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J. Biol. Chem., Vol. 279, Issue 34, 36112-36120, August 20, 2004

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Characterization of the Signaling Capacities of the Novel gp130-like Cytokine Receptor^*

Alexandra Dreuw, Simone Radtke, Stefan Pflanz, Barbara E. Lippok, Peter C. Heinrich, and Heike M. Hermanns¶

From the Institut für Biochemie, Universitätsklinikum der Rheinisch-Westfälischen Technischen Hochschule Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany

Received for publication, February 2, 2004 , and in revised form, June 1, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (OSMR). 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 OSMR but also in a homomeric receptor complex. Most interesting, unlike OSMR 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The recently identified novel cytokine receptor gp130-like monocyte receptor/gp130-like receptor (GPL)¹ (1, 2) 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 (3). 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 (2), 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 (4–6). The longest isoform (745 amino acids) contains three intracellular tyrosine motifs; the first and third tyrosine is conserved between human and murine GPL (2). Besides the transmembrane isoforms, a soluble GPL has been proposed (CRL3; GenBank^TM accession number AF106913 [GenBank] -1).

Transcripts for GPL have been found in all cells of the myelomonocytic lineage (2) and of the epithelium from skin, lung, and prostate as well as in activated CD4+ and CD8+ T cell subsets (7). Additionally, GPL is highly expressed in tissues involved in reproduction, particularly in testis (1, 2).

The closest mammalian relative of GPL is gp130 (8), the common receptor subunit of the interleukin(IL)-6-type cytokines; GPL and gp130 share 28% sequence homology (2). The GPL gene (gpl) is located in tandem to the gp130 gene (gp130) on chromosome 5 with opposite transcriptional orientations (1, 2). The common intron/exon organization of both genes (2, 9) 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 (1, 2). 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 (10, 11). 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 (7, 12). IL-31 seems most closely related to oncostatin M, leukemia inhibitory factor, and cardiotrophin-1 (CT-1) (7), all of which belong to the family of IL-6-type cytokines (13). Besides GPL (IL-31R), the signaling receptor complex for IL-31 contains the oncostatin M receptor (OSMR), another signaling receptor subunit of the IL-6-type cytokines (14).

In the present study we characterize some signaling properties of GPL. In a homomeric as well as heteromeric receptor complex with OSMR 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 (15, 16). Subsequently, we demonstrate that GPL can only activate ERK1/2 when oligomerized with OSMR or gp130, but not in a homomeric arrangement.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
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 (17). 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 2x 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^TM 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. (2). 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 /gp130, /gp130-B1/2, /OSMR1, /OSMR-B1/2, and /gp130-YFFFFF has been described previously (16, 18, 19). The expression vectors pSVL-huIL-5R/huGPL-(530–745) (/GPL-(530–745)) and pSVL-huIL-5R/huGPL-(530–745) (/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. (2) (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-/GPL-(530–745) or pSVL-/GPL-(530–745) as a template and the QuikChange® site-directed mutagenesis kit (Stratagene, La Jolla, CA). The truncated construct pSVL-/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-/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 /GPL-(530–745) and /GPL-(530–745) were inserted into XhoI/BglII-digested pCAGGS expression vector (20). The expression plasmid for Jak1 was kindly provided by Dr. I. M. Kerr (London Research Institute, Cancer Research UK, London, UK).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Schematic representation of the chimeric receptors used in this study. The extracellular domains of the IL-5R or IL-5R were fused to the indicated transmembrane and cytoplasmic receptor regions. Tyrosine residues in the intracellular regions are indicated as lines. The box1 and box2 regions are depicted as hatched boxes.

Antibodies—The antibodies against IL-5R (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); -Tyr(P) antibody (Tyr(P)-99), -STAT1 (E-23), -STAT5B (C-17), -SHP2 (C-18), and -IL-5R (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 -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—₂M(-215)-luciferase contains the promoter region, -215 to +8, of the rat ₂-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 (21) 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 -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 (22). 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 -galactosidase expression vector pCH110 (Amersham Biosciences). HepG2 cells were transfected with 6 µg of luciferase reporter construct, 2 µg of -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 -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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
GPL Initiates Signal Transduction in Combination with gp130, OSMR, 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 (7) to constitute in combination with OSMR 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, OSMR, 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 OSMR fused to the extracellular domain of the interleukin-5 or receptor, respectively.

