Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation
© Zhong and Pandey; licensee BioMed Central Ltd. 2010
Received: 20 April 2009
Accepted: 8 February 2010
Published: 8 February 2010
Thymic stromal lymphopoietin (TSLP) is an interleukin-7 (IL-7) like cytokine, which plays an important role in the regulation of immune responses to allergens. TSLP binds to a heterodimeric receptor complex composed of the IL-7 receptor α chain (IL-7Rα) and the TSLP receptor (TSLPR, also known as CRLF2). It has previously been suggested that the lone tyrosine residue in the mouse TSLPR cytoplasmic domain is required for cell proliferation using chimeric receptor systems. Also the role of tyrosine residues in the IL-7Rα cytoplasmic domain in TSLP signaling has not yet been investigated. We undertook a systematic analysis to test the role of tyrosine residues of both the IL-7Rα and the TSLPR in inducing cell proliferation in a growth factor dependent cell line, Ba/F3.
A multiple sequence alignment of the IL-7Rα and TSLPR cytoplasmic domains revealed conservation of most, but not all, cytoplasmic tyrosine residues across several species. Our site-directed mutagenesis experiments revealed that the single tyrosine residue in human TSLPR was not required for TSLP-dependent cell proliferation. It has previously been reported that Y449 of human IL-7Rα is required for IL-7 dependent proliferation. Interestingly, in contrast to IL-7 signaling, none of tyrosine residues in the human IL-7Rα cytoplasmic domain were required for TSLP-dependent cell proliferation in the presence of a wild type TSLPR. However, the mutation of all cytoplasmic four tyrosine residues of human IL-7Rα and human TSLPR to phenylalanine residues abolished the proliferative ability of the TSLP receptor complex in response to TSLP.
These results suggest that TSLP requires at least one cytoplasmic tyrosine residue to transmit proliferative signals. Unlike other members of IL-2 cytokine family, tyrosine residues in IL-7Rα and TSLPR cytoplasmic domains play a redundant role in TSLP-mediated cell growth.
Thymic stromal lymphopoietin (TSLP) was first identified as a growth factor in the conditioned medium supernatant from the Z210R.1 thymic stromal cell line to support B cell proliferation in vitro [1, 2]. TSLP is now known to play a key role in the initiation of asthma [3, 4]. TSLP shares IL-7Rα with IL-7. TSLP signaling is mediated by a heterodimeric receptor complex, which consists of the interleukin-7 receptor α chain (IL-7Rα) and a unique TSLP-binding receptor (TSLPR), to transmit proliferative signals in cells [5–7]. IL-7 binds to a heterodimeric receptor complex, the IL-7Rα and the cytokine receptor common gamma chain (γc), which is shared by IL-2, 4, 7, 9, 15 and 21. Both TSLP and IL-7 can activate the transcription factor STAT5. In the IL-7 receptor complex, IL-7Rα binds to Jak1 and γc binds to Jak3 upon addition of IL-7. However, none of Jak kinases have been reported to be phosphorylated by the binding of TSLP to its receptor . Previous studies showed that Y449 in the IL-7Rα cytoplasmic domain provides a docking site for PI-3K and STAT5 and is required for a proliferative signal by IL-7 signaling [8–10]. Further, the tyrosine residues of γc are not required for IL-7-mediated cell proliferation . Isaksen and colleagues observed that the lone tyrosine residue of the mouse TSLPR cytoplasmic domain is required for TSLP-mediated cell proliferation using chimeric receptors composed of the human GM-CSFR α chain extracellular domain fused to the mouse TSLPR transmembrane and cytoplasmic domains and the human GM-CSFR β chain extracellular domain fused to the mouse IL-7Rα transmembrane and cytoplasmic domains . Brown et al. showed that anti-IL-7Rα antibodies inhibited TSLP-mediated proliferation of pre B-leukemia , indicating that both IL-7Rα and TSLPR contribute to TSLP-dependent cell proliferation.
We aligned the protein sequences of IL-7Rα and TSLPR cytoplasmic domains and observed that Y449 and Y456 of human IL-7Rα and Y368 of human TSLPR were conserved across the species examined while Y401 of human IL-7Rα was not conserved. Because the role of tyrosine residues in the context of the 'native' form TSLP receptor complex in TSLP-mediated cell proliferation has not been previously investigated, we took a systematic approach to evaluate the role of cytoplasmic tyrosine residues of TSLP receptor complex in mediating TSLP-induced cell proliferation. Our data show that the cytoplasmic tyrosine residues of either human IL-7Rα or human TSLPR can mediate TSLP-induced cell proliferation and that mutation of all the four cytoplasmic tyrosine residues of human IL-7Rα and human TSLPR to phenylalanine residues is required to abolish TSLP-dependent cell proliferation.
