INTRODUCTION

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Asthma is a complex inflammatory disorder that is characterized by periodic airway obstruction, wheezing, and bronchial hyperresponsiveness (BHR)¹ (1). Clinical studies of asthma have revealed that the pathology of this disease is associated with widespread narrowing of the airways due to edema and infiltration of multiple inflammatory cells into the lung epithelia (2). These cell types include mast cells, eosinophils, B-cells, and TH2-lymphocytes as the predominant cell mediators of this disorder (3, 4). While these inflammatory cells appear to be important, their precise role in producing BHR is unclear and still under investigation. Nevertheless, the response to antigen and subsequent release of chemical inflammatory mediators (histamines, leukotrienes, prostaglandins, etc.) by a number of these cells has been documented (5).

Genetic studies have suggested that BHR is a multigenic process (6-11). Recently, we have identified interleukin-9 (IL9) as a factor in regulating airway hyperresponsivess in inbred strains of mice (12). C57BL/6 mice, which show low airway responsiveness, had undetectable levels of IL9 in activated splenocytes and in the lung, while DBA/2 mice, which have airway hypperresponsiveness, expressed robust levels of IL9 in both tissues. A role for IL9 in asthma and allergy has been supported by the findings that it has pleiotropic activities on cell types associated with these diseases such as TH2 lymphocytes, B-cells, mast cells, and eosinophils (13-19). In particular, IL9 has been shown to act as a growth and differentiation factor for mast cells and to enhance the release of IgE from B-cells. These two activities as well as the effect of IL9 on controlling BHR in mice suggest that it and its associated pathway(s) are involved in the pathogenesis of allergy and asthma. Most recently, Holroyd et al.² have found genetic linkage of asthma and BHR to the IL9 receptor (hIL9R) locus in humans, supporting the notion that this pathway is involved in the disease process in humans.

Because of the genetic linkage of the IL9 receptor locus to allergy and asthma susceptibility, we explored the possibility that variations in hIL9R structure/function or gene expression may exist. Here we report the identification of an abundantly expressed receptor splice variant (referred to as Q receptor) that lacks codon 173. This variant is unable to bind IL9 and transduce its signal to effector cells. These data suggest a mechanism for modulating IL9 signaling in effector cells.

	EXPERIMENTAL PROCEDURES

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Purification of Human PBMCs and Mitogen Stimulation-- Peripheral blood was obtained by consent from 50 unrelated donors. Venipuncture was performed, and blood was collected into vacutainer tubes containing EDTA. An equal volume of PBS (Mg²⁺/Ca²⁺-free) was added to whole blood. Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation over Ficoll-Paque gradients (Pharmacia Biotech 17-1440-02) (21). Purified cells were grown in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (Life Technologies, Inc.), 5 µg/ml phorbol 12-myristate 13-acetate, and 1 µg/ml phytohemagglutinin (Sigma) and incubated at 37 °C in 5% CO₂ for 6 days.

Nucleic Acid Isolation and Reverse Transcriptase-PCR-- Cytoplasmic RNA and genomic DNAs were isolated from phorbol 12-myristate 13-acetate/phytohemagglutinin-treated PBMCs as described previously (22). RNAs were reverse transcribed and amplified by PCR as described (23). Primers used to amplify the entire coding region of the hIL9R (GenBank^TM accession no. M84747) were as follows: 5'-GCT GGA CCT TGG AGA GTG-3' (R8 forward, centered at nucleotide 208 of the cDNA) and 5'-GTC TCA GAC AAG GGC TCC AG-3' (R1801 reverse, centered at nucleotide 1822 of the cDNA) (15, 24).