In earlier studies we have demonstrated that chimeras containing the full-length cytoplasmic region of the OSMR (/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 (/OSMR1) (19), maintaining STAT recruitment and MAPK-activating sites. Compared with all other constructs used in our study, /OSMR1 displayed similar surface expression levels. A schematic representation of all receptor constructs used is presented in Fig. 1.

We cotransfected expression vectors for /GPL-(530–745), /OSMR1, /LIFR, or /gp130 along with /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 ₂-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 /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 - and -chimera need to be present containing at least the Jak-binding box1 regions to initiate a signaling cascade (18). The /OSMR1+/GPL-(530–745) (Fig. 2A, lane 2) and /gp130+/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 /LIFR+/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 ₂M promoter (Fig. 2A, lane 1).

View larger version (28K): [in this window] [in a new window]   FIG. 2. Heteromerization of the cytoplasmic parts of GPL with gp130, OSMR, or LIFR leads to induction of STAT-responsive reporter genes in HepG2 cells. HepG2 hepatoma cells were transiently cotransfected with pCAGGS-based expression vectors encoding the chimeric receptors /GPL, /OSMR1, /LIFR, or /gp130 together with /GPL as indicated along with different STAT-responsive luciferase reporter gene constructs. A, 2M promoter-Luc; B, IRF1-tk-Luc; C, casein-tk-Luc. In the case of casein-tk-Luc, STAT5B was cotransfected. One day after transfection cells were stimulated with IL-5 (10 ng/ml) (gray) for 16 h or left untreated (white). Cellular extracts were prepared, and fire-fly luciferase activity was measured. The data represent the average of triplicate independent determinations of firefly luciferase activity normalized to the activity of cotransfected -galactosidase (mean ± S.D.). The relative luciferase activities and -fold inductions (relative to untreated cells) are representative of three independent experiments.

GPL Cytoplasmic Regions Are Sufficient to Induce Jak/STAT Activation When Dimerized with the OSMR 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-5R or IL-5R (data not shown). Only experiments with comparable expression of all different receptors were evaluated.

View larger version (40K): [in this window] [in a new window]   FIG. 3. Heteromerization of the cytoplasmic parts of OSMR and GPL (A) or gp130 and GPL (B) leads to STAT1 and STAT3 activation in HEK293T cells. HEK293T cells were transiently cotransfected with expression vectors encoding the chimeric receptor constructs as indicated. 48 hours after transfection, cells were stimulated with IL-5 (20 ng/ml) for 15 min or left untreated before cellular lysates were prepared. Immunoprecipitates (IP) of Jak1 and aliquots of cellular lysates were separated by SDS-PAGE. Western blots (WB) were developed with the phosphotyrosine-specific antibody PY99 to reveal Jak1 phosphorylation or with antisera specific for tyrosine-phosphorylated STAT1 or STAT3. The blots were stripped and reprobed with specific antibodies against Jak1, STAT1, and STAT3.

Similar results were obtained when examining signaling by the /gp130+/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 (/gp130+/gp130) (Fig. 3B, upper panel, lane 1). In this case, STAT1 phosphorylation upon /gp130+/GPL-(530–745) complex formation was easily detectable but was weaker than the corresponding signal generated by /gp130+/gp130 (Fig. 3B, 3rd panel, compare lanes 1 and 2).

To confirm these findings, we next expressed /GPL-(530–745) together with an OSMR construct that is deleted after its box1/2 domain (/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 /OSMR-B1/2+/gp130 complex served as a positive control because it had been shown earlier that gp130 tyrosine motifs can initiate STAT tyrosine phosphorylation (19). Additionally, we examined signaling induced by /GPL-(530–745)+/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 relatively weak compared with gp130 (Fig. 3A, 5th panel, compare lanes 3 and 4). This becomes even more evident in Fig. 3B when comparing the STAT1 activation by the /GPL-(530–745)+ /GPL-(530–745) homomer with the STAT1 activation mediated by the /gp130+/gp130 homomer (3rd panel, lanes 1 and 4). Taken together, if compared with gp130, GPL is quite an efficient STAT3 but a rather weak STAT1 activator.

