An Interleukin (IL)-13 Receptor Lacking the Cytoplasmic Domain Fails to Transduce IL-13-Induced Signals and Inhibits Responses to IL-4
1997; Elsevier BV; Volume: 272; Issue: 36 Linguagem: Inglês
10.1074/jbc.272.36.22940
ISSN1083-351X
AutoresPatricia L. Orchansky, Sheila D. Ayres, Douglas J. Hilton, John W. Schrader,
Tópico(s)Heat shock proteins research
ResumoInterleukin (IL)-13 is a pleiotropic immunoregulatory cytokine that shares many, although not all, of the biological activities of IL-4. The overlapping biological properties of IL-4 and IL-13 appear to be due to the existence of shared components of the receptors, and we and others showed that the IL-4 receptor-α is involved in signal transduction paths activated by both. We show here that expression of the IL-13 receptor-α in two factor-dependent cell lines, the premyeloid FD5 and the T lymphoid CT4.S, conferred the ability to grow continuously in response to IL-13; to respond to IL-13 with tyrosine phosphorylation of JAK1, Tyk2, IL-4Rα, IRS-2, and STAT6; and to respond to IL-4 with tyrosine phosphorylation of Tyk2 in addition to those induced in parental cell lines. Expression of a truncated IL-13 receptor-α that lacked the cytoplasmic domain demonstrated that this domain was essential for IL-13-dependent growth and phosphorylation of the above substrates. Expression of this truncated IL-13 receptor also resulted in an inhibition of biochemical and biological responses to IL-4 that was exacerbated by the presence of IL-13. These dominant inhibitory effects indicate that the extracellular domain of the truncated IL-13 receptor competes with γc for complexes of IL-4 and the IL-4 receptor-α, or, when itself bound to IL-13, competes with IL-4 for the IL-4 receptor-α. Interleukin (IL)-13 is a pleiotropic immunoregulatory cytokine that shares many, although not all, of the biological activities of IL-4. The overlapping biological properties of IL-4 and IL-13 appear to be due to the existence of shared components of the receptors, and we and others showed that the IL-4 receptor-α is involved in signal transduction paths activated by both. We show here that expression of the IL-13 receptor-α in two factor-dependent cell lines, the premyeloid FD5 and the T lymphoid CT4.S, conferred the ability to grow continuously in response to IL-13; to respond to IL-13 with tyrosine phosphorylation of JAK1, Tyk2, IL-4Rα, IRS-2, and STAT6; and to respond to IL-4 with tyrosine phosphorylation of Tyk2 in addition to those induced in parental cell lines. Expression of a truncated IL-13 receptor-α that lacked the cytoplasmic domain demonstrated that this domain was essential for IL-13-dependent growth and phosphorylation of the above substrates. Expression of this truncated IL-13 receptor also resulted in an inhibition of biochemical and biological responses to IL-4 that was exacerbated by the presence of IL-13. These dominant inhibitory effects indicate that the extracellular domain of the truncated IL-13 receptor competes with γc for complexes of IL-4 and the IL-4 receptor-α, or, when itself bound to IL-13, competes with IL-4 for the IL-4 receptor-α. Interleukin (IL) 1The abbreviations used are: IL, interleukin; IL-13Rα, IL-13 receptor α chain; IL-4Rα, IL-4 receptor α chain; γc, γ common chain of IL-2, IL-4, IL-7, IL-9, and IL-15 receptors; IRS-2, insulin receptor substrate-2; STAT6, signal transducer and activator of transcription-6; LCCM, L cell conditioned media; IL-4-CM, IL-4-containing conditioned medium; CSF, colony-stimulating factor; GM-CSF, granulocyte-macrophage colony-stimulating factor; JAK, Janus kinase; Ab, antibody; mAb, monoclonal antibody; kb, kilobase pair(s); FCS, fetal calf serum; FITC, fluorescein isothiocyanate.-13, a pleiotropic immune regulatory cytokine, shares structural and biological characteristics with IL-4. 