Plasmids, Subcloning, and Sequencing Procedures-- For the structural analysis of hIL9R, reverse transcriptase-PCR products containing the entire coding region of the hIL9R from 50 donors were cloned into pCR2.1 T-tailed vectors (Invitrogen). At least 10 recombinant clones from each individual were completely sequenced. Recombinant expression vectors were engineered by subcloning the relevant cDNAs from the original cloning vector as EcoRI fragments into the EcoRI restriction site of the LXSN retroviral vector (25) that contains a neomycin resistance gene as a selectable marker. The GH8 (the only combination of codons 344 and 410-418 that has not been reported by others nor observed in our IL9R structure analysis) and GR9 (see Fig. 2A) expression plasmids were engineered by first subcloning the GR8 and GH9 forms of the hIL9R cDNA into the EcoRI site of pZeoSV2 (Invitrogen) to produce pZeoGR8 and pZeoGH9, respectively. The GH8 and GR9 cDNAs were then engineered by exchanging XmaI fragments of these plasmids (5' site is located in the pZeoSV2 polylinker, and the 3' site is located at nucleotide 1328 (between codons 344 and 410) of the receptor cDNA) to generate pZeoGH8 and pZeoGR9. Finally, GH8 and GR9 cDNAs were subcloned as EcoRI fragments from pZeoSV2 into the EcoRI site of LXSN vector. pZeoRR9 was obtained by replacing the BstEII/NotI fragment of pZeoGH9 containing base pairs 791-1822 of the hIL9R cDNA with the BstEII/NotI fragment from ph9RA6 (26). Successively, the EcoRI/XhoI fragment from pZeoRR9 containing the RR9 version of the hIL9R cDNA was subcloned into the EcoRI/XhoI site of LXSN. Constructs were sequenced using the ABI Prism DNA sequencing kit (Perkin-Elmer), and reactions were run in a model 377 DNA automated sequencer (ABI Prism, Perkin-Elmer).

Cell Lines and Cell Culture-- TS1 is an IL9-dependent murine T cell line (15). It was maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies, Inc.) containing 10% fetal bovine serum in the presence of 25 ng/ml of murine IL9 (R & D Systems, Minneapolis, MN). Mo7e is a cytokine-dependent human megakaryoblastic leukemia cell line that proliferates in response to IL3, granulocyte-macrophage colony-stimulating factor, IL9, or stem cell factor. These cells were maintained in RPMI medium containing 20% fetal bovine serum in the presence of 50 ng/ml human IL9 or 10 ng/ml IL3. COS7 cells were purchased from ATCC and were grown in DMEM plus 10% fetal bovine serum. All cell lines were cultured at 37 °C in a 5% CO₂ humidified incubator.

Generation of Cell Lines-- TS1 cells stably expressing various forms of hIL9R variants were generated by electroporation and Geneticin (G418) selection. Briefly, 3 × 10⁶ cells were resuspended in 0.3 ml of RPMI 1640 (Life Technologies) containing 10 µg of the appropriate plasmid, pulsed (260 V/1000 microfarads) in a GenePulse II electroporator (Bio-Rad), and resuspended in growth medium. After 24 h, cells were selected in 1 mg/ml G418 (Life Technologies) for approximately 14 days, at which time mock-transfected cells died. Selected cells were maintained in growth medium supplemented with 25 ng/ml murine IL9 and 0.4 mg/ml G418. Expression of hIL9R was periodically assessed by Western blot.

Cell Proliferation Assay and Cytokine Stimulation-- Interleukin-9-induced cell proliferation was performed as follows. TS1 transfectants were washed three times with PBS and resuspended in DMEM, 10% fetal bovine serum. Cells were seeded at 10³ cells/well in 96-well microplates and grown in the presence of 5 ng/ml recombinant human IL9 or murine IL9. Proliferation was assayed after 7 days using a modified protocol of the acid phosphatase assay (CLONTECH). Briefly, 50 µl of a buffer containing 0.1 M sodium acetate (pH 5.5), 0.1% Triton X-100, and 10 mM p-nitrophenyl phosphate (Sigma 104 phosphatase substrate) was added per well containing 0.2 ml of growth medium and incubated for 1.5 h at room temperature. Reactions were terminated by the addition of 0.05 N sodium hydroxide and quantified by absorbance at 410 nm using a Dynatech model MR600 plate reader. All experiments were performed in at least triplicate.