GPL Itself Does Not Mediate ERK1/2 Phosphorylation—Besides activating the Jak/STAT pathway, many cytokine receptors have been shown to initiate the MAPK cascade. In order to investigate whether GPL like its close relative gp130 can lead to activation of ERK1 and ERK2, we coexpressed the same receptor combinations as used in Fig. 3, A and B. Again, similar cell surface expression of the various receptor combinations was confirmed by FACS analysis (data not shown). Transfected HEK293T cells were treated with IL-5 for 15 min, and cell lysates were prepared. Most interesting, the cytoplasmic region of GPL can contribute to activation of ERK1/2 when combined with the intracellular part of OSMR1 (Fig. 4A, lane 2) or gp130 (Fig. 4B, lane 2). However, this ERK1/2 activation is substantially weaker as if initiated by the /OSMR1+ /gp130 heteromer or /gp130+/gp130 (Fig. 4, A and B, lane 1). The combination /OSMR-B1/2 + /gp130 (Fig. 4A, lane 3) shows that the presence of one MAPK-activating receptor is sufficient for activation of ERK1/2. It therefore seemed possible that GPL contributes only indirectly by providing the essential second Janus kinase via its box1 region, whereas physical recruitment of the important MAPK-activating scaffold exclusively relies on the signal-transducing partner receptor. Indeed, when the tyrosine motifs of either the OSMR (OSMR-B1/2; Fig. 4A, lane 4) or gp130 (gp130-B1/2; Fig. 4B, lane 3) are deleted, phosphorylation of ERK1/2 disappears, indicating that in a heteromeric arrangement the ERK1/2 activation is mediated via tyrosine residues in the cytoplasmic parts of gp130 or the OSMR, respectively, but not by GPL. In accordance with this, a combination of /GPL-(530–745)+/GPL-(530–745) was insufficient to activate ERK1/2 (Fig. 4, A, lane 5 and B, lane 4) even though the same combination can induce a strong tyrosine phosphorylation of STAT3 and to a limited extent of STAT1 (Fig. 3).

View larger version (32K): [in this window] [in a new window]   FIG. 4. GPL by itself is unable to activate ERK1/2. A and B, HEK293T cells were transiently cotransfected with vectors encoding the GPL/OSMR combinations (A) or GPL/gp130 combinations (B) as indicated. 48 h after transfection, cells were stimulated with IL-5 (20 ng/ml) for 15 min or left untreated before cellular lysates were prepared. Proteins were resolved by SDS-PAGE, and Western blots (WB) were stained with an antiserum specific for phospho-ERK1/2 and restained with a mixture of ERK1- and ERK2-specific antisera. C, HEK293T cells were transiently cotransfected with expression vectors for Jak1 and the chimeric receptors as indicated. 24 h after transfection, cellular lysates were prepared, and immunoprecipitates (IP) of the IL-5R chain (right) and aliquots of cellular lysates (left) were separated by SDS-PAGE. Western blots (WB) were developed with antibodies against SHP-2 (upper panel), Shc (2nd panel), STAT3 (3rd panel), Jak1 (4th panel), and IL-5R (bottom panel on the right side).

