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• W2020199484 abstract "The gp130-like receptor (GPL) is a recently cloned member of the family of type I cytokine receptors. The name reflects its close relationship to gp130, the common receptor subunit of the interleukin (IL)-6-type cytokines. Indeed, the recently proposed ligand for GPL, IL-31, is closely related to the IL-6-type cytokines oncostatin M, leukemia inhibitory factor, and cardiotrophin-1. The second signal transducing receptor for IL-31 seems to be the oncostatin M receptor β (OSMRβ). The present study characterizes in depth the molecular mechanisms underlying GPL-mediated signal transduction. GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identify tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of GPL (GPL745) as the major STAT5- and STAT3-activating sites, respectively. Additionally, we demonstrate Jak1 binding to GPL and its activation in heteromeric complexes with the OSMRβ but also in a homomeric receptor complex. Most interesting, unlike OSMRβ and gp130, GPL is insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of recruitment of the tyrosine phosphatase SHP-2 or the adaptor protein Shc to the cytoplasmic domain of GPL. The gp130-like receptor (GPL) is a recently cloned member of the family of type I cytokine receptors. The name reflects its close relationship to gp130, the common receptor subunit of the interleukin (IL)-6-type cytokines. Indeed, the recently proposed ligand for GPL, IL-31, is closely related to the IL-6-type cytokines oncostatin M, leukemia inhibitory factor, and cardiotrophin-1. The second signal transducing receptor for IL-31 seems to be the oncostatin M receptor β (OSMRβ). The present study characterizes in depth the molecular mechanisms underlying GPL-mediated signal transduction. GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identify tyrosine residues 652 and 721 in the cytoplasmic region of the longest isoform of GPL (GPL745) as the major STAT5- and STAT3-activating sites, respectively. Additionally, we demonstrate Jak1 binding to GPL and its activation in heteromeric complexes with the OSMRβ but also in a homomeric receptor complex. Most interesting, unlike OSMRβ and gp130, GPL is insufficient to mediate ERK1/2 phosphorylation. We propose that this is due to a lack of recruitment of the tyrosine phosphatase SHP-2 or the adaptor protein Shc to the cytoplasmic domain of GPL. The recently identified novel cytokine receptor gp130-like monocyte receptor/gp130-like receptor (GPL) 1The abbreviations used are: GPL, gp130-like receptor; gp130, glycoprotein 130; LIFR, leukemia inhibitory factor receptor; OSMR, oncostatin M receptor; Jak, Janus kinase; STAT, signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase; α2M, α2-macroglobulin; IRF, interferon regulatory factor; SH2, Src homology; SIE, sis-inducible element; CIS, cytokine inducible SH2 protein; IL, interleukin; FACS, fluorescence-activated cell sorter.1The abbreviations used are: GPL, gp130-like receptor; gp130, glycoprotein 130; LIFR, leukemia inhibitory factor receptor; OSMR, oncostatin M receptor; Jak, Janus kinase; STAT, signal transducer and activator of transcription; MAPK, mitogen-activated protein kinase; ERK, extracellular signal regulated kinase; α2M, α2-macroglobulin; IRF, interferon regulatory factor; SH2, Src homology; SIE, sis-inducible element; CIS, cytokine inducible SH2 protein; IL, interleukin; FACS, fluorescence-activated cell sorter. (1Ghilardi N. Li J. Hongo J.A. Yi S. Gurney A. de Sauvage F.J. J. Biol. Chem. 2002; 277: 16831-16836Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) belongs to the family of type I cytokine receptors. It shares their common structural motifs, i.e. the cytokine-binding module with two pairs of conserved cysteine residues and a WSXWS motif in the extracellular region (3Bazan