 nM IL-13Ralpha 2 (B). The amount of receptor (expressed in RUs) binding to biotinylated IL-13 on the sensor chip decreased significantly only when IL-13 was present during preincubation. When shIL-13Ralpha 1 was preincubated with shIL-4Ralpha , a slight decrease in the dissociation rate (indicated by the arrow) was observed leading to an increase in affinity.

When these experiments were repeated using shIL-13Ralpha 2 (Fig. 4), the presence of IL-13 reduced the amount of receptor binding to the sensor chip surface, but preincubation with IL-4Ralpha had no effect on either the amount of receptor bound or the affinity constants.

Analysis of Receptor Complexes-- The complex of IL-13Ralpha 1 and IL-4Ralpha is known to bind IL-13 with high affinity. To characterize the kinetic and binding properties of this complex, sensor chip surfaces were prepared using both shIL-13Ralpha 1 and IL-4Ralpha bound to protein A. In this way we were able to prepare homogeneous surfaces at controlled densities of either IL-13Ralpha 1 or IL-4Ralpha , as well as mixed co-receptor surfaces with varying ratios of each subunit. The binding kinetics of individual receptor subunits were initially evaluated and compared with the results obtained from the IL-13-immobilized chip (Table I). Fitting the data to a single-site model provided constants for shIL-13Ralpha 1 and shIL-4Ralpha that were consistent with those previously determined for the biotinylated IL-13 surface (Table I), indicating that immobilization of either the ligand or the receptor on the chip surface was equivalent.

                              
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Table I
Comparison of dissociation constants under different experimental conditions
Constants were determined as described under "Experimental Procedures." IL-13 surface refers to SA sensor chip coated with biotinylated IL-13. Receptor surface indicates were shIL-13Ralpha 1, shIL-13Ralpha 2, or hIL-4Ralpha were immobilized to the sensor surface via Protein A and IL-13 injections.

We then carried out more critical studies focusing on the functional IL-13Ralpha 1·IL-4Ralpha high affinity site. The surface was coated with either 1200 RU of shIL-13Ralpha 1 and 600 RU of IL-4Ralpha (high IL-13Ralpha 1) or 600 RU of shIL-13Ralpha 1 and 1200 RU of IL-4Ralpha (low IL-13Ralpha 1). The resultant sensograms obtained after injecting a series of concentrations of IL-13 were evaluated using a two-site model (Table II). Where IL-13Ralpha 1 was in excess, two interactions were apparent: one of high affinity (KD of 495 ± 125 pM) and a second of lower affinity (KD of 38 ± 3.2 nM). Because the affinity of the second interaction was comparable with that determined for IL-13Ralpha 1 alone, it most likely represented the interaction of IL-13 with excess IL-13Ralpha 1 subunits that have not formed part of the high affinity site. Thus, the high affinity interaction most likely represented binding of IL-13 to IL-13Ralpha 1·IL-4Ralpha . Where IL-13Ralpha 1 was limiting, two interactions were also observed: a high affinity site (KD = 492 ± 158 pM) identical to that observed with high IL-13Ralpha 1 and a very low affinity site (KD > 400 nM). Because the latter site is most likely due to nonspecific binding of IL-13 to IL-4Ralpha and/or the sensor chip surface, we conclude that the high affinity site is best explained by binding of IL-13 to heterodimeric complexes of IL-13Ralpha 1 and IL-4Ralpha ; the mechanism of IL-13 binding to this high affinity site was then investigated using the kinetic data obtained from a series of injections of IL-13 over a mixed receptor surface. The ka value obtained for these experiments was similar to that obtained for the binding of IL-13 to IL-13Ralpha 1. However, the dissociation phase was remarkably slower thus indicating a stabilization of the complex and a sequential mechanism of binding. The ability of IL-4Ralpha to stabilize the binding of IL-13 to IL-13Ralpha 1 was also observed in equilibrium binding studies. Thus, although we were unable to obtain any binding data for Scatchard analysis using IL-13Ralpha 1 on its own, in the presence of IL-4Ralpha a dissociation constant of ~10 nM was determined (data not shown).