Because GPL itself is insufficient to activate ERK1/2, we postulated that the receptor does not recruit any of these proteins. To investigate this hypothesis, we coexpressed /OSMR-B1/2, /OSMR1, /gp130, and /GPL-(530–745) along with Jak1 in HEK293T cells. We then immunoprecipitated the tyrosine-phosphorylated -chimeras with an antibody to the extracellular IL-5R domain (Fig. 4C, bottom panel, lanes 5–8) and stained the Western blots for coprecipitated proteins. As expected, phosphorylated /gp130 can coimmunoprecipitate SHP-2 but not Shc (Fig. 4C, 1st and 2nd panels, lane 7). Vice versa, phosphorylated /OSMR1 can coimmunoprecipitate Shc but not SHP-2 (Fig. 4C, 1st and 2nd panels, lane 6). As expected, phosphorylated /GPL-(530–745) precipitates neither SHP-2 nor Shc (Fig. 4C, 1st and 2nd panels, lane 8) just like the negative control /OSMR-B1/2, which lacks the whole C-terminal cytoplasmic part of the box1/2 region (Fig. 4C, 1st and 2nd panels, lane 5). As anticipated from the previous experiments, we could precipitate STAT3 not only with /gp130 and /OSMR1 but also with /GPL-(530–745) (Fig. 4C, 3rd panel, lanes 6–8), whereas the negative control /OSMR-B1/2 does not show a STAT3 recruitment (lane 5). Jak1, however, could be precipitated with all four receptor constructs, because all of them contain the box1 region (Fig. 4C, 4th panel, lanes 5–8). Examination of the total cell lysates proved that all coprecipitated proteins were present in the lysates to similar amounts (Fig. 4C, lanes 1–4).

Different Recruitment Sites for STATs in the Cytoplasmic Region of GPL—In order to delineate which of the three tyrosine motifs within the cytoplasmic region of GPL is responsible for STAT activation, we individually replaced them by phenylalanine. We then transfected pSVL-based expression vectors for the respectively mutated /GPL-(530–745) and /GPL-(530–745) into HepG2 cells along with the ₂M promoter-Luc reporter gene construct monitoring STAT-mediated gene expression (Fig. 5A; GPL-YYY, GPL-YYF, GPL-YFY, and GPL-FYY). It became evident that the most C-terminal tyrosine motif (Tyr-721) is required for full transcriptional activity of this reporter construct (Fig. 5A, lane 2). Mutation of the remaining tyrosine residues did not prevent reporter gene induction (Fig. 5A, lanes 3 and 4). Because the ₂M promoter is mainly inducible by STAT3, we postulated that Tyr-721 might be the recruitment site for this STAT factor to the cytoplasmic region of GPL.

View larger version (64K): [in this window] [in a new window]   FIG. 5. GPL mediates activation of STAT1, -3, and -5 via different cytoplasmic tyrosine residues. A, HepG2 hepatoma cells were transiently cotransfected with pSVL-based expression vectors encoding the indicated receptor combinations along with the 2M promoter-Luc. One day after transfection, cells were stimulated with IL-5 (10 ng/ml) (gray) for 16 h or left untreated (white). Luciferase activities of lysates were measured, and data were analyzed as described previously in the legend to Fig. 2. B, HEK293T cells were transiently transfected with pSVL-based expression vectors for the indicated receptor combinations (to monitor STAT5 activation, we additionally cotransfected an expression plasmid for STAT5B). 48 h after transfection, cells were stimulated with IL-5 (20 ng/ml) for 15 min or left untreated before cellular lysates were prepared. Western blots (WB) were immunodetected with either a STAT3-Tyr(P)-705-specific (upper panel) or a STAT5-Tyr(P)-694-specific (3rd panel) antibody and reprobed with a STAT3-(2nd panel) or STAT5B-specific antibody (bottom panel). C, HEK293T cells were transiently transfected with the same expression vectors used in B, but here an expression vector for SIE-tk-Luc was also cotransfected. One day after transfection cells were stimulated with IL-5 (10 ng/ml) (gray) for 16 h or left untreated (white). Reporter gene assays were carried out as described in the legend to Fig. 2. D, HEK293T cells were transiently transfected with expression vectors for the indicated receptor combinations, and additionally expression vectors for cis promoter-Luc and STAT5B were cotransfected. One day after transfection, cells were stimulated with IL-5 (10 ng/ml) (gray) for 16 h or left untreated (white). Reporter gene assay was carried out as described in the legend to Fig. 2. E, HEK293T cells were transiently cotransfected with expression vectors encoding Jak1 and the chimeric receptors indicated. 24 h after transfection, cell lysates were prepared, and immunoprecipitations (IP) using an antibody specific for the IL-5R chain were performed (right). Immunoprecipitates and aliquots of cellular lysates were resolved by SDS-PAGE. Western blots were immunodetected with antibodies against STAT3 (1st panel), Jak1 (2nd panel), and IL-5R (bottom panel on the right side). F, HEK293T cells were transiently transfected with pSVL-based expression vectors for the indicated receptor combinations. 48 h after transfection, cells were stimulated with IL-5 (20 ng/ml) for 15 min or left untreated before cellular lysates were prepared. Equal amounts of cellular proteins were separated by SDS-PAGE. Western blots were immunodetected with a STAT1-Tyr(P)-701-specific antibody (upper panel) and reprobed with antibodies against STAT1 (bottom panel).