Results and Discussion
Most, but not all, cytoplasmic tyrosine residues of IL-7Rα and TSLPR are conserved across species
The lone cytoplasmic tyrosine residue in TSLPR is not required for TSLP-dependent cell proliferation
Human TSLPR contains only one cytoplasmic tyrosine residue (Y368) very close to the carboxyl terminus (Figure 1A). To determine whether this residue is required for TSLP-mediated cell proliferation, it was replaced by a phenylalanine residue (Y368F). A Ba/F3 cell line expressing both hTSLPR (Y368F) and hIL7Rα (WT) was established using retrovirus-based infection. As shown in Figure 2D and 2E, mutation of this tyrosine residue failed to abolish the proliferative response to TSLP. On day 3 of culture, 100 ng/ml TSLP induced ~25% more proliferation in Ba/F3 cells expressing hTSLPR (Y368F)/hIL-7Rα (WT) than Ba/F3 cells expressing hTSLPR (WT)/hIL-7Rα (WT) (p-value < 0.01) (Figure 2D). To examine this in greater detail, we carried out a dose response study (Figure 2E). We observed that in contrast to what was observed at 100 ng/ml, Ba/F3 cells expressing the hTSLPR (Y368F)/hIL-7Rα (WT) pair grew at a slower rate (p-value < 0.01) than Ba/F3 cells expressing the hTSLPR (WT)/hIL-7Rα (WT) in response to low doses of TSLP (1 ng/ml and 10 ng/ml). There was no statistical difference between the two at 0.01 ng/ml and 0.1 ng/ml. The cell surface expression of human TSLPR and human IL-7Rα was similar across the cell lines as confirmed by FACS using anti-human TSLPR and anti-human IL-7Rα antibodies (data not shown). Nevertheless, in contrast to a previous report using a chimeric receptor system , our data showed that the lone tyrosine residue in the TSLPR cytoplasmic domain is not required for TSLP-dependent cell proliferation. Our findings suggest that studies about TSLP-mediated signaling pathways should be carried out in the context of the native TSLP receptor complex. Intriguingly, these data suggest that Y368 in the hTSLPR cytoplasmic domain may play an inhibitory role in TSLP signaling in response to high doses of TSLP while playing a positive role at lower doses.
The cytoplasmic tyrosine residues in IL-7Rα are required for IL-7 but not TSLP-dependent cell proliferation
TSLP-dependent cell proliferation requires the presence of at least one tyrosine residue
To test if any tyrosine residues were required for TSLP-induced proliferation, we mutated all tyrosine residues in the TSLP receptor complex (TSLPR (Y368F)/IL-7Rα (Y401F/Y449F/Y456F)). As shown in Figure 5G and 5H, this abolished the ability of the human TSLP receptor complex to drive cell proliferation. Taken together, our data suggest that TSLP signaling requires at least one tyrosine residue to support cell growth while all the four tyrosine residues in human TSLP receptor complex play various roles in TSLP-mediated cell proliferation.
STAT5 phosphorylation requires at least one cytoplasmic tyrosine residue in the human TSLP receptor complex
Here we report that mutation of all four cytoplasmic tyrosine residues in the TSLP receptor complex abolishes the proliferative response to TSLP as well as STAT5 phosphorylation. Mutation of individual tyrosine residues in the cytoplasmic domain of human IL-7Rα or TSLPR was not sufficient to abolish this proliferative response although it did impair the response at low doses of TSLP (1 ng/ml and 10 ng/ml). Intriguingly, these mutations led to an increase in the proliferation rate in response to high doses of TSLP (100 ng/ml). Further experimentation will be required to elucidate the mechanistic basis of the signals that are mediated by the TSLP receptor complex. In this regard, it is interesting to note that the cytosolic tyrosine residues in IL-7Rα seem to be dispensable for TSLP, but not IL-7, signaling.
PE-conjugated streptavidin, PE-conjugated anti-human CD4, PE-conjugated anti-human γc, and Alexa Fluor®647 conjugated anti-human IL-7Rα antibodies were from BD Biosciences Pharmingen (San Jose, CA, USA). Anti-phosphotyrosine antibodies (HRP-conjugated) were from Millipore (Billerica, MA, USA) and anti-Stat5a antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Recombinant mouse IL-3, recombinant human IL-7, recombinant human TSLP and biotinylated anti-human TSLPR antibodies were from R&D Systems (Minneapolis, MN). RPMI 1640, fetal bovine serum (FBS), L-glutamine, and antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). QuikChange XL-II mutagenesis kit was from Stratagene (La Jolla, CA, USA). Sequencing services were performed by the DNA and Peptide Synthesis and Sequencing facility at Johns Hopkins University School of Medicine using dye terminator chemistry. All other reagents used in this study were from Fisher Scientific (Pittsburgh, PA, USA).