Immunoprecipitations, Immunoblotting, and Antibodies-- 2-5 × 10⁷ cells were harvested and lysed in 1 ml of RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride, 50 mM sodium fluoride, 1 mM sodium orthovanadate, and 1× "Complete" protease inhibitors mixture (Boehringer Mannheim) and then incubated for 45 min on ice. Lysates were cleared of debris by centrifugation for 20 min at 4 °C, and supernatants were transferred to a fresh tube. For immunoprecipitations, 1-5 µg of antibody was added to the lysate and incubated overnight at 4 °C. The next day, 20 µl of Protein A + G-agarose-conjugated beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was added to the samples and incubated at 4 °C for 2 h. Samples were then centrifuged for 1 min, and beads were washed four times. After the final wash, beads were resuspended in 50 µl of Laemmli buffer and boiled for 3 min before electrophoresis on 8% Tris-glycine gels (Novex). Proteins were electroblotted onto Immobillon-P membrane (Millipore Corp.) in 48 mM Tris base, 40 mM glycine, 0.037% SDS, 20% methanol and blocked overnight in Tris-buffered saline plus 0.05% Tween 20 (TBS-T) (Bio-Rad) and 5% condensed milk (or 1% bovine serum albumin for anti-phosphotyrosine). Membranes were probed using specific primary antibodies (listed below) followed by a secondary horseradish peroxidase-conjugated antibody and chemiluminescence (Pierce). Specific antibodies for murine and human IL9R (sc698) and murine Jak1, Irs1, Irs2, Stat1, Stat2, Stat3, Stat4, Stat5, and phosphotyrosine were all purchased from Santa Cruz Biotechnology. Anti-Jak3 and monoclonal anti-hIL9R MAB290 were purchased from Upstate Biotechnology and R & D Systems, respectively. The anti-N-terminal hIL9R antibodies AH9R-1, -2, and -7 have been previously described (26).

Immunostaining and FACS Analysis-- COS7 cells were plated onto glass coverslips after electroporation with the appropriate plasmids. After 48 h, cells were washed with PBS and incubated with 5 µg/ml of the anti-N-terminal hIL9R monoclonal antibody MAB290 (R & D Systems) in PBS containing 1% bovine serum albumin for 1 h at room temperature. Cells were then washed three times with PBS and incubated with a 1:100 dilution of anti-mouse IgG Texas Red-conjugated antibody (Sigma) for 30 min. Cells were then fixed for 15 min in 4% paraformaldehyde/PBS containing 0.1% Triton X-100. For intracellular staining, cells were fixed and blocked for 30 min in 5% bovine serum albumin/PBS. Cells were then incubated with 2 µg/ml of the carboxyl-terminal specific hIL9R polyclonal antibody sc698 (Santa Cruz Biotechnology) followed by incubation with 1:100 dilution of anti-rabbit IgG Texas Red-conjugated antibody (Sigma). Cells were washed three times with PBS and then counterstained with 1 µg/ml of 4,6-diamidino-2-phenylindole. Samples were analyzed using an Olympus AX-70 microscope, and micrographs were taken and printed using a Sony camera DKC5000/Sony printer UP-D8800 system. FACS analysis was performed as described previously (26).

Binding Assay-- Recombinant human IL9 (4 µg) was iodinated using IODO-GEN-bead (Pierce) yielding 20,000 CPM/ng of protein as described previously (15). 5 × 10⁶ cells were in 100 µl of binding buffer (DMEM, 25 mM Hepes, 1% bovine serum albumin, 10 mM sodium azide) containing 5 nM of ¹²⁵I-IL9 for 3 h at 4 °C. Cells were layered over 800 µl of a 1.5:1 mixture of dibutyl- and dioctylphtalate oils and centrifuged at 750 × g for 1 min. Cell pellets were counted in a Gamma 5500 counter (Beckman) to obtain counts of bound ¹²⁵I-IL9. Nonspecific binding was determined by the addition of 200-fold excess of unlabeled IL9. Data represent the mean of triplicate experiments.

Cellular Fractionation-- Cellular fractionation was performed as described (28). Briefly, 4 × 10⁶ cells were washed once in PBS, resuspended in 0.2 ml of hypotonic buffer (10 mM Tris-HCl, pH 7.5, 10 mM NaCl, 1.5 mM MgCl₂, 0.2 mM CaCl₂, 1× "Complete" protease inhibitors mixture (Boehringer Mannheim)). Cells were disrupted through a 25-gauge needle and further incubated for 30 min on ice. Cell lysates were sequentially centrifuged at 700 × g for 7 min, 3500 × g for 10 min, and 16,000 × g for 30 min at 4 °C. The supernatant (cell content) was recovered, and the membrane pellet was washed once in hypotonic buffer. Membrane proteins were extracted by solubilization in CHAPS buffer (PBS, 8 mM CHAPS, complete protease inhibitor) and cleared of debris by centrifugation at 16,000 × g for 20 min at 4 °C. Total cell extracts were obtained by RIPA buffer extraction as described above.