As described previously (1) GPL can also strongly induce tyrosine phosphorylation of STAT5; in contrast to /gp130-YFFFFF+/gp130 (Fig. 5B, 3rd panel, lane 1), the combination of /gp130-YFFFFF with /GPL-YYY results in a strong STAT5 activation (lane 2). Most interesting, Tyr-721 in GPL is not required for STAT5 tyrosine phosphorylation (/GPL-YYF, Fig. 5B, lane 3) just like Tyr-683 (/GPL-YFY, Fig. 5B, lane 4). However, mutation of the first tyrosine residue (Tyr-652) to phenylalanine (/GPL-FYY, lane 5) completely abrogates STAT5 activation. Comparable expression of the different GPL mutants was measured by FACS analysis (not shown), and equal loading of the different cellular lysates was shown by restaining the blot with an antibody recognizing STAT5B irrespective of its phosphorylation status (Fig. 5B, bottom panel).

Defective STAT5 activation of GPL-FYY resulted in a failure to induce gene expression of a cis promoter-driven reporter gene (Fig. 5D, lane 5). The gene for the suppressor of cytokine signaling family member CIS has been described to be highly STAT5-responsive (22). In comparison to the OSMR, GPL seemed to be a weaker inducer of STAT5, because the cis promoter-Luc reporter gene was induced only about 3-fold (Fig. 5D, lane 2), whereas the combination of /gp130-YFFFFF and /OSMR1 led to an almost 7-fold stimulation (Fig. 5D, lane 1). The isolated cis promoter seems to be also responsive to STAT3 because IL-5 stimulation of /gp130-YFFFFF+/GPL-YYF resulted in a reduced luciferase expression (Fig. 5D, lane 3); STAT5 tyrosine phosphorylation, however, was unaffected (Fig. 5B, 3rd panel, lane 3).

Abrogation of binding of STAT3 to GPL after mutation of Tyr-721 was additionally verified by coexpressing /GPL-(530–745), /GPL-YYF, /OSMR-B1/2, and /OSMR1 along with Jak1 in HEK293T cells. The Jak1-mediated phosphorylation of the various receptor constructs resulted in binding of STAT3 to /GPL-(530–745) and /OSMR1 (Fig. 5E, upper panel, lanes 5 and 8) but not to /GPL-YYF or the negative control /OSMR-B1/2 (Fig. 5E, upper panel, lanes 6 and 7). Again Jak1 could be coimmunoprecipitated with all receptor constructs (Fig. 5E, 2nd panel, lanes 5–8). Equal amounts of the various receptor chimeras were precipitated (Fig. 5E, lower panel, lanes 5–8). Analysis of total cell lysates verified equal amounts of proteins in all extracts (Fig. 5E, lanes 1–4).

By using the different GPL point mutants, we also monitored STAT1 tyrosine phosphorylation mediated by this novel cytokine receptor. As observed before (Fig. 3) GPL was a weaker activator of STAT1 than gp130 (Fig. 5F, lanes 1 and 2). Whereas STAT3 and STAT5 activation depended on a particular tyrosine residue in the cytoplasmic region of GPL, all three tyrosines seemed to contribute equally to STAT1 tyrosine phosphorylation. In comparison to /gp130-YFFFFF+/GPL-YYY (Fig. 5F, lane 2), the three mutants /GPL-YYF (lane 3), /GPL-YFY (lane 4), and /GPL-FYY (lane 5) led to a similarly reduced STAT1 activation when oligomerized with /gp130-YFFFFF.