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We report here the crucial role of IL-13Rα, not only in IL-13- but also in IL-4-induced responses. Transfection of IL-13Rα cDNA in FD5, a premyeloid cell line, or CT4.S, a T cell line, resulted in continuous IL-13-dependent growth and IL-13-dependent phosphorylation of JAK1, Tyk2, IL-4Rα, IRS-2, and STAT6. In contrast, a truncated IL-13Rα, encoding the extracellular and transmembrane domains but lacking the cytoplasmic domain, failed to mediate detectable biological or biochemical responses to IL-13. Moreover, cells overexpressing this truncated IL-13Rα showed a greatly decreased response to IL-4 in terms of JAK1, JAK3, Tyk2, IL-4Rα, IRS-2, and STAT6 phosphorylation and proliferation. This effect was exacerbated by the presence of IL-13 and reflected sequestration of the IL-4Rα into sterile complexes of the truncated IL-13Rα and IL-4 or IL-13. These results indicate that the cytoplasmic domain of IL-13Rα is necessary for signaling by IL-13 and in some cases IL-4 and that the extracellular domain of the IL-13Rα interacts with the extracellular domain of the IL-4Rα in the presence of either IL-4 or IL-13. FD5 cells (41Gliniak B.C. Rohrschneider L.R. Cell. 1990; 63: 1073-1083Abstract Full Text PDF PubMed Scopus (84) Google Scholar) are a subclone of the premyeloid cell line FDMACII that are dependent upon IL-3, IL-4, GM-CSF, or CSF-1 for proliferation. They were passaged in RPMI 1640 medium, supplemented with 10% fetal bovine serum, 20 μm 2-mercaptoethanol, 100 units of penicillin/streptomycin, and either 3% (v/v) IL-4-containing conditioned medium (IL-4-CM) or 2% L cell conditioned medium (LCCM) containing CSF-1. CT4.S cells, an IL-2-responsive derivative of the original IL-4-dependent CT4.S cell line (42Hu L. Ohara J. Watson C. Tsang W. Paul W.E. J. Immunol. 1989; 142: 800-807PubMed Google Scholar), were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 20 μm2-mercaptoethanol, 100 units of penicillin/streptomycin, and either 5% (v/v) IL-4-CM or 2% (v/v) IL-2-containing conditioned media (IL-2-CM), derived in each case from AgX063 cells transfected with cDNA encoding the respective cytokine (43Karasuyama H. Melchers F. Eur. J. Immunol. 1988; 18: 97-104Crossref PubMed Scopus (1081) Google Scholar). Crude preparations of chemically synthesized IL-4, IL-13, and GM-CSF were kindly provided by Dr. I. Clark-Lewis (The Biomedical Research Center, University of British Columbia, Vancouver, Canada). Antiserum against IRS-2 was a generous gift of Dr. J. H. Pierce (NIH, Bethesda, MD). Antiserum against STAT6 and Tyk2 were obtained from Santa Cruz Biotechnology. Antiserum against JAK1, JAK2, and JAK3 and mAb against phosphotyrosine (4G10) were obtained from Upstate Biotechnology Inc. Anti-murine IL-4Rα mAb was purchased from Genzyme, and anti-FLAG mAb was purchased from Eastman Kodak Co. Electrophoretic chemicals were obtained from Bio-Rad, and restriction enzymes were from New England BioLabs. The full-length cDNA sequence encoding the mouse IL-13Rα was cloned into the mammalian expression vector pEF-BOS (44Mizushima S. Nagata S. Nucleic Acids Res. 1990; 18: 5322Crossref PubMed Scopus (1499) Google Scholar) (pEB-13R) under the transcriptional control of the elongation factor-1α promoter and poly(A) adenylation signal from human granulocyte colony-stimulating factor. The coding sequence was preceded by the IL-3 signal sequence and an N-terminal FLAG epitope tag sequence (16Hilton D.J. Zhang J.G. Metcalf D. Alexander W.S. Nicola N.A. Willson T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 497-501Crossref PubMed Scopus (390) Google Scholar). The cDNA encodes the extracellular domain of IL-13Rα (amino acids 27–340), the transmembrane region (amino acids 341–364), and the intracellular domain (amino acids 365–424) (Fig.1 A). The receptor sequence encoding the cytoplasmic domain was removed from pEB-13R by digestion with AflII. Three fragments were generated, one of 4.2 kb encoding most of the vector, another of 1.9 kb encoding a small region of the vector together with the extracellular and transmembrane receptor