	RESULTS

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Structural Analysis of hIL9R from Human PBMCs-- In order to analyze hIL9R nucleotide structure in randomly ascertained individuals, we established a cohort of 50 unrelated volunteers. These volunteers were asked to report their past medical history as being nonallergic, allergic, or asthmatic. Using this criterion, 29 out of 50 volunteers were determined to be allergic and/or asthmatic. PBMCs were purified from these individuals, mitogen-stimulated, and cultured for 6 days, at which time maximal IL9R expression is found.³ Cells were then harvested, and cytoplasmic RNAs and genomic DNA were isolated. Full-length hIL9R cDNAs (see Fig. 1 for a schematic representation of the hIL9R cDNA) were generated from each sample by reverse transcription and amplified by PCR. Products were then cloned into T-tailed vectors and sequenced. A comparison of sequences from an average of 10 clones from each individual enabled us (i) to determine if nucleotide variations were natural or due to Taq polymerase-induced mutations; (ii) to analyze transcription of both alleles; and (iii) to identify post-transcriptionally altered forms. Table I summarizes the frequency of the more common variants identified in our transcript analyses. Several alternative splice forms involving exons 3, 4, 5, and 8 were found as minor species. Two exon 5 splice variants were identified in which the first 5 or 29 nucleotides of the exon were deleted, resulting in a frameshift. These alternative splice variants (containing total deletion of exon 3, total deletion of exon 4, or partial deletion of exon 5) could all result in potential soluble receptors, because the transmembrane domain of the receptor is encoded by exon 7. In contrast, the splice variant lacking exon 8 could produce a receptor retaining the transmembrane domain but lacking the intracellular signaling domain encoded by the sequences of exons 8 and 9. 

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the hIL9R cDNA. The boxes depict exons 2-9 that encompass the coding region (relative size is in scale, except the 3'-untranslated portion of exon 9, outlined by a dashed line). The extracellular region is encoded by exons 2-6, the transmembrane region by exon 7, and the intracellular domain by exons 8 and 9. The arrows indicate polymorphisms or aberrant splices that affect partial sequence of the exon: deletion of the first 5 or 29 nucleotides in exon 5 (a); deletion of the first 3 nucleotides in exon 6 (codon 173) (b); Arg/Gly polymorphism at codon 310 (c); Arg/His polymorphism at codon 344 (d); polymorphism at codon 410-417/410-418 consisting of either 8 or 9 serines (e). *, complete deletion of exon 3, 4, or 8.

Expression of Human IL9 Receptor Variants in a Murine IL9-dependent Cell Line-- Cell lines were generated that expressed the various receptor isoforms in order to study receptor function (see Fig. 2A for details on constructs). Each of the receptor variants was subcloned into the LXSN mammalian expression vector that contains a murine moloney leukemia viral long terminal repeat for constitutive expression and a neomycin resistance gene as a selectable marker. Each construct was then electroporated into the murine TS1 cell line. This cell line was derived from primary murine T-lymphocytes and was found to be dependent on murine IL9 for growth and survival, but it is not responsive to human IL9 (30). Transfectants were then analyzed by Western blot for hIL9R expression. As shown in Fig. 2B, comparable hIL9R expression was found for all isoforms except for GR8, which showed 3-fold less expression. Interestingly, we noticed that GR8 protein ran slightly faster than GH9 when a higher electrophoretic resolution could be obtained (Fig. 2C). The addition of human IL9 to the cells did not result in change of the migration pattern between the GR8 and GH9 receptors (lanes 4 and 6). To address the possibility that this migratory difference was due to cell-specific post-translational modifications, we transiently transfected the simian derived COS7 cells with the GR8, GR9, GH8, and GH9 expression plasmids. Cells were grown for 48 h and then harvested for protein and analyzed for IL9 receptor migration by Western blot. In these conditions, GR8 and GH8 ran faster than GH9 and GR9 (data not shown), indicating that the serine repeat, and not the Arg³⁴⁴/His³⁴⁴ polymorphism, was critical for this shift in electrophoretic mobility. Studies are in progress to understand the nature of this differential mobility.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   TS1 cells expressing hIL9R variants. A, hIL9R expression constructs. The first column shows the name of each construct. Each row indicates the amino acid variation and the corresponding codon. Gln, deletion of glutamine (Q). B, TS1 cells were stably transfected with the various hIL9R expression constructs, and expression was assessed by Western blot using a C-terminal specific anti-hIL9R antibody. C, differential mobility of GR8 and GH9 receptor types. TSLXSN, TSGR8, and TSGH9 cells were starved for 6 h and then stimulated for 5 min with (+) or without () human IL9. Cell extracts were immunoprecipitated with the C-terminal anti-hIL9R antibody and immunoblotted using the same antibody. h.c., immunoglobulin heavy chain.