J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6934-6938Crossref PubMed Scopus (1878) Google Scholar). It has a single transmembrane domain and an intracellular region without apparent intrinsic enzymatic activity. Four different membrane-spanning splice variants of the receptor have been described (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), three of which contain a conserved proline-rich sequence (box 1) in their membrane-proximal cytoplasmic parts, required in most cytokine receptors for binding of tyrosine kinases of the Janus family (4Tanner J.W. Chen W. Young R.L. Longmore G.D. Shaw A.S. J. Biol. Chem. 1995; 270: 6523-6530Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 5Goldsmith M.A. Xu W. Amaral M.C. Kuczek E.S. Greene W.C. J. Biol. Chem. 1994; 269: 14698-14704Abstract Full Text PDF PubMed Google Scholar, 6Lebrun J.J. Ali S. Ullrich A. Kelly P.A. J. Biol. Chem. 1995; 270: 10664-10670Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). The longest isoform (745 amino acids) contains three intracellular tyrosine motifs; the first and third tyrosine is conserved between human and murine GPL (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Besides the transmembrane isoforms, a soluble GPL has been proposed (CRL3; GenBank™ accession number AF106913-1). Transcripts for GPL have been found in all cells of the myelomonocytic lineage (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) and of the epithelium from skin, lung, and prostate as well as in activated CD4+ and CD8+ T cell subsets (7Gross J.F. Sprecher C. Hammond A. Dillon S. Dasovich M. Kuijper J. Kramer J. Grant F. Presnell S. Gao Z. Whitmore T. Kuestner R. Maurer M. Bilsborough J. Harder B. Rosenfeld-Franklin M. Mudri S. Johnson J. Clegg C. Ren H.-P. Waggie K. Shea P. Dong D. Bukowski T.R. Parrish-Novak J. Foster D. Eur. Cytokine Netw. 2003; 14 (A308): 109PubMed Google Scholar). Additionally, GPL is highly expressed in tissues involved in reproduction, particularly in testis (1Ghilardi N. Li J. Hongo J.A. Yi S. Gurney A. de Sauvage F.J. J. Biol. Chem. 2002; 277: 16831-16836Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The closest mammalian relative of GPL is gp130 (8Hibi M. Murakami M. Saito M. Hirano T. Taga T. Kishimoto T. Cell. 1990; 63: 1149-1157Abstract Full Text PDF PubMed Scopus (1097) Google Scholar), the common receptor subunit of the interleukin(IL)-6-type cytokines; GPL and gp130 share 28% sequence homology (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The GPL gene (gpl) is located in tandem to the gp130 gene (gp130) on chromosome 5 with opposite transcriptional orientations (1Ghilardi N. Li J. Hongo J.A. Yi S. Gurney A. de Sauvage F.J. J. Biol. Chem. 2002; 277: 16831-16836Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The common intron/exon organization of both genes (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 9Szalai C. Toth S. Falus A. Gene (Amst.). 2000; 243: 161-166Crossref PubMed Scopus (4) Google Scholar) may suggest evolution of this cytokine receptor by a gene duplication event. Like gp130, the extracellular domain organization of GPL displays five predicted fibronectin type III-like domains (D1–D5); D1 and D2 comprise the cytokine-binding module. However, it lacks the Ig-like domain present at the N terminus of gp130 (1Ghilardi N. Li J. Hongo J.A. Yi S. Gurney A. de Sauvage F.J. J. Biol. Chem. 2002; 277: 16831-16836Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Studies on the IL-6/IL-12 family of cytokine receptors demonstrated that the N-terminal Ig-like domain contributes to binding of many cytokines (10Grötzinger J. Biochim. Biophys. Acta. 2002; 1592: 215-223Crossref PubMed Scopus (70) Google Scholar, 11Boulanger M.J. Chow D.C. Brevnova E.E. Garcia K.C. Science. 