coding sequences, and a third fragment of 0.5 kb encoding the cytoplasmic domain sequence and part of the poly(A). The fragments of 4.2 and 1.9 kb were purified from 0.8% agarose gels; blunt-ended with Klenow polymerase, creating a new in-frame stop codon downstream of the transmembrane sequence; and ligated to each other. The new plasmid (pEB-13RΔCD) encodes the entire extracellular and transmembrane receptor sequences and the two membrane-proximal amino acid residues of the cytoplasmic domain (Lys, Arg) (Fig. 1 B). The identity of the construct was confirmed by restriction mapping and sequencing. FD5 and CT4.S cells, grown in RPMI containing 10% FCS, 3% IL-4-CM, or Dulbecco's modified Eagle's medium containing 10% FCS, 5% IL-4-CM, respectively, were washed and resuspended in transfection buffer (25 mm Hepes, 0.75 mmNa2HPO4, 140 mm KCl, 5 mm NaCl, 2 mm MgCl2, 0.5% Ficoll 400) at a concentration of 1.3 × 107 cells/ml. For each transfection, 1 × 107 cells were mixed with 1 μg of pPGK/Neo, a plasmid conferring neomycin resistance, alone or together with 10 μg of either pEB-13R or pEB-13RΔCD cDNA and were subjected to electroporation using a Bio-Rad gene pulser at 960 microfarads and 280 V or 300 V for FD5 or CT4.S, respectively. In parallel, 107 cells were electroporated without DNA to subsequently monitor neomycin-induced death. After transfection, these groups of cells were cultured in the appropriate media for 48 h and then transferred to selection media in 96-well plates at 104 cells/well. FD5 cells were selected in RPMI containing 10% FCS, 100 μg/ml G418, and 2% LCCM, to avoid selection for responsiveness to IL-4. CT4.S cells were selected in Dulbecco's modified Eagle's medium containing 10% FCS, 1 mg/ml G418, and 2% IL-2-CM to avoid inhibitory effects of IL-4. Individual colonies of neomycin-resistant cells were cloned and propagated for assays. Cells of individual clones were tested for expression of full-length (FD5–13R or CT4.S-13R) or truncated IL-13Rα (FD5–13RΔ or CT4.S-13RΔ) by FACScan analysis using M2, a mAb against FLAG, and goat-anti-mouse IgG-FITC and confirmed by immunoprecipitation and immunoblotting with anti-FLAG mAb. A representative clone from each cell population was recloned by limit dilution and used for further experiments. Cytokine-induced proliferation of cells was assessed by [3H]thymidine incorporation intode novo synthesized DNA (45Schrader J.W. Ziltener H.J. Leslie K.B. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 2458-2462Crossref PubMed Scopus (33) Google Scholar), cell counting, and microscopic visualization. For assessment of DNA synthesis, cells were washed three times with Hanks' buffered salts supplemented with 2% (v/v) FCS. CT4.S or their transfectants were plated at 500 cells/Terisaki well, and varying concentrations of factor were added from a dilution series. Each point was repeated in triplicate. Cells were incubated at 37 °C for 5 days, pulsed for 6 h with [3H]thymidine at a final concentration of 15 μCi/ml, harvested, and counted on a scintillation counter. Results were expressed as a percentage of the maximal [3H]thymidine incorporation observed in cultures stimulated with 5% IL-2-CM. To determine long term growth and morphological changes, parental and transfected cells were plated at low density with different factors and counted, and in some cases photographed, at regular intervals. To investigate the biochemical effects of stimulation with different cytokines, selected clones of FD5, FD5–13R, and FD5–13RΔ were placed in RPMI, 10% FCS, 0.2% LCCM for 16 h, washed twice with serum/factor-free media containing 20 mmHepes, and incubated at 1–3 × 107 cells/ml of the same media at 37 °C for 1 h. Cells were then stimulated at 37 °C with either synthetic IL-4 (20 μg/ml), synthetic IL-13 (20 μg/ml), synthetic GM-CSF (10 μg/ml) for 10 min, recombinant insulin (5 μg/ml) for 2 min, or left untreated as control. Cells were lysed in lysis buffer (20 mm Tris, pH 7.6, 150 mmNaCl, 