Effect of Human IL9 on Proliferation and Survival of TS1 Cells Expressing Different Forms of hIL9R-- TS1 survival and proliferation depend on the presence of murine IL9 and are not supported by the human cytokine. Ectopic expression of the human receptor renders these cells responsive to the human ligand (15). In order to further assess the biological activity of the receptor isoforms, we investigated whether they differed in their ability to transduce an effective growth signal upon human IL9 stimulation. TS1 transfectants were treated with either murine or human IL9 and plated as described under "Experimental Procedures." As shown in Fig. 3, all transfectants grew in the presence of murine IL9. Additionally, TS1 cells that expressed GR8, GH9, RR9, and GR9 (from hereon referred to as TSGR8, TSGH9, TSRR9, and TSGR9, respectively) responded to human IL9 treatment, while TSGR8 showed a lower proliferative response. This was most likely due to the lower expression of the receptor as compared with the other cell lines (see Fig. 2B, lane 2). Indeed, another set of TSGR8 and TSGH9 transfectants were generated later that expressed equal levels of receptor and showed similar growth rates (data not shown). In contrast, TS1 cells expressing QGR8 and QGH9 (hereafter referred to as TSQGR8 and TSQGH9, respectively) as well as those transfected with vector alone (referred to as TSLXSN) failed to respond to the human ligand. These data indicate that the absence of the glutamine at codon 173 suppressed the ability of the receptor to transduce a growth signal.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Proliferation of TS1 cells expressing different hIL9R variants. Cells were treated with no cytokine, 5 ng/ml murine IL9 (dotted bars), or human IL9 (shaded bars). The rate of proliferation was assessed 7 days later by determining the ratio between the number of treated and untreated cells (percentage of control).