2003; 300: 2101-2104Crossref PubMed Scopus (475) Google Scholar). Thus it seems unlikely that GPL functions in a homomeric receptor complex like the erythropoietin or the thrombopoietin receptor. Indeed, recently published work describes GPL as part of the receptor complex for a novel four-helix bundle cytokine, IL-31 (7Gross J.F. Sprecher C. Hammond A. Dillon S. Dasovich M. Kuijper J. Kramer J. Grant F. Presnell S. Gao Z. Whitmore T. Kuestner R. Maurer M. Bilsborough J. Harder B. Rosenfeld-Franklin M. Mudri S. Johnson J. Clegg C. Ren H.-P. Waggie K. Shea P. Dong D. Bukowski T.R. Parrish-Novak J. Foster D. Eur. Cytokine Netw. 2003; 14 (A308): 109PubMed Google Scholar, 12Dillon S. Haugen H. Kim K. Bontadelli K. Cutler D. Mudri S. Bilsborough J. Waggie K. Ren H.-P. Maurer M. Harder B. Rosenfeld-Franklin M. Sprecher C. Parrish-Novak J. Hammond A. Dasovich M. Johnston J. Lockwood L. Chen Z. Clegg C. Foster D. Gross J. Eur. Cytokine Netw. 2003; 14: 223Google Scholar). IL-31 seems most closely related to oncostatin M, leukemia inhibitory factor, and cardiotrophin-1 (CT-1) (7Gross J.F. Sprecher C. Hammond A. Dillon S. Dasovich M. Kuijper J. Kramer J. Grant F. Presnell S. Gao Z. Whitmore T. Kuestner R. Maurer M. Bilsborough J. Harder B. Rosenfeld-Franklin M. Mudri S. Johnson J. Clegg C. Ren H.-P. Waggie K. Shea P. Dong D. Bukowski T.R. Parrish-Novak J. Foster D. Eur. Cytokine Netw. 2003; 14 (A308): 109PubMed Google Scholar), all of which belong to the family of IL-6-type cytokines (13Heinrich P.C. Behrmann I. Haan S. Hermanns H.M. Müller-Newen G. Schaper F. Biochem. J. 2003; 374: 1-20Crossref PubMed Scopus (2503) Google Scholar). Besides GPL (IL-31Rα), the signaling receptor complex for IL-31 contains the oncostatin M receptor β (OSMRβ), another signaling receptor subunit of the IL-6-type cytokines (14Mosley B. De Imus C. Friend D. Boiani N. Thoma B. Park L.S. Cosman D. J. Biol. Chem. 1996; 271: 32635-32643Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar). In the present study we characterize some signaling properties of GPL. In a homomeric as well as heteromeric receptor complex with OSMRβ or gp130, GPL is a strong activator of STAT3 and STAT5, whereas STAT1 is only poorly tyrosine-phosphorylated. We identify tyrosines 652 and 721 in the cytoplasmic region of GPL as the activation sites for STAT5 and STAT3, respectively. GPL recruits Jak1, which is strongly tyrosine-phosphorylated upon receptor activation. However, the receptor fails to recruit the adaptor molecules SHP-2 or Shc, recently shown to be involved in gp130- and OSMR-mediated MAPK activation, respectively (15Stahl N. Farruggella T.J. Boulton T.G. Zhong Z. Darnell Jr., J.E. Yancopoulos G.D. Science. 1995; 267: 1349-1353Crossref PubMed Scopus (866) Google Scholar, 16Hermanns H.M. Radtke S. Schaper F. Heinrich P.C. Behrmann I. J. Biol. Chem. 2000; 275: 40742-40748Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). Subsequently, we demonstrate that GPL can only activate ERK1/2 when oligomerized with OSMRβ or gp130, but not in a homomeric arrangement. Cell Culture and Transient Transfection—Human hepatoma cells (HepG2) were maintained in Dulbecco's modified Eagle's medium/F-12 and HEK293T cells in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 100 mg/liter streptomycin, and 60 mg/liter penicillin. Cells were grown at 37 °C in a water-saturated 5% CO2 atmosphere. HepG2 cells were transiently transfected with 10–20 μg of plasmid DNA by using the calcium phosphate method as described (17Schaper F. Gendo C. Eck M. Schmitz J. Grimm C. Anhuf D. Kerr I.M. Heinrich P.C. Biochem. J. 1998; 335: 557-565Crossref PubMed Scopus (142) Google Scholar). Briefly, cells were washed twice with phosphate-buffered saline 1 h before transfection, and their culture medium was changed to Dulbecco's modified Eagle's medium. For transfection, 62 μl of 2 m CaCl2 was added to the DNA. The solution was then mixed with 500 μl of 2× HBS (10g/liter Hepes, 16 g/liter NaCl, 0.74 