                              
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Table II
Kinetic rate constants for the shIL-13Ralpha 1·hIL-4Ralpha co-surface: the high affinity site

We also used the BIAcore experimental protocol to investigate the possibility of a similar mechanism when IL-4 binds to an IL-4 receptor complex consisting of IL-4Ralpha and IL-13Ralpha 1. Sensor chip surfaces were prepared with high and low concentrations of IL-4Ralpha and IL-13Ralpha 1. Under both conditions, fitting of binding data either to a one- or two-site model indicated no high affinity sites greater than that observed for IL-4Ralpha . Thus, the presence of IL-13Ralpha 1 had no detectable effect on the binding of IL-4 to IL-4Ralpha (data not shown).

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have used SPR to determine the binding kinetics of the two IL-13 binding proteins, IL-13Ralpha 1 and IL-13Ralpha 2. This represents the first detailed biosensor analysis of this receptor-ligand interaction. Our results confirmed those obtained in a more complex cell-based assay by showing that IL-13Ralpha 2 has a higher affinity for IL-13 than IL-13Ralpha 1. The data obtained by SPR were similar, but not identical, to previous values obtained from equilibrium binding assays in that the measured affinities were less than those previously reported. The results may reflect real differences inherent to the SPR biosensor method or simply errors in determinations, because the kinetic rates are approaching the limitations of the respective experimental procedures. The difference is unlikely to be due to biotinylation of IL-13, because similar results were obtained when IL-13Ralpha 1 and IL-13Ralpha 2 were immobilized on the sensor chip surface and IL-13 was injected. However, our findings with the IL-13R extracellular domains are not unprecedented: similar results have been observed for the epidermal growth factor receptor where the binding affinities of the extracellular domain, determined either by SPR (24) or titration calorimetry (25), yield binding constants that are less than those obtained in binding assays with cells or membranes. This appears to be due, in part, to the use of the extracellular domain of the receptor and the consequent loss of stabilizing interactions derived from the transmembrane and intracellular domains. Kawakami et al. (20, 21) have suggested that the transmembrane domain of IL-13 receptors are required to stabilize the binding of IL-13, this is the most likely explanation for the differences observed in the biosensor measurements. Although this identifies a potential limitation of use of isolated extracellular domains, the value of this SPR method is that it enables ligand-receptor interactions to be measure in real-time without the confounding influence of other protein-protein interactions. This property is highlighted by our attempts to estimate the binding affinity of shIL-13Ralpha 1 using conventional equilibrium binding studies. Due to its comparatively low affinity, we were unable to detect significant binding at equilibrium. From the kinetic data derived from the SPR measurements, it is most likely that this is due to the high off-rate of IL-13Ralpha 1, causing substantial loss of ligand during the wash procedures that are required for measurement of bound ligand. The advantage of SPR is that we can monitor these interactions in real-time, thus giving an opportunity to evaluate low binding affinities that are not amenable to more conventional methods.

Using SPR we were able to dissect the binding contributions of individual IL-13 receptor chains and the influence of IL-4Ralpha on these. In the initial assays where IL-13 was immobilized on the sensor surface, only the presence of IL-13 appeared to affect the binding of shIL-13Ralpha 1or shIL-13Ralpha 2 to the surface. However, we noted that, when IL-4Ralpha was co-injected with shIL-13Ralpha 1, there appeared to be a decrease in the rate of dissociation compared with that obtained for shIL-13Ralpha 1 on its own. This therefore represented a higher affinity interaction and was a closer approximation to the binding constants observed in intact cells. This observation led us to develop experimental protocols to try to examine this high affinity site further. To determine the kinetics of binding of IL-13 to this heterocomplex we prepared surfaces comprising a combination of shIL-13Ralpha 1 and IL-4Ralpha . Because these surfaces contained more than a single class of binding sites, we prepared relatively low density surfaces to analyze the high affinity site while minimizing any mass diffusion effects during the association phase. When concentrations of IL-13 were applied to the surface, a two-site model gave KD values of 495 ± 125 pM and 34.2 ± 6.9 nM, indicating the presence of a high affinity site.