Human IL9-activated Signal Transduction Cascade in TS1 Cells Expressing hIL9R Variants-- Part of the IL9R signal transduction cascade has been well established (26, 31). The interaction of IL9 with its receptor results in phosphorylation of the receptor itself and the activation of Jak1, Jak3, Stat1, Stat3, Stat5, Irs1, and Irs2 signaling molecules. These previous studies all employed the RR9 form of the receptor (26). We examined whether the GR8 and GH9 receptors varied in their ability to activate these proteins. To rule out possible interference that the endogenous receptor may cause in this analysis, we treated TS1 cells with either murine or human IL9 and analyzed the tyrosine phosphorylation of murine IL9 receptor. As shown in Fig. 4A, the murine IL9 receptor could be immunoprecipitated by an anti-phosphotyrosine antibody from cells treated with murine IL9 but not from cells untreated or treated with human IL9, thus demonstrating the inability of human IL9 to react with the murine IL9 receptor. In a similar experiment, TSGR8 and TSGH9 cells were cytokine-starved for 6 h and then treated for 5 min with human IL9. Since both cell lines express the endogenous murine receptor, murine IL9 treatment was included as an internal control (31). After stimulation, total proteins were extracted; immunoprecipitated using various Jak-, Stat-, and Irs-specific antibodies; and then Western blotted onto nylon membranes. Blots were probed with an anti-phosphotyrosine antibody that specifically detects tyrosine-phosphorylated proteins. TS1 cells grown in the absence of cytokine had undetectable amounts of tyrosine-phosphorylated proteins (Fig. 4B, lanes 1 and 4). Treatment of both TSGR8 and TSGH9 cells with murine IL9 resulted in tyrosine phosphorylation of Jak, Stat, and Irs proteins described above. Representative analyses of these experiments are shown for Jak1, Stat1, and Irs1 in Fig. 4B (lanes 2 and 5). Similarly, human IL9 treatment resulted in an identical profile as in the murine IL9-treated cells, while no differences were observed between the GR8 and GH9 receptors (Fig. 4B, lanes 3 and 6, and data not shown). Analysis of GR8 and GH9 receptor tyrosine phosphorylation found both to be equally phosphorylated after human IL9 treatment (data not shown), thus suggesting that these residues have no detectable role in receptor phosphorylation or phosphorylation of downstream substrates. Next, we examined whether failure of TSQGR8 or TSQGH9 cells to proliferate in response to human IL9 reflected an altered signal transduction function of these receptors. Again, we analyzed the tyrosine phosphorylation of members of the Irs, Jak, and Stat families in these cell lines upon treatment with either murine or human IL9. Immunoprecipitation-Western blot analysis revealed that neither QGR8 nor QGH9 receptors were capable of ligand-induced activation of any Jak, Stat, or Irs family member (Fig. 4C, lanes 3 and 6) as compared with the GH9 receptor that was used as positive control (Fig. 4C, lane 9). Furthermore, human IL9 treatment did not lead to phosphorylation of the receptor itself (data not shown). The TSQGR8 and TSQGH9 cells treated with murine IL9 showed activation of all proteins tested (Fig. 4C, lanes 2 and 5), thus demonstrating the integrity of the IL9 signaling cascade in these cells.

View larger version (56K): [in this window] [in a new window]   Fig. 4.   IL9-induced activation of Jak, Stat, and Irs proteins by hIL9R variants. A, human IL9 does not induce phosphorylation of murine IL9 receptor. TS1 cells were starved for 6 h and then treated for 5 min with no cytokine (), murine IL9 (m), or human IL9 (h). Cell extracts were immunoprecipitated with an antibody specific for tyrosine-phosphorylated (-PY) proteins, blotted, and probed with an anti-murine IL9 receptor antibody (-mIL9R). B, GR8 and GH9 receptors activate Jak, Stat, and Irs proteins upon IL9 binding. TSGR8 and TSGH9 cells were treated as in A, and lysates were immunoprecipitated using antibodies specific for various Jak, Stat, and Irs proteins as indicated. Immunoblots were first probed with an anti-phosphotyrosine antibody (-PY) to detect only tyrosine-phosphorylated proteins, stripped and reprobed with the same antibody used for the immunoprecipitation. C, Q receptor does not activate Jak, Stat, and Irs proteins upon IL9 treatment. TSQGR8, TSQGH9, and TSGH9 cells were treated as in A, and proteins were immunoprecipitated and immunoblotted as in B. I.P.: -, antibody used for immunoprecipitation.

Ligand-binding Analysis, Cellular Localization, and Immunoreactivity of the Q Receptor-- We have demonstrated that deletion of the glutamine at codon 173 results in loss of hIL9R signaling. This loss of function could be explained by an impaired binding of the ligand to the receptor. To test this hypothesis, we performed ligand-binding analysis on TSLXSN, TSGR8, TSGH9, TSQGR8, and TSQGH9 cells using iodinated human IL9 protein (¹²⁵I-hIL9). As shown in Fig. 5A, TSGR8 and TSGH9 cells were capable of binding ¹²⁵I-hIL9, whereas binding was undetectable in TSLXSN, TSQGR8, or TSQGH9 cells. The TSLXSN, TSGH9, and TSQGH9 cells were next fractionated in order to assess the cellular localization of the Q receptor. Protein fractions as well as total cell extracts were run in SDS-polyacrylamide gel electrophoresis, transferred onto a nitrocellulose membrane, and probed with antibodies specific for actin (marker for cytoplasm (32)), proliferating cell nuclear antigen (marker for cytoplasm and nucleus (33)), and hIL9R. As shown in Fig. 5B, actin could be detected only in the cytoplasmic/nuclear fraction and in total extracts. Likewise, most of the proliferating cell nuclear antigen was present in the cytoplasmic/nuclear fraction, although a small portion could be found in the membrane fraction. In contrast, both GH9 and QGH9 were exclusively detected in the membrane fraction at comparable levels. Longer exposures failed to detect either receptor in the cytoplasmic/nuclear fraction (data not shown), suggesting that both receptor forms are predominantly localized to the plasma membrane.