In order to clarify if the remaining STAT1 phosphorylation is mediated directly by the Janus kinases, we generated a new GPL construct, /GPL-(530–626). This construct lacks all tyrosine motifs but retains the Jak-binding region. Stimulation of /gp130-YFFFFF+/GPL-(530–626), however, did not result in STAT1 tyrosine phosphorylation (Fig. 5F, lane 6).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
In the present study we have characterized signaling capacities of GPL both in a homomeric as well as in a heteromeric arrangement. We were able to show that the cytoplasmic region of GPL can initiate Jak/STAT-mediated signaling in HepG2 hepatoma cells as well as in HEK293T cells. These results are consistent with findings by Ghilardi et al. (1), who homomerized chimeric GPL (gp130-like monocyte receptor) in 32D cells, but are in contrast to the results of Diveu et al. (2), who found that induced homomerization of GPL was signaling incompetent in COS7 cells. Because Diveu et al. (2) used similar chimeric constructs based on the IL-5 receptor complex, we tested our constructs in the same cells. Indeed, we were also unable to detect signaling by the homomerized GPL in COS7 cells (data not shown), implying that the signaling capacity of homomerized GPL cytoplasmic parts depends to some degree on the cellular context. Nevertheless, when oligomerized with /gp130-YFFFFF, /GPL-(530–745) induces STAT3 as well as STAT1 tyrosine phosphorylation (not shown). Therefore, also in COS7 cells, the intracellular tyrosine residues of GPL are sufficient to activate STAT transcription factors. However, signaling in the homomeric arrangement is very unlikely to occur in vivo because GPL lacks an N-terminal Ig-like domain. For the IL-6/IL-12 family of cytokines, it is well established that they interact with their cognate receptors through three binding sites (I–III) (10) with site III binding to the Ig-like domain of one receptor subunit. It seems likely that GPL contributes the binding interfaces for interaction with either site I or II but that the full receptor complex requires an additional receptor subunit contributing an Ig-like domain (2).

By using chimeric constructs, we demonstrated that GPL is able to signal in combination with its close relatives gp130 and LIFR, and additionally, we showed for the first time that GPL leads to STAT activation when combined with the OSMR. This finding is of special interest because GPL together with OSMR constitutes the signaling receptor complex for the recently discovered cytokine IL-31 (7). Because our experiments show particularly strong activation of signaling upon combination of GPL and gp130 cytoplasmic domains, it could be interesting to study whether this receptor pair can be activated by a different cytokine.

Recent studies on GPL demonstrated that GPL in a homomeric (1) as well as heteromeric receptor complex with gp130 or the LIFR (2) is able to activate STAT3. However, Diveu et al. (2) postulated that GPL might not directly activate STAT3 due to the fact that its cytoplasmic domain lacks a canonical YXXQ motif identified as a docking site for STAT3 in the signal transducing receptors of the IL-6-type cytokine family gp130, LIFR, and OSMR (15, 26, 27). Here we have shown that the GPL tyrosine motifs are sufficient to activate STAT factors, STAT3 and STAT5 in particular (Figs. 3 and 5).

We further identified Tyr-721 as part of the most C-terminal tyrosine motif to be responsible for STAT3 recruitment and activation. Point mutation of this residue to phenylalanine not only abrogated the recruitment of STAT3 to phosphorylated GPL (Fig. 5E) but also phosphorylation of STAT3 detectable in cellular lysates (Fig. 5B, upper panel). Therefore, GPL recruits STAT3 via the nonclassical tyrosine motif ⁷²¹YLKN.