Having established the presence of a high affinity site on the sensor surface, we turned our attention to the mechanism of binding. There are three possible outcomes to the binding of IL-13 to the mixed receptor surface made up of IL-13Ralpha 1 and IL-4Ralpha ; first, that IL-13 will bind to IL-13Ralpha 1 and lL-4Ralpha independently, second, that IL-13Ralpha 1 and IL-4Ralpha pre-associate to form a high affinity site, or third, that a sequential binding mechanism occurs so that lL-13 first binds to IL-13Ralpha 1, which then recruits IL-4Ralpha to form a high affinity complex. Because we had demonstrated that the high affinity site existed, we eliminated the first possibility. By analyzing the kinetic data, we determined whether the high affinity site was pre-associated or sequential. Binding to a pre-associated site would result in association and dissociation constants that were different from the individual subunits, whereas for a sequential interaction the association constant would be unchanged but the dissociation rate constant value would change. The results of the data analysis yielded the best fit to a model in which IL-13 bound to IL-13Ralpha 1 and then recruited IL-4Ralpha to stabilize the interaction and give a high affinity complex. The association rate constant was essentially the same as that observed for IL-13Ralpha 1 alone, whereas the dissociation constant was 2-fold slower yielding an equilibrium constant of 495 ± 125 pM. This experimental procedure was repeated using IL-4 in an attempt to see if a similar mechanism existed within this complex. No increase in affinity or any evidence of a high affinity site was observed throughout these experiments. Although this was slightly surprising, due to the similarity in protein structure and common receptor subunit, it may be that the contribution of IL-13Ralpha 1 to the IL-4 receptor complex is so small that it is not detectable using this experimental approach.

A sequential mechanism has been proposed for binding of IL-4 to an alternative IL-4R made of IL-4Ralpha and the common gamma c chain (26). First, IL-4 binds to IL-4Ralpha and then recruits gamma c to form a signaling heterodimer. The results from our binding studies have shown that a similar mechanism may be apparent with the interaction of IL-13 with its receptor. Following the sequential mechanism suggested for IL-4, IL-13 would appear to bind first to IL-13Ralpha 1. The resulting complex then recruits IL-4Ralpha to form a signaling heterodimer. Binding to IL-13Ralpha 1 may induce a conformational change that presumably then allows it to bind to IL-4Ralpha . It may be this rearrangement that is responsible for the overall increase in affinity of the IL-13·IL-13Ralpha 1·IL-4Ralpha complex. This would then account for the observed decrease in the dissociation rate in our combined experiments.

Analysis of the kinetic data shows that shIL-13Ralpha 1 binds to IL-13 and disassociates rapidly. However, shIL-13Ralpha 2 owes much of its high affinity to a very slow dissociation rate. It has been suggested that IL-13Ralpha 2 may act as a decoy receptor regulating the action of IL-13. The kinetic data may give some support to this model, because a high on-rate and very slow dissociation rate are characteristic of a negative regulator.

We have employed SPR to determine the kinetic binding constants for the simple shIL-13Ralpha 1, shIL-13Ralpha 2, and IL-4Ralpha receptor subunits. In addition, we were able to analyze a complex system in which two subunits were present on the same surface and establish that these proteins bind IL-13 in a sequential manner to give a high affinity complex. This model mimics the high affinity IL-13 receptor found on the cell surface, providing a useful system for the screening and analysis of novel antagonistic molecules that may have therapeutic potential.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Sandy Goldman of the Genetics Institute (Cambridge, MA) for generously providing shIL-13Ralpha 1.Fc and shIL-13Ralpha 2.Fc for these studies and to Phil Buckle (BIAcore) for his advice and assistance.

    FOOTNOTES

* This work was supported in part by the Medical Research Council (MRC), United Kingdom (Grant G4500010).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 An MRC Training Fellow.

§ To whom correspondence should be addressed. Tel.: 23-80-798-523; Fax: 23-80-777-996; E-mail: donnad@soton.ac.uk.

Published, JBC Papers in Press, September 26, 2002, DOI 10.1074/jbc.M209560200

    ABBREVIATIONS

The abbreviations used are: IL, interleukin; shIL-13Ralpha 1, soluble human IL-13 receptor alpha  chain 1; shIL-13Ralpha 2, soluble human IL-13 receptor alpha  chain 2; sIL-4Ralpha , soluble human IL-4 receptor; SPR, surface plasmon resonance, RU resonance units; STAT, signal transducers and activators of transcription; SA chip, streptavidin-coated sensor chip.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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