View larger version (36K): [in this window] [in a new window]   Fig. 5.   Q receptor is localized in the plasma membrane but does not bind hIL9. A, binding analysis of 125I-labeled hIL9 on TSLXSN, TSGR8, TSGH9, TSQGR8, and TSQGH9 cells. Specific binding (dotted bar) is compared with nonspecific binding (black bar) in the presence of a 200-fold excess of unlabeled human IL9. B, localization of GH9 and QGH9 receptors. TSLXSN, TSGH9, and TSQGH9 cells were lysed, and cytoplasmic/nuclear and membrane fractions were isolated. Protein fractions and total protein extracts were run in SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Immobilized proteins were sequentially reacted with antibodies specific for hIL9R, actin, and proliferating cell nuclear antigen (PCNA).

View larger version (123K): [in this window] [in a new window]   Fig. 6.   Immunofluorescence micrographs of COS7 cells expressing wild type or Q receptors. a, cells were transiently transfected with LXSN (A and B), GR8 (C and D), GH9 (E and F), QGR8 (G and H), or QGH9 (I and J) constructs. 48 h after transfection, cells were sequentially incubated with MAB290 neutralizing antibody (N-terminal specific) and anti-mouse IgG Texas Red-conjugated antibody (B, D, F, H, and J) and fixed as described under "Experimental Procedures." Cells were counterstained with 4,6-diamidino-2-phenylindole (A, C, E, G, and I) to visualize every cell in the photographed field. b, same as in a except that cells were first fixed/permeabilized and then incubated with a C-terminal specific antibody (sc698) followed by incubation with anti-rabbit IgG Texas Red-conjugated antibody. Bar, 10 µm.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Antibodies specific to the extracellular domain do not detect Q receptor. A, TSLXSN, TSGR8, TSGH9, TSQGR8, and TSQGH9 cells (see Fig. 2A) were sequentially incubated with a biotinylated N-terminal specific antibody (AH9R7) and phycoerythrin-labeled streptavidin. Specific fluorescence signal (thick line) was evaluated by FACS analysis. For autofluorescence controls (nonspecific), the primary antibody was omitted (thin line). B, TSLXSN, TSGH9, and TSQGH9 cells were lysed, and total proteins were incubated with sc698 (C-terminal specific) or with AH9R2 (top), AH9R7 (middle), and MAB290 (bottom) N-terminal specific antibodies and immunoprecipitated using Protein A + G-agarose-conjugated beads. Proteins were run in SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, and probed with sc698 antibody. Arrows indicate specific bands for hIL9R protein.

	DISCUSSION

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The interleukin-9 pathway has been implicated in the pathogenesis of asthma (12, 19).² This study was undertaken in an attempt to identify hIL9R structure variants that may explain the linkage to this locus in determining resistance or susceptibility to asthma and/or allergy. The data presented here describe the identification of several hIL9R variants. We identified numerous nucleotide changes within the intracellular coding region of the receptor (codons 310, 344, and 410-417/410-418) that did not affect proliferation or signal transduction induced by hIL9R (26, 31, 34). Furthermore, analysis of these variants in assays used to assess IL9-induced gene activation and anti-apoptosis activity (as described in Ref. 26) again failed to identify any functional differences among these isoforms (data not shown). However, these experiments do not preclude the involvement of these residues in receptor signaling functions for other IL9-induced biological processes not evaluated here, such as cell type-specific differentiation, antiapoptosis, or induced gene expression. Several studies have shown that IL9 has a direct role on the differentiation of blood precursors (35) and mast cells (16, 17), while others have demonstrated its stimulatory activity on B-lymphocytes (13, 14). In addition to cell type specificity, these receptor variants may also exert a differential activity on IL9-induced gene expression. The reporter assay used in our studies employed a reporter element that is dependent upon Stat activation. The effect of these receptor variants has not yet been tested on Stat-independent, IL9-induced gene expression. The finding that IL9 receptor mutants, which abolish Stat1, Stat3, or Stat5 activation, under certain circumstances are still able to induce cellular proliferation⁵ suggests that IL9 receptor may signal through motifs other than Jak and Stat domains. A differential phosphorylation of the SER8 and SER9 receptor variants could account for the electrophoretic pattern seen in these receptors and might serve as a regulatory mechanism in signaling events such as gene expression. In addition, two human transformed cell lines have been found to contain receptor variants (Arg³⁴⁴-SER9) not found in the >500 transcripts analyzed in this study. These alleles may have evolved from mutational event(s) that occurred during transformation in which the activation of IL9R is required for clonal expansion. This possibility is supported by the finding that IL9 overexpression is associated with T-cell lymphomas in IL9 transgenic mice (36).