Crystal structures of phosphotyrosine peptides bound to SH2 domains of different proteins have shown that the five immediate C-terminal amino acid neighbors of the phosphotyrosine are most important for the interaction (28–31). These five amino acid residues also appear to determine binding specificity (32, 33), and a recent study (34) specified the sequence requirements for the STAT1 and STAT3 SH2 domain. STAT3 preferentially binds to peptides displaying the motif phosphotyrosine-(basic or hydrophobic)-(proline or basic)-glutamine. From that point of view, the GPL recruitment site for STAT3, ⁷²¹YLKN, can be considered sufficiently related in sequence. On the basis of these data, intracellular tyrosine motifs closely resembling but not exactly matching the YXXQ consensus may require experimental reevaluation with respect to their potential to activate STAT3.

Furthermore, we were able to show that activation of STAT5 is independent of Tyr-721 but requires Tyr-652 (Fig. 5B, 3rd panel). Like Tyr-721, this tyrosine residue is conserved between human and murine GPL, implying a crucial role for GPL-mediated signaling. The consensus binding sites for STAT5 in various cytokine receptors are not as well conserved as the STAT3 recruitment site; however, the GPL motif ⁶⁵²YVTC resembles quite well the binding sites for STAT5 in the IL-2R (⁵¹⁰YLSL) (35).

GPL was a weak activator of STAT1 especially when compared with gp130 (Fig. 3B). Indeed, gp130, unlike GPL or OSMR, fulfils the sequence requirements for STAT1 binding quite well because it contains two tyrosine motifs matching the consensus sequence YXPQ. The proline residue seems to be important (34, 36) to allow efficient STAT1 activation, and this is absent in GPL and OSMR. This explains why an /gp130+/GPL-(530–745) heteromer is a better STAT1 activator than the /OSMR1+/GPL-(530–745) heteromer (compare Fig. 3, A and B). It is also obvious why the /GPL-(530–745)+/GPL-(530–745) homomer is a stronger activator of STAT1 than the /OSMR-B1/2+/GPL-(530–745) heteromer, because the weak GPL activation sites for STAT1 are doubled in the homomeric arrangement. Most interesting, all three tyrosine residues in the cytoplasmic domain of GPL seem to contribute equally to the full STAT1 tyrosine phosphorylation (Fig. 5F).

GPL exists at least in five different isoforms as follows: a soluble variant containing 509 amino acids (CRL3, GenBank^TM accession number AF106913 [GenBank] -1) and four membrane-spanning forms generated by alternative splicing and therefore differing in length and sequence beyond the Asn-560 residue: GPL₅₆₀, GPL₆₁₀, GPL₆₂₆, and GPL₇₄₅ (2). Only the longest version of GPL contains the STAT5- and STAT3-activating sites: Tyr-652 and Tyr-721, respectively. Thus, among the four membrane-spanning isoforms of GPL, only GPL₇₄₅ should activate STAT3 and -5. In fact, only the two isoforms GPL₇₄₅ and GPL₆₁₀ contain any tyrosine motifs. Whereas GPL₇₄₅ contains three tyrosine motifs, two conserved between mouse and human (Tyr-652 and Tyr-721), GPL₆₁₀ contains only a single tyrosine motif. This tyrosine, however, is unlikely to recruit proteins with an SH2 domain, because it has been described that for SH2 domain binding the tyrosine +3 position is very important (37). The only intracellular tyrosine residue of GPL₆₁₀, Tyr-608, is missing the +3 position and is therefore unlikely to represent a STAT recruitment site.

Within an OSMR-GPL heteromeric receptor complex, two STAT-activating receptor subunits are present, because STAT5 and STAT3 activation are also mediated via the OSMR subunit. Contributions of both cytokine receptors might be important to obtain a sufficient STAT activation level for efficient induction of gene expression.