The interleukin-3 (IL-3)-dependent pre B cell line, Ba/F3, was grown in RPMI 1640 supplemented with FBS, L-glutamine, penicillin, streptomycin, and mouse recombinant IL-3 (10 ng/ml). Stably transfected Ba/F3 cell lines were grown in RPMI 1640 supplemented with heat-inactivated FBS, L-glutamine, penicillin, streptomycin and mouse recombinant IL-3 (10 ng/ml). Human embryonic kidney 293T (HEK293T) cells were cultured in Dulbecco modified essential medium, high glucose, supplemented with FBS, L-glutamine, penicillin, and streptomycin. Cell lines were maintained in the exponential growth phase unless indicated otherwise.
Plasmids and expression vectors
The human TSLPR was obtained from Dr. James Ihle . The human IL-7Rα was subcloned into a bicistronic retrovirus vector pMX-IRES-GFP  that expresses GFP while the human TSLPR and γc were subcloned into a bicistronic retrovirus vector pMX-IRES-hCD4  that expresses the human CD4 antigen. The human IL-7Rα, γc and TSLPR mutants were generated by site-directed mutagenesis and confirmed by sequencing.
Generation of stable cell lines using retroviruses
HEK293T cells were transfected with the retroviral vector constructs (12 μg) together with the helper virus pCL-ECO (12 μg) (Imegenex, San Diego, CA, USA) by using Lipofectamine 2000 (Invitrogen). Twenty-four hours after transfection, the supernatants were harvested and filtered through a 0.45-μm filter. Ba/F3 cells were infected with the pairs of the human IL-7Rα in pMX-IRES-GFP and the human TSLPR or γc in pMX-IRES-hCD4. 48 hours after infection, Ba/F3 cells were stained by PE-conjugated anti-human CD4 antibodies and then sorted for GFP and the human CD4 expression.
Receptors cell surface expression
After sorting, exponentially growing Ba/F3 cells expressing the pair of the wild type/mutated TSLP receptor complex were washed twice, stained with biotinylated anti-human TSLPR antibodies followed by PE-conjugated streptavidin and Alexa Fluor®647 conjugated anti-human IL-7Rα and analyzed by flow cytometry. Similarly, exponentially growing Ba/F3 cells expressing the pair of the wild type/mutated IL-7 receptor complex were washed twice, stained with PE-conjugated anti-human γc, and Alexa Fluor®647 conjugated anti-human IL-7Rα antibodies and analyzed by flow cytometry.
Immunoprecipitation and Western blotting
Exponentially growing Ba/F3 cells expressing different combinations of receptors were washed three times with RPMI 1640, deprived of IL-3 for 16 h, and then left untreated or stimulated with recombinant human TSLP or mouse IL-3 for 10 minutes at 37°C. Cells were lyzed in modified RIPA buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, and 1 mM sodium orthovanadate in the presence of protease inhibitors) followed by centrifugation. The supernatant was subjected to immunoprecipitation using anti-Stat5a antibodies. After washing, the immunoprecipitates were resolved by SDS-PAGE and assayed by Western blotting with anti-phosphotyrosine antibodies (4G10) followed by reprobing with anti-Stat5a antibodies.
The growth of cells expressing different combination of receptors was examined in 3 independent sets of experiments. In each experiment, exponentially growing Ba/F3 cells (in triplicate) expressing different combinations of receptors were washed three times with RPMI 1640, deprived of IL-3 for 16 h, and then stimulated with recombinant human TSLP or IL-7. Living cells were counted using a Beckman Coulter Z1 (Beckman Coulter, Fullerton, CA, USA). The results (except where indicated) are expressed as increase in cell number (stimulation [n-fold]) as compared to the number of cells plated on day 0. The mean and S.E.M were calculated for each independent experiment. The results from the three independent experiments were similar in each case and one representative experiment is shown in the figures.
Protein sequence alignments
The protein sequence alignments were performed using ClustalX version 2.0.10 using the BLOSUM62 matrix.
Data were expressed as mean ± SEM. Differences were examined by Student's t-test between two groups. p < 0.05 was considered significant.
List of abbreviations
thymical stromal lymphopoietin
interleukin-7 receptor α chain
interleukin-2 receptor γ chain/common γ chain.
We thank Dr. James Ihle for providing the human TSLP receptor and Dr. Xuedong Liu for providing pMX-IRES-GFP and pMX-IRES-hCD4 plasmids. Cell sorting was performed by Lee Blosser and Ada Tam in flow cytometry core facility at Johns Hopkins University School of Medicine.
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