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


CONCLUSIONS
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.
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.
Jun
J
Zhong
Akhilesh
A
Pandey
D000818:Animals; D049109:Cell Proliferation; D016207:Cytokines / Q000378:metabolism; D003593:Cytoplasm; D006801:Humans; D007136:Immunoglobulins; D015851:Interleukin-7 / Q000276:immunology; D046912:Multiprotein Complexes; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018121:Receptors, Cytokine / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020395:Receptors, Interleukin-7 / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D016415:Sequence Alignment; D015398:Signal Transduction; D014443:Tyrosine; D000094632:Thymic Stromal Lymphopoietin
D016207:Cytokines; D007136:Immunoglobulins; D015851:Interleukin-7; D046912:Multiprotein Complexes; D018121:Receptors, Cytokine; D020395:Receptors, Interleukin-7; C410591:Tslpr protein, mouse; C415350:interleukin-7 receptor, alpha chain; D014443:Tyrosine; D000094632:Thymic Stromal Lymphopoietin