BMC Immunol. 2010 Feb 8;11:5. doi: 10.1186/1471-2172-11-5.

Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7Ralpha and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation.

Source

McKusick-Nathans Institute of Genetic Medicine and Departments of Biological Chemistry, Oncology and Pathology, Johns Hopkins University School of Medicine, 733 N Broadway, Baltimore, Maryland 21205, USA.

Abstract

BACKGROUND:

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 alpha chain (IL-7Ralpha) 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-7Ralpha 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-7Ralpha and the TSLPR in inducing cell proliferation in a growth factor dependent cell line, Ba/F3.

RESULTS:

A multiple sequence alignment of the IL-7Ralpha 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-7Ralpha is required for IL-7 dependent proliferation. Interestingly, in contrast to IL-7 signaling, none of tyrosine residues in the human IL-7Ralpha 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-7Ralpha and human TSLPR to phenylalanine residues abolished the proliferative ability of the TSLP receptor complex in response to TSLP.

CONCLUSION:

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-7Ralpha and TSLPR cytoplasmic domains play a redundant role in TSLP-mediated cell growth.

PMID:: 20144186; [PubMed - indexed for MEDLINE]
PMCID:: PMC2836284

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Figure 2

The lone cytoplasmic tyrosine residue of human TSLPR is not required for TSLP-dependent cell proliferation. (A) A schematic illustration of the human TSLP receptor complex composed of the human IL-7Rα and the human TSLPR. Y denotes the cytoplasmic tyrosine residues. (B) Exponentially growing Ba/F3 cells were starved and then treated with human TSLP (100 ng/ml). The data are represented as fold changes with the number of cells seeded on day 0 (for TSLP stimulation) representing the baseline. The mean of experiments done in triplicate for each time point and each cell line is shown. Error bars indicate S.E.M. (C) Surface expression of the wild type human TSLPR and IL-7Rα in the cell lines used in (B). Exponentially growing infected Ba/F3 cells were analyzed by flow cytometry for cell surface expression of human TSLPR and IL-7Rα. (D) TSLP-induced proliferation of Ba/F3 cells co-expressing the indicated were examined as described in (B). Statistical significance of the proliferative responses in the different cells at the same time point was evaluated using unpaired Student's t-test (* indicates a significant difference with p-value < 0.01). (E). Exponentially growing Ba/F3 cells were starved and then stimulated with the indicated concentration of human TSLP for 3 days. Cell numbers were counted after 3 days. The data are represented as fold changes with the number of cells seeded on day 0 (for TSLP stimulation) representing the baseline. Statistical significance of the proliferative responses in the different cells in response to the same dose of TSLP was evaluated using unpaired Student's t-test (* indicates a significant difference with p-value < 0.01). (The growth of cells was examined in 3 independent sets of experiments and found to be similar. The results from one representative experiment are shown).

Site-directed mutagenesis reveals a unique requirement for tyrosine residues in IL-7R? and TSLPR cytoplasmic domains in TSLP-dependent cell proliferation

BMC Immunol. 2010;11:5-5.