The intracellular region of GPL contains a proline-rich sequence resembling the well conserved box1, required for binding of Janus kinases. Yet it was unknown which Jak can be activated by GPL. Studies on IL-6-type cytokines, the closest relatives of IL-31, have revealed that Jak1 plays an important role in their signaling process (38, 39). Therefore, it was a promising candidate for binding to GPL. Here we demonstrated that the OSMR/GPL heteromer as well as the GPL homomer can activate Jak1 (Fig. 3A). We could also precipitate Jak1 when coexpressed with /GPL indicating that this Jak is recruited to GPL (Fig. 4C). Three of the four membrane-anchored isoforms of GPL contain the box1 region. Therefore, all of them can be expected to bind and activate Jaks. Because the shortest isoform of GPL does not contain a box1 region, it has to be regarded as signaling incompetent and potentially dominant-negative. Considering the different GPL isoforms lacking distinct signaling entities, it is tempting to speculate that alternative splicing of GPL could serve as a mechanism for modulation of IL-31-mediated signals. In this context, it will be interesting to characterize the expression pattern of GPL isoforms in different cell types.

In this study we addressed the question of MAPK activation by GPL, another signaling pathway activated by many cytokines, for the first time. Most interesting, unlike its closest relatives gp130 or OSMR, GPL cannot directly support ERK1/2-activation. When heteromerized with OSMR or gp130, a well detectable activation of ERK1/2 could be observed in HEK293T cells (Fig. 4, A and B, lane 2). However, when compared with the ERK1/2 activation induced by either /OSMR1+/gp130 (Fig. 4A, lane 1) or /gp130+/gp130 (Fig. 4B, lane 1), it becomes apparent that the induced phosphorylation was much weaker. Deletion of the tyrosine residues in OSMR or gp130 necessary for ERK1/2 activation (15, 16) (/OSMR-B1/2, /gp130-B1/2) resulted in a receptor complex that was unable to activate ERK1/2 (Fig. 4, A, lane 4 and B, lane 3), confirming that the tyrosine residues in GPL cannot trigger MAPK activation. Consistently, /GPL-(530–745)+/GPL-(530–745) was incapable of mediating ERK1/2 phosphorylation.

We identified a molecular mechanism for this signaling incompetence by showing that tyrosine-phosphorylated GPL was unable to recruit the tyrosine phosphatase SHP-2 or the adaptor protein Shc (Fig. 4C, lane 8). Both molecules have been shown to be involved in MAPK activation by numerous cytokine receptors. Especially the failure to recruit SHP-2 is of interest, because this protein has additionally been shown to be an important negative regulator of cytokine-induced signaling (40).

Taken together the present study has elucidated several important molecular mechanisms underlying GPL₇₄₅-mediated signal transduction. GPL is a strong activator of STAT3 and additionally STAT5, whereas STAT1 is only marginally tyrosine-phosphorylated. We identified Tyr-652 and Tyr-721 as the major STAT5- and STAT3-activating sites in GPL₇₄₅, respectively, and in addition demonstrated Jak1 binding and activation. Unlike OSMR and gp130, GPL is unable to mediate ERK1/2 phosphorylation. Finally, we provide a potential mechanism by showing that GPL cannot recruit either SHP-2 or Shc.

	FOOTNOTES

 
^* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 542 (Bonn, Germany) and by the Fonds der Chemischen Industrie (Frankfurt, Germany). 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.

Present address: Micromet AG, Staffelseestrasse 2, 81477 München, Germany.

To whom correspondence may be addressed. Tel.: 49-241-8088831; Fax: 49-241-8082428; E-mail: heinrich{at}rwth-aachen.de. ¶ To whom correspondence may be addressed. Tel.: 49-241-8088868; Fax: 49-241-8082428; E-mail: hermanns{at}rwth-aachen.de.

¹ The 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; ₂M, ₂-macroglobulin; IRF, interferon regulatory factor; SH2, Src homology; SIE, sis-inducible element; CIS, cytokine inducible SH2 protein; IL, interleukin; FACS, fluorescence-activated cell sorter.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. Robert Kastelein, Fernando Bazan, and Jackie Timans for their fundamental support inthe initial state of this study; Dr. Jan Tavernier for the plasmids encoding the human IL-5R - and -chains; Dr. Ian M. Kerr for the plasmid encoding the Jak1; Dr. Hugues Gascan for the SIE-tk-Luc, and Dr. Akihiko Yoshimura for the cis promoter-Luc plasmids. We also thank Drs. Serge Haan, Gerhard Müller-Newen, and Fred Schaper for critical reading of the manuscript.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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