cDNA Cloning and Characterization of the Human Interleukin 13 Receptor α Chain

Abstract We have cloned cDNAs corresponding to the human interleukin 13 receptor α chain (IL-13Rα). The protein has 76% homology to murine IL-13Rα, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13Rα; however, the combination of both IL-13Rα and IL-4Rα resulted in substantial binding activity, with a Kd of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13Rα serves as an alternative accessory protein to the common cytokine receptor γ chain (γc) for IL-4 signaling, it could not replace the function of γc in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13Rα and γc are similar, and like γc, IL-13Rα is located on chromosome X.

We have cloned cDNAs corresponding to the human interleukin 13 receptor ␣ chain (IL-13R␣). The protein has 76% homology to murine IL-13R␣, with 95% amino acid identity in the cytoplasmic domain. Only weak IL-13 binding activity was found in cells transfected with only IL-13R␣; however, the combination of both IL-13R␣ and IL-4R␣ resulted in substantial binding activity, with a K d of approximately 400 pM, indicating that both chains are essential components of the IL-13 receptor. Whereas IL-13R␣ serves as an alternative accessory protein to the common cytokine receptor ␥ chain (␥ c ) for IL-4 signaling, it could not replace the function of ␥ c in allowing enhanced IL-2 binding activity. Nevertheless, the overall size and length of the cytoplasmic domain of IL-13R␣ and ␥ c are similar, and like ␥ c , IL-13R␣ is located on chromosome X.
Interleukin 13 (IL-13) 1 is a T-cell-derived cytokine that exhibits a broad range of activities in the regulation of inflammatory and immune responses. Many of the actions of IL-13 are also exhibited by another T-cell-derived cytokine, IL-4, with which it shares approximately 30% amino acid sequence identity. These shared activities include down-regulation of inflammatory cytokines (1), induction of expression of the IL-1 receptor antagonist (2), induction of surface expression of class II major histocompatibility complex (3), induction of CD23 and IgE expression on B cells (4), costimulation with anti-CD40 antibodies (5), and the inhibition of IL-2-induced proliferation of chronic lymphocytic leukemia cells of B-cell origin (6). Although almost all functions of IL-13 are shared by IL-4, IL-4 additionally exerts unique effects, including the ability to induce responses on certain cell types, including T cells, which do not respond to IL-13 (7). Although the receptor for IL-4 on T cells was found to contain both the 140-kDa IL-4-binding protein (denoted IL-4R or IL-4R␣) and the common cytokine receptor ␥ chain (␥ c ) (8,9), it was subsequently demonstrated that IL-4 could also induce actions on nonhematopoietic cells such as COS-7 cells and renal cell carcinoma lines that ex-pressed IL-4R␣ but not ␥ c or the ␥ c -associated signaling molecule Janus kinase 3 (10,12). These data indicated that there are two classes of IL-4 receptor: the type I IL-4 receptor (containing IL-4R␣ and ␥ c ), which is expressed on a number of lineages, including T cells, B cells, and monocytes; and the type II IL-4 receptor (containing IL-4R␣ and another protein originally denoted ␥Ј), which is not expressed on T cells but is expressed on certain other cell types (10). Although it remains unclear whether the exact signals induced by these two types of IL-4 receptors are identical, it is clear that IL-4 can activate signal transducers and activators of transcription 6 via either type of receptor (10). Since antibodies to IL-4R␣ could block IL-13 action even though IL-13 could not bind to IL-4R␣ (10,11), we hypothesized that the ␥Ј of the type II IL-4 receptor was actually the IL-13R (10). This hypothesis is consistent with biochemical data that IL-13 can partially compete for binding of IL-4 to cells responding to both IL-4 and IL-13 (11), with the ability of both IL-4 and IL-13 to chemically cross-link to ϳ65 kDa proteins (12,13), and with the ability of both IL-4 and IL-13 to induce tyrosine phosphorylation of IL-4R␣ (14,15). The ability of IL-4 and IL-13 to act through a shared type II IL-4 receptor (10) was confirmed with the cloning of murine IL-13R␣ (16). It was demonstrated that overexpression of murine IL-13R␣ in COS cells showed specific binding of 125 Ilabeled murine IL-13 that could not be competed by murine IL-4 (a finding likely explained by the inability of murine IL-4 to bind to the primate IL-4R␣ constitutively expressed on COS cells); however, when CTLL-2 cells were transfected with murine IL-13R␣, both IL-4 and IL-13 could cross-compete with each other for binding to the cells (16). We now report the cloning of a human IL-13R␣ cDNA, which together with IL-4R␣ allows high affinity IL-13 binding.