1% Nonidet P-40, 2 mm EDTA, 1 mm sodium orthovanadate, 1 mm sodium molybdate, 10 mmsodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 0.7 μg/ml pepstatin, 10 μg/ml aprotinin). Lysates were cleared by centrifugation and incubated with antisera against STAT6, IRS-2, JAK1, JAK2, JAK3, or Tyk2 or with mAbs against IL-4Rα or FLAG for 2 h at 4 °C followed by an additional hour with Protein A- or Protein G-Sepharose. The beads were washed three times with cold lysis buffer containing decreasing concentrations of detergent and heated in Laemmli sample buffer (46Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207529) Google Scholar) containing dithiothreitol at a final concentration of 40 mm. The samples were subjected to SDS-polyacrylamide gel electrophoresis using 7.5 or 8.5% polyacrylamide gels. The gels were then equilibrated in transfer buffer (20 mm Tris, 150 mm glycine, 20% methanol) and transferred to polyvinylidene difluoride membranes by electrophoresis toward the anode at 200 mA for 2 h in a Bio-Rad transblot apparatus (47Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44939) Google Scholar). The polyvinylidene difluoride membranes were blocked in 4% bovine serum albumin in TBST (10 mm Tris, pH 7.5, 150 mm NaCl, 0.1% Tween 20) for 4–16 h at room temperature. The blots were then washed twice with TBST and incubated for 2 h with anti-Tyr(P) mAb (0.25 μg/ml) or anti-FLAG mAb (1 μg/ml) in TBST, 1% bovine serum albumin. The blots were thoroughly washed with TBST and incubated for 1 h with goat anti-mouse IgG antibody-conjugated to horseradish peroxidase (DAKO A/S, Denmark) diluted 1:10,000 in TBST. The blots were thoroughly washed and subsequently developed with the enhanced chemiluminescence assay as described by the manufacturer (ECL kit, Amersham Corp.). Afterward, blots were stripped with erasing buffer (62.5 mm Tris-Cl, pH 6.8, 2% SDS, 100 mm 2-mercaptoethanol) for 1 h at 55 °C, washed twice with TBST, blocked with 4% bovine serum albumin in TBST, and incubated for 2 h with antisera against STAT6 (1:2000), IRS-2 (1:2000), JAK1 (1:1000), JAK2 (1:2000), JAK3 (1:1000), or Tyk2 (1:1000) in TBST, 1% bovine serum albumin. The blots were washed several times in TBST and incubated for 1 h in goat anti-rabbit IgG antibody-conjugated to horseradish peroxidase (DAKO A/S, Denmark), 1:10,000 in TBST. Proteins were detected as described previously. The expression plasmid pEB-13R containing the coding sequence for the full-length IL-13Rα cDNA (16Hilton D.J. Zhang J.G. Metcalf D. Alexander W.S. Nicola N.A. Willson T.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 497-501Crossref PubMed Scopus (390) Google Scholar) (Fig.1 A) was digested withAflII to create the expression plasmid pEB-13ΔCD containing the extracellular, transmembrane and two membrane-proximal amino acid sequences of IL-13Rα cDNA under the transcriptional control of the elongation factor-1α promoter (Fig. 1 B). The truncated and full-length receptor expression plasmids were used to transfect FD5 and CT4.S cells, both of which are unresponsive to IL-13 but grow continuously in IL-4. Transfectants were grown in LCCM or IL-2-CM, respectively, rather than IL-4, to avoid selection for IL-4 responsiveness, which could complicate interpretation of results. Twelve neomycin-resistant colonies from each of the transfections with cDNA encoding the full-length or the truncated IL-13Rα were individually analyzed for IL-13Rα expression by fluorometry using anti-FLAG mAb and FITC conjugated to goat-anti-mouse antibody. Of the colonies of FD5 transfectants, 7 of 12 were positive for expression of the full-length IL-13Rα, and 5 of 12 were positive for expression of the truncated IL-13Rα. Both forms of the IL-13Rα were expressed as surface proteins, although in all five clones expressing IL-13RΔCD, the transfected receptor was expressed at levels that were 10–20-fold higher than those seen in all seven cl
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