g/liter KCl, 0.25 g/liter NaH2PO4·H2O, 2 g/liter glucose, pH 7.05) and added to 10 ml of Dulbecco's modified Eagle's medium. The reaction mixture was dispensed onto a 6-well plate. HEK293T cells were transiently transfected with 3.5–15 μg of plasmid DNA using FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. Cloning of GPL—5′ and 3′ GPL fragments were cloned separately from human ovary Marathon-Ready™ cDNA by rapid amplification of cDNA ends using the Advantage cDNA polymerase (Clontech, Palo Alto, CA) and touchdown PCR. After the full open reading frame, including 3′- and 5′-nontranslated sequences, had been determined by rapid amplification of cDNA ends, gene-specific 3′- and 5′-primers were used to clone full-length GPL cDNA. The primers used amplified the longest possible open reading frame of the receptor, M1KLSP5-738PEH-TKGEV745 (GPL745). The obtained sequence for GPL745 matches the one recently published by Diveu et al. (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The GPL cDNA was then cloned into PCR2.1-TOPO (Invitrogen). For transfer to expression vectors, the following oligonucleotides were used: 5′-GTTGTAAAGCTTCCTGATACatgaagctctctccc-3′ (sense); 5′-GCAGCAGAATTCttagacttctcccttggtgtgctctg-3′ (antisense). The coding sequence is written in lowercase letters. Expression Constructs—The construction of the pSVL-based expression vectors for the IL-5 receptor-based chimeras β/gp130, β/gp130-B1/2, β/OSMRΔ1, β/OSMR-B1/2, and α/gp130-YFFFFF has been described previously (16Hermanns H.M. Radtke S. Schaper F. Heinrich P.C. Behrmann I. J. Biol. Chem. 2000; 275: 40742-40748Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 18Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 19Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar). The expression vectors pSVL-huIL-5Rα/huGPL-(530–745) (α/GPL-(530–745)) and pSVL-huIL-5Rβ/huGPL-(530–745) (β/GPL-(530–745)) were constructed by exchanging the cDNA for the transmembrane and intracellular region of gp130 by the corresponding sequence for GPL (amino acids 530–745), which was obtained using standard PCR and the oligonucleotides 5′-CCGGAATTCgtctttgagattatcctc-3′ (sense) and 5′-CGCGGATCCttagacttctcccttgg-3′ (antisense). The coding sequence is written in lowercase letters; the recognition sequences for the restriction enzymes EcoRI and BamHI are underlined. Our chimeric IL-5R/GPL constructs differ slightly from the ones recently described by Diveu et al. (2Diveu C. Lelievre E. Perret D. Lak-Hal A.H. Froger J. Guillet C. Chevalier S. Rousseau F. Wesa A. Preisser L. Chabbert M. Gauchat J.F. Galy A. Gascan H. Morel A. J. Biol. Chem. 2003; 278: 49850-49859Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) (GPL-(524–745)). The point mutated constructs containing the amino acid substitutions Y652F, Y683F, and Y721F (Fig. 1) were generated by PCR using the appropriate oligonucleotides with either the cDNA for pSVL-β/GPL-(530–745) or pSVL-α/GPL-(530–745) as a template and the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). The truncated construct pSVL-β/GPL-(530–626) was obtained by PCR by using a 3′-oligonucleotide incorporating an in-frame termination codon followed by the recognition site for BamHI. The resulting PCR product was inserted into the EcoRI- and BamHI-digested expression plasmid pSVL-β/GPL-(530–745). The integrity of all constructs was verified by DNA sequence analyses using an ABI PRISM 310 Genetic Analyzer (PerkinElmer Life Sciences). For better expression in transfected HepG2 cells, XhoI/BamHI fragments comprising the cDNA encoding α/GPL-(530–745) and β/GPL-(530–745) were inserted into XhoI/BglII-digested pCAGGS expression vector (20Nakajima K. Yamanaka Y. Nakae K. Kojima H. Ichiba M. Kiuchi N. Kitaoka T. Fukada T. Hibi M. Hirano T. EMBO J. 1996; 15: 3651-3658Crossref PubMed Scopus (521) Google Scholar). The expression plasmid for Jak1 was