MATERIALS AND METHODS
Isolation of cDNA Clones Encoding Human IL-13R␣-A cDNA library in Zap-II (Stratagene, La Jolla CA) was prepared from mRNA from human T-cell lymphotropic virus-I-transformed MT-2 cells. Based on the sequence of the murine IL-13R␣ cDNA (16), primers were selected and evaluated for their ability to generate by PCR from DNA from this library a band that was appropriate in size for IL-13R␣. In this fashion, we found that the MT-2 cDNA library contained human IL-13R␣. A total of 6 ϫ 10 5 phage clones were plated on LB plates using XL1-Blue MRFЈ cells as plating bacteria, and plaques were transferred onto nylon membranes (Amersham Corp.) for hybridization. As described under "Results," a 431-bp probe was generated by PCR by using primers from two Expressed Sequence Tags clones that appeared to correspond to human IL-13R␣. This probe was labeled with [ 32 P]dCTP by random priming (Boehringer Mannheim DNA labeling kit) and hybridized with membranes in 5 ϫ saline/sodium phosphate/EDTA containing 0.1% SDS, 5 ϫ Denhardt's solution, and 100 g/ml salmon sperm DNA at 65°C. Secondary screening was performed with the * 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. eluted primary plaques as described above to obtain pure positive plaques.
The pBluescript phagemid was excised in vivo from the positive phage clones using Ex-Assist helper phage and XL1-Blue MRFЈ bacteria according to the manufacturer's instructions (Stratagene). The phagemids were propagated in XLOLR cells, plasmid DNA was purified, and DNA from each clone was sequenced both on an Applied Biosystems 431 automated sequencer and by manual sequencing using Sequenase (U. S. Biochemical Corp.).
Cell Lines and Transfections-293T ϩ and COS-7 cells were cultured in Dulbecco's modified Eagle's medium (Biofluids) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C and 5% CO 2 . Full-length IL-13R␣ and IL-4R␣ cDNAs were subcloned into the mammalian expression vector pME18S. 293T ϩ cells were transfected at 50% confluence using calcium phosphate precipitation reagents (5 Prime-3 Prime, Inc.). Briefly, 61 l of 1 M CaCl 2 was mixed with 5 g of plasmid DNA plus H 2 0 to 500 l. DNA precipitation buffer (500 l) was added gently, and the mixture was added to 293 T ϩ cells covered with 30 ml of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. The medium was changed after 16 h, and cells were harvested after 24 -36 h. COS-7 cells were transfected with DEAE-dextran by standard methods.
Binding and Affinity-labeling Assays-125 I-Labeled human IL-4 (927 Ci/mmol) and 125 I-labeled human IL-2 (885 Ci/mmol) were from Amersham. Human IL-13 was labeled to a specific activity of 1135 Ci/mmol with 125 I (Amersham) and IODO-GEN reagent (Pierce) as described previously (12). Binding was performed for the indicated times on ice in RPMI 1640 medium supplemented with 25 mM HEPES, pH 7.4, containing 0.5% bovine serum albumin (binding buffer) and the appropriate unlabeled and iodinated ligands (IL-4, IL-13, or IL-2). For affinitylabeling experiments, cells to which 125 I-labeled cytokines had been bound were pelleted and resuspended in phosphate-buffered saline, pH 8.0, containing 1 mM MgCl 2 . The bifunctional water-soluble chemical cross-linker BS 3 (Pierce) was added to a final concentration of 1 mM. Cells were then further incubated for 20 min on ice. After termination of the reaction with 40 mM Tris-HCl, pH 7.5, cells were lysed in lysis buffer (10 mM Tris, pH 7.5, containing 1.25% Nonidet P-40, 0.875% Brij, 150 mM NaCl, and 2 mM EDTA) and lysates were analyzed on 7.5% SDS-polyacrylamide gel electrophoresis. For binding assays, after the incubation period, cells were centrifuged through 20% olive oil and 80% dibutyl phthalate, tubes were frozen on dry ice, and bound and free fractions were separated by cutting the tubes and counted on a ␥ counter. Binding data were analyzed using the LIGAND computer program (17).