Several alternative splice variants were also identified from our analyses. One class of alternative splice variants potentially encodes for soluble receptors due to alternatively spliced out exon 3, 4, or 5. These splice variants bear frameshifts and premature stop codons that in turn truncate the predicted polypeptide upstream of the residues encoded by exon 7, which encodes for the transmembrane domain. An alternative splice deletion of exon 8 was also identified that results in a putative membrane-bound form of receptor lacking the intracellular signaling domain sequences. These alternative splice forms may all be involved in serving as IL9 antagonists, as is the case of other cytokine receptors (such as IL4 and IL5 receptors), although the binding affinity of these receptors is greatly reduced (37-39).⁶ In addition to the splice variants described above, the abundant Q transcript was also identified. It was found expressed in 47 out of 50 individuals and ranged up to 50% of the total transcript within individual samples. The encoded protein was found to be present at the cell surface when transfected into a murine T-cell line but was unable to bind its known ligand. The cause for its decreased ligand affinity appears to be due to a conformational change as determined by the fact that it lacked reactivity to three different N-terminal antibodies that only recognize the native receptor. A potential role of the Q receptor may be to down-regulate IL9 receptor function. Negative regulation via alternative splicing has been previously documented for the human growth hormone and the thyroid hormone receptors in controlling molecular signaling of the wild type versions of these proteins (40, 41). Alternatively, a dominant negative model can be proposed. As demonstrated by studies on IL4 and IL6 receptors, ligand-induced homodimerization of hemopoietin receptor subunits may serve to co-approximate receptor-bound protein tyrosine kinases such as the members of the Jak family (42, 43). This dimerization then results in trans-phosphorylation and activation of Jak and receptor subunits, which in turn initiate the signal transduction cascade. The IL9 receptor could undergo a similar dimerization. The co-expression of the nonfunctional Q and wild type receptor subunits might lead to the formation of a complex incapable of eliciting an effective trans-phosphorylation and activation of the protein-tyrosine kinases. Since ligand binding seems to be required for subunit dimerization and the Q receptor lacks this property, this conjecture cannot be supported at the present time. However, it would be worthwhile to determine the effect of the ectopic co-expression of these receptors to address this hypothesis. These complex studies are now being attempted.

The aim of this study was to assess the variability in hIL9R structure in PBMCs derived from a cohort of unrelated individuals because of the recent findings that the hIL9R locus is linked to asthma and allergy susceptibility.² In this report, we have described the identification of multiple structural changes within the hIL9R transcript. While no biological differences were found in the receptors bearing amino acid substitutions, a clear difference was found in the Q splice variant. The abundance of Q transcript appeared to be variable among individuals within our cohort (0-50%; see Table I). Moreover, the frequency of Q transcript was found to be statistically less in the allergic and/or asthmatic group as compared with the nonallergic group (18.3 and 28.7%, respectively (p < 0.05)). However, we believe that studies on additional populations will be required to further validate the significance of these results. We are currently analyzing the hIL9R locus for genomic changes that may influence the overall rate of Q splicing and account for the genetic linkage data. Nucleotide sequences within distal and proximal intronic sequences as well as in 5'-flanking sequences have been documented to influence splicing (20, 45). The identification of a polymorphism that might affect the Q splicing will grant us the use of a more quantitative genetic approach to study large populations of nonallergic and allergic and/or asthmatic individuals. This will allow us a better evaluation of the potential association of this receptor type with asthma and/or allergy.