kindly provided by Dr. I. M. Kerr (London Research Institute, Cancer Research UK, London, UK). Cell Lysis, Immunoprecipitations, and Western Blotting—Transfected HEK293T cells were stimulated for 15 min with 20 ng/ml recombinant human IL-5 (Cell Concepts, Umkirch, Germany). Immediately after stimulation, cells were lysed in Triton lysis buffer (20 mm Tris, pH 7.5, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 10 mm NaF, 1 mm Na3VO4, 1 mm phenylmethylsulfonyl fluoride, 5 μg/ml aprotinin, 3 μg/ml pepstatin, and 5 μg/ml leupeptin), scraped off the dish, and left on ice for 30 min. Lysates were centrifuged with 14,000 rpm for 10 min at 4 °C. Equal amounts of cellular protein were separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane. Antibodies raised against Jak1 and IL-5Rβ were used for immunoprecipitation. As reported earlier, cotransfection of Jak1 (1 μg) led to a ligand-independent tyrosine phosphorylation of the cytoplasmic regions of the various receptors (16Hermanns H.M. Radtke S. Schaper F. Heinrich P.C. Behrmann I. J. Biol. Chem. 2000; 275: 40742-40748Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar). After incubation overnight at 4 °C, immune complexes were collected on protein A-Sepharose (Amersham Biosciences) during a 60-min incubation, washed twice with washing buffer (as lysis buffer, but with only 0.1% Triton X-100), and boiled for 5 min in Laemmli buffer at 95 °C. Immune complexes were analyzed further by 7.5% SDS-PAGE. Western blot analysis was conducted using the indicated antibodies and the enhanced chemiluminescence kit (Amersham Biosciences). Before reprobing, blots were stripped in 2% SDS, 100 mm β-mercaptoethanol in 62.5 mm Tris-HCl, pH 6.7, for 20 min at 75 °C. Antibodies—The antibodies against IL-5Rβ (S-16) and Jak1 (HR-785) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used for immunoprecipitation, and the Jak1 antibody was additionally used for Western blots. The antibodies against Shc and STAT3 were purchased from Transduction Laboratories (Lexington, KY); α-Tyr(P) antibody (Tyr(P)-99), α-STAT1 (E-23), α-STAT5B (C-17), α-SHP2 (C-18), and α-IL-5Rβ (N-20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibodies raised against active ERK1/2, tyrosine-phosphorylated STAT1(Tyr(P)-701), STAT3(Tyr(P)-705), STAT5 (Tyr(P)-694), as well as the α-ERK1/2 were obtained from Cell Signaling Technology (Beverly, MA). The horseradish peroxidase-conjugated secondary antibodies were purchased from Dako (Hamburg, Germany). Reporter Gene Assays—α2M(-215)-luciferase contains the promoter region, -215 to +8, of the rat α2-macroglobulin gene upstream of the luciferase-encoding sequence of plasmid pGL3 basic (Promega, Madison, WI). The SIE-tk-Luc construct contains two copies of the STAT consensus binding sequence from the c-fos promoter upstream of a thymidine kinase minimal promoter (21Coqueret O. Gascan H. J. Biol. Chem. 2000; 275: 18794-18800Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and was kindly provided by Dr. H. Gascan (INSERM, Angers, France). The IRF1-tk-Luc construct contains the STAT1-responsive element of the irf1 promoter, and the casein-tk-Luc construct includes six repeated STAT-binding elements of the α-casein promoter upstream of a thymidine kinase minimal promoter cloned into the pGL3 vector (Promega, Madison, WI). The cis promoter-Luc reporter construct was kindly provided by Dr. A. Yoshimura (Kyushu University, Fukuoka, Japan) and has been described recently (22Matsumoto A. Masuhara M. Mitsui K. Yokouchi M. Ohtsubo M. Misawa H. Miyajima A. Yoshimura A. Blood. 