Chromosomal Localization Using Somatic Cell Hybrids-The NIGMS somatic cell hybrid mapping panel 2 was obtained from the Coriell Institute (Camden, NJ). Human-specific IL-13R␣ PCR primers were designed (sense, nucleotides 957-979; antisense, nucleotides 1104 -1077; see Fig. 1), and these primers were used to analyze DNA from cell hybrids to specify the chromosome bearing the human IL-13R␣ gene.

RESULTS AND DISCUSSION
Isolation of Full-length cDNA for Human IL-13R␣-The sequence for murine IL-13R␣ cDNA (16) was used to search for homologous sequences in the data base of the Expressed Sequence Tags, and two partial human clones of unknown function were identified. One clone (GenBank accession number H57074) exhibited an 81% match to nucleotides 406 -550 of murine IL-13R␣, whereas another clone (GenBank accession number H89334) was 76% identical to nucleotides 804 -985. Primer pairs based on these human sequences were prepared. Using plasmid DNA made from MT-2 and peripheral blood lymphocyte cDNA libraries as templates, we identified primer pairs that could amplify human cDNA fragments of the size anticipated based on the murine IL-13R␣ sequence. A 431-bp PCR product was gel purified and sequenced. The sequence showed 80% homology to murine IL-13R␣, indicating that it corresponded to human IL-13R␣. This PCR product was then used to screen a Zap-II library made from MT-2 cells, and 11 hybridizing clones were sequenced. A full-length clone of 1572 bp was identified; it contained an 84-bp-long 5Ј-untranslated region, an open reading frame encoding 427 amino acids (calculated molecular weight of 48,774), and a 204-bp-long 3Јuntranslated region (Fig. 1). The sequence around the predicted ATG start codon had Kozak consensus sequences typical of translation start sites (18). At the N terminus was a hydrophobic region consistent with a signal peptide. We analyzed residues 15-34 as potential signal peptide cleavage sites by summing the weight matrices for positions Ϫ12 to ϩ2 for each of these residues; these positions most accurately predict the site of cleavage (19). The highest score was obtained for glycine 21, with a total weight matrix of 7.52. A cleavage site after this glycine also fulfills the other suggested criteria for the signal peptide cleavage site, including a small amino acid in position Ϫ1, no aromatic or charged residue at Ϫ3, and the absence of proline from positions Ϫ3 to ϩ1 (20). Thus, we predict that the first 21 amino acids represent the signal peptide. This prediction is consistent with a hydrophobicity plot of the IL-13R␣ amino acid sequence using the Kyte-Doolittle algorithm (data not shown). Based on the hydrophobicity plot, the transmembrane domain was predicted to span amino acids 344 -367. Like other members of the cytokine receptor superfamily (21), a WSXWS motif and four conserved cysteine residues were found in the extracellular domain of IL-13R␣ (Fig. 1, boxed). A total of 11 potential N-linked carbohydrate addition sites (Asn-X-Ser/ Thr) are present in the extracellular domain, including one that is contained within the WSXWS motif. The open reading frame of human IL-13R␣ has 81% nucleotide and 76% amino acid identity with the published murine IL-13R␣ sequence (16) and contains 3 additional amino acids (Fig. 2). Interestingly, 57 of the 60 amino acids in the cytoplasmic domain (95%) are identical in human and murine IL-13R␣, underscoring the likely significance of this region in signal transduction.
Human IL-13R␣ mRNA Is Expressed in Multiple Tissues-The expression pattern of human IL-13R␣ was examined by Northern blot analysis (Fig. 3). Two species of mRNA of approximately 4.0 and 2.0 kilobases were detected (Fig. 3). Expression of IL-13R␣ mRNA was detected in all tissues examined, with the highest levels in heart, liver, skeletal muscle, and ovary and the lowest levels in brain, lung, and kidney. The high level of expression in skeletal muscle was interesting, since murine IL-13R␣ mRNA was not detected in this tissue (16). Like murine IL-13R␣, human IL-13R␣ also exhibits two principal transcripts (5.2 and 2.2 kilobases for murine IL-13R␣; Ref. 15; 4.0 and 2.0 kilobases for human IL-13R␣), presumably reflecting alternative polyadenylation in each case. As expected, the human mRNAs are each longer than our full-length cDNA, which did not contain a consensus AATAAA polyadenylation sequence and, therefore, presumably does not contain the entire 3Ј-untranslated region.
Coexpression of IL-4R␣ and IL-13R␣ Is Required to Generate High Affinity IL-13 Binding-To examine the binding specificity of human IL-13R␣, we performed affinity-labeling experiments in which 125 I-labeled human IL-13 or IL-4 was bound and cross-linked to 293T ϩ fibroblasts that had been transfected with IL-13R␣, IL-4R␣, or both IL-13R␣ and IL-4R␣. No signal was detected except in cells transfected with both IL-4R␣ and IL-13R␣ (Fig. 4A). In cells transfected with both cDNAs, a major 75-80-kDa band was seen, presumably reflecting the cross-linking of 125 I-labeled human IL-13 (12-13 kDa) to IL-13R␣, thus resulting in an apparent molecular mass of approximately 65 kDa for IL-13R␣ (although the calculated molecular weight for this protein is 48,774, the presence of multiple glycosylation sites presumably explains its slower migration on gels). In addition, a larger band corresponding to affinity-labeled IL-4R␣ was also seen. Both bands were competed when a 500-fold molar excess of either unlabeled IL-13 and IL-4 were added in the binding phase (Fig. 4A). In contrast, 125 I-labeled human IL-4 was cross-linked mainly to the 140-kDa IL-4R␣ chain in a fashion that was independent of coexpression of IL-13R␣ (Fig. 4B). In cells transfected with both IL-4R␣ and IL-13R, in addition to affinity-labeled IL-4R␣, a weak 75-80-kDa band corresponding to affinity-labeled IL-13R␣ was also observed. The appearance of both of these affinity- IL-4R␣. These results were confirmed in binding assays. Corresponding to the affinity-labeling experiments, significant specific binding of 125 I-IL-13 was observed only when both receptor chains were expressed on 293T ϩ cells and either excess IL-4 or IL-13 could compete the binding. The minimal IL-13 binding activity detected when cells were transfected with only IL-13R␣ (Fig. 5A) is consistent with the low binding affinity of murine IL-13R␣ for murine IL-13 (2-10 nM; Ref. 15). In contrast, expression of IL-4R␣ resulted in significant binding of 125 I-IL-4, and additional expression of IL-13R␣ only slightly increased this binding (Fig. 5B). Consistent with the affinity-labeling experiments, unlabeled IL-13 could not com-pete with the binding of 125 I-IL-4 in the absence of IL-13R␣. In cells transfected with both IL-4R␣ and IL-13R␣, however, IL-13 displaced approximately 80% of specifically bound 125 I-IL-4 (Fig. 5B), consistent with previously published results in COS cells (11).
Binding Equilibrium of 125 I-IL-13 and Scatchard Plot Analysis-To determine the kinetics of binding of IL-13 to its receptor, we incubated 293T ϩ cells cotransfected with IL-13R␣ and IL-4R␣ with 125 I-IL-13 in the presence or absence of 500-fold excess unlabeled IL-13 and analyzed the bound radioactivity at different times. As shown in Fig. 6A, the specific binding of 125 I-IL-13 steadily increased over 6 h, after which time binding equilibrium was achieved. Based on this slow on rate, displacement experiments for Scatchard analysis were performed after 6 h of binding at 4°C. Consistent with a previous report (11), IL-4 bound more rapidly, with equilibrium being achieved by 2 h (data not shown). We also examined the dissociation of the ligand receptor complex for IL-13. Cells were incubated with 125 I-IL-13 for 6 h, washed twice, resuspended in binding buffer, and incubated on ice. After 5 h, only 20% of the bound radioactivity was released into the medium (data not shown). These data indicate that IL-13 has slower on and off rates than does IL-4. Formal Scatchard analysis revealed that IL-13 bound with a K d of approximately 400 pM to cells transfected with both IL-4R␣ and IL-13R␣ (Fig. 6, B and C).