1997; 89: 3148-3154Crossref PubMed Google Scholar). It contains ∼540 bases of the upstream region of the cis gene including four STAT5-responsive mammary gland factor boxes. Luciferase activity values were normalized to transfection efficiency monitored by the cotransfected β-galactosidase expression vector pCH110 (Amersham Biosciences). HepG2 cells were transfected with 6 μg of luciferase reporter construct, 2 μg of β-galactosidase control plasmid, and expression vectors for each receptor construct (1 μg of pCAGGS-based vectors; 6 μg of pSVL-based vectors) using the calcium phosphate transfection method. In the case of the casein-tk and the cis promoter reporter gene, 2 μg of STAT5B expression vector were additionally added. HEK293T cells were transfected with 6 μg of the SIE-tk-Luc or the cis promoter-Luc construct, 2 μgof β-galactosidase control plasmid, and 2.5 μg of each receptor expression vector. Transient transfection was carried out with FuGENE 6 transfection reagent (Roche Applied Science) as described in the manufacturer's instructions. Twenty four hours after transfection, cells were stimulated with 10 ng/ml recombinant human IL-5 (Cell Concepts, Umkirch, Germany) for 16 h. Cell lysis and luciferase assays were performed using the Promega luciferase assay system (Promega, Madison, WI). GPL Initiates Signal Transduction in Combination with gp130, OSMRβ, and LIFR—The novel cytokine receptor GPL displays the highest homology to gp130, the common signal transducing receptor of the IL-6-type cytokine family. GPL has been described recently (7Gross J.F. Sprecher C. Hammond A. Dillon S. Dasovich M. Kuijper J. Kramer J. Grant F. Presnell S. Gao Z. Whitmore T. Kuestner R. Maurer M. Bilsborough J. Harder B. Rosenfeld-Franklin M. Mudri S. Johnson J. Clegg C. Ren H.-P. Waggie K. Shea P. Dong D. Bukowski T.R. Parrish-Novak J. Foster D. Eur. Cytokine Netw. 2003; 14 (A308): 109PubMed Google Scholar) to constitute in combination with OSMRβ a functional receptor for the novel cytokine IL-31. Therefore, we first studied whether dimerization of GPL with any of the signal transducing receptors of the IL-6 family (gp130, OSMRβ, or LIFR) generates a signaling-competent receptor complex. To become independent of endogenous receptors, we used chimeric receptor constructs. These contain the transmembrane and intracellular regions of the longest isoform of GPL (amino acids 530–745), gp130, LIFR, or OSMRβ fused to the extracellular domain of the interleukin-5 α or β receptor, respectively. In earlier studies we have demonstrated that chimeras containing the full-length cytoplasmic region of the OSMRβ (β/OSMR) are poorly expressed on the cell surface. Hence, we had to use a truncated chimeric construct that lacks the C-terminal 28 amino acids (β/OSMRΔ1) (19Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar), maintaining STAT recruitment and MAPK-activating sites. Compared with all other constructs used in our study, β/OSMRΔ1 displayed similar surface expression levels. A schematic representation of all receptor constructs used is presented in Fig. 1. We cotransfected expression vectors for α/GPL-(530–745), α/OSMRΔ1, α/LIFR, or α/gp130 along with β/GPL-(530–745) into HepG2 hepatoma cells, known to be highly responsive to IL-6-type cytokines. As a read out for STAT3-mediated gene expression, we cotransfected an α2-macroglobulin promoter-driven luciferase reporter gene. As shown in Fig. 2A, GPL initiated signaling in combination with all three receptor chains (lanes 2–4), whereas the β/GPL-(530–745) chimera alone was not sufficient for signaling, as expected (lane 5). This is in accordance with our earlier studies, which demonstrated that both the α- and β-chimera need to be present containing at least the Jak-binding box1 regions to initiate a signaling cascade (18Behrmann I. Janzen C. Gerhartz C. Schmitz-Van de Leur H. Hermanns H. Heesel B. Graeve L. Horn F. Tavernier J. Heinrich P.C. J. Biol. Chem. 1997; 272: 5269-5274Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). The α/OSMRΔ1+β/GPL-(530–745) (Fig. 2A, lane 2) and α/gp130+β/GPL-(530–745) (lane 4) combinations were most potent, leading to a 60- and 35-fold