The binding and affinity-labeling experiments together con- firm that a receptor complex consisting of human IL-4R␣ and IL-13R␣ can efficiently bind either IL-4 or IL-13, and that in this context IL-4 and IL-13 can compete for the same binding sites. All known cytokines of the cytokine receptor superfamily use multimerized forms (homodimers, heterodimers, or higher order oligomers) of receptors for signaling. According to the current understanding, one receptor chain functions as a primary ligand-binding protein, and the accessory receptor chain(s) are then recruited to form the functional multimerized receptor complex, which has a higher affinity for the ligand than the primary binding subunit. For IL-4 and IL-13, it is clear from the affinity-labeling experiments that IL-4 more efficiently cross-links to IL-4R␣, whereas IL-13 more efficiently cross-links to IL-13R␣. The weak binding of IL-13 to cells expressing human IL-13R␣ was therefore unexpected, since IL-4 shows substantial binding to IL-4R␣. In contrast, human IL-13 needs the expression of both IL-13R␣ and IL-4R␣ for efficient binding. The relatively poor binding of IL-13 for IL-13R␣ suggests that IL-4R␣ and IL-13R␣ may have certain affinity for each other and that a heterodimer of these chains might exist in the absence of ligand. The development of antibodies will facilitate the ability to address this question. In this regard, it is interesting that in addition to ligand binding sites, the growth hormone receptor also contains a receptor-receptor dimerization surface (site 3) (21). Although site 3 is not sufficient by itself to allow growth hormone receptor dimerization, in the presence of ligand it significantly stabilizes the receptor dimer. It remains to be determined whether IL-4R␣ and IL-13R␣ can interact in the absence of ligand.
It is useful to also consider the possibility that the receptor I-IL-13 with and without a 500-fold molar excess of IL-13 for the indicated times at 4°C. Specific binding was calculated by subtracting the bound counts in presence of excess IL-13 from the counts bound in its absence. Binding for Scatchard plot analysis was performed at 4°C for 6 h; bound and free radioactivity were determined and analyzed using the LIGAND computer program. A displacement curve (B) and Scatchard transformation (C) representative of four experiments are shown. In the experiment shown, the K d was 400 pM; the range was approximately 300 -600 pM.
FIG. 7. IL-13R␣ does not cooperate with IL-2R␤ to bind IL-2. COS-7 cells were transfected with the indicated plasmids, incubated with 2 nM 125 I-IL-2 in the presence or absence of a 500-fold molar excess of unlabeled IL-2 or IL-13, and cross-linked with BS 3 , and affinitylabeled proteins were then analyzed on SDS gels. Gels were visualized on a PhosphorImager.
FIG. 8. Localization of the human IL-13R␣ gene to chromosome X. A panel of 24 DNA samples from mostly monochromosomal somatic cell hybrids were analyzed by PCR using primers specific for human IL-13R␣. A specific band of 148 bp was amplified reproducibly from two hybrids, one containing only human chromosome X and the other containing both human chromosomes 1 and X, indicating that the gene encoding human IL-13R␣ is located on human chromosome X. The human chromosomes contained in each hybrid are indicated. As controls, human genomic DNA (hu.gen.DNA) and murine (Mu) and human (Hu) IL-13R␣ cDNAs were included as templates for the PCR reactions.
for IL-13 may contain a third, as of yet unrecognized, receptor component. A precedent for such a model is provided by analogy to the IL-2 receptor system in which IL-2 binds very poorly to IL-2R␤ (22,23), even though this binding is a prerequisite for interaction of the common ␥ c . However, IL-2 binding to IL-2R␤ is greatly facilitated by its binding to IL-2R␣ (24), a receptor-binding protein that is not part of the cytokine receptor superfamily (25) but which has a much faster on rate for IL-2 than IL-2R␤ (26,27). It is conceivable that such an additional receptor component could also exist for IL-13, although at present, no definitive biochemical data indicate the existence of such a protein.
IL-4R␣ can form a functional IL-4 receptor in combination with ␥ c (8,9). The fact that both IL-13R␣ and ␥ c can interact with IL-4R␣ to form functional IL-4 receptors implies that IL-13R␣ and ␥ c may share at least one epitope for IL-4 binding, even though the extracellular domains of ␥ c and IL-13R␣ share only 18% sequence identity (overall homology is 14%). Since IL-2 and IL-4 share related structures containing four ␣ helices in an "up-up-down-down" configuration (28), and it is reasonable to assume that they contact similar regions of ␥ c , we asked whether IL-13R␣ might also share an epitope with ␥ c for IL-2 binding. However, affinity-labeling studies showed that 125 I-IL-2 could not bind to COS cells when these cells were transfected with IL-13R␣ together with IL-2R␤, although, as expected, it could affinity label cells transfected with both IL-2R␤ and ␥ c (Fig. 7). These data show that IL-13R␣ binding is specific for IL-4 and IL-13. However, in addition to their being similar in size, it is noteworthy that the genes encoding ␥ c and IL-13R␣ are both located on the X chromosome (Fig. 8). In view of the expression of IL-13R␣ in multiple tissues, it is conceivable that mutations in IL-13R␣ may result in a severe or even lethal phenotype. This will be clarified by targeted deletion of IL-13R␣ in mice.