induction of the luciferase activity, respectively (right side). The α/LIFR+β/GPL-(530–745) heteromer appeared to have the weakest signaling capacity (Fig. 2A, lane 3). Most interesting, the GPL homodimer also initiated a strong activation of the α2M promoter (Fig. 2A, lane 1). Experiments using an irf1 promoter-based reporter gene construct (Fig. 2B), known to be responsive to STAT1 and STAT3, and an α-casein promoter-based reporter (Fig. 2C) as a mainly STAT5-driven promoter resulted in a similar activation pattern. GPL Cytoplasmic Regions Are Sufficient to Induce Jak/STAT Activation When Dimerized with the OSMRβ or gp130 —The reporter gene assays shown in Fig. 2 have clearly demonstrated that GPL-containing receptor complexes are capable of inducing STAT-dependent promoter activity. To corroborate these findings, we next analyzed the signaling capacities of the GPL/OSMR heteromer (Fig. 3A) and the GPL/gp130 heteromer (Fig. 3B) at a molecular level. In order to obtain higher expression levels of transfected receptor chimeras, we used HEK293T cells for these experiments. In general, cell surface expression levels of the various chimeric receptor constructs were measured by FACS analysis using antibodies recognizing the extracellular parts of IL-5Rα or IL-5Rβ (data not shown). Only experiments with comparable expression of all different receptors were evaluated. Like α/OSMRΔ1+β/gp130, a mimic for the functional oncostatin M receptor, the combination α/OSMRΔ1+β/GPL-(530–745) induced Jak1 as well as STAT3 phosphorylation upon IL-5 stimulation, albeit less pronounced (Fig. 3A, left side, 1st and 3rd panels, lanes 1 and 2). The STAT1 phosphorylation induced by α/OSMRΔ1+β/GPL-(530–745) was barely detectable when compared with the level of STAT1 phosphorylation by α/OSMRΔ1+β/gp130 (Fig. 3A, left side, 5th panel, lanes 1 and 2). Similar results were obtained when examining signaling by the α/gp130+β/GPL-(530–745) heteromer; stimulation of this receptor complex triggered substantial STAT3 tyrosine phosphorylation (Fig. 3B, upper panel, lane 2) only slightly weaker than STAT3 activation induced by the homomerized cytoplasmic regions of gp130 (α/gp130+β/gp130) (Fig. 3B, upper panel, lane 1). In this case, STAT1 phosphorylation upon α/gp130+β/GPL-(530–745) complex formation was easily detectable but was weaker than the corresponding signal generated by α/gp130+β/gp130 (Fig. 3B, 3rd panel, compare lanes 1 and 2). To confirm these findings, we next expressed β/GPL-(530–745) together with an OSMR construct that is deleted after its box1/2 domain (α/OSMR-B1/2). In the resulting heteromer, STATs can only be recruited by the GPL tyrosine motifs as all STAT recruitment sites in the OSMR are missing. The α/OSMR-B1/2+β/gp130 complex served as a positive control because it had been shown earlier that gp130 tyrosine motifs can initiate STAT tyrosine phosphorylation (19Hermanns H.M. Radtke S. Haan C. Schmitz-Van de Leur H. Tavernier J. Heinrich P.C. Behrmann I. J. Immunol. 1999; 163: 6651-6658PubMed Google Scholar). Additionally, we examined signaling induced by α/GPL-(530–745)+β/GPL-(530–745), which obviously completely relies on STAT recruitment by GPL. As shown in Fig. 3A (right section), all three dimers were functional; a similar Jak1 phosphorylation was obtained upon stimulation; likewise, a similar STAT3 phosphorylation can be detected in all three cases (Fig. 3A, 1st and 3rd panels, lanes 3–5). Again, GPL is clearly capable of activating STAT1. However, the GPL-mediated STAT1 phosphorylation is relat" @default.
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• W2020199484 date "2004-08-01" @default.
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• W2020199484 title "Characterization of the Signaling Capacities of the Novel gp130-like Cytokine Receptor" @default.
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