Abstract
The cytokine interleukin-3 (IL-3) is a critical regulator of inflammation and immune responses in mammals. IL-3 exerts its effects on target cells via receptors comprising an IL-3-specific alpha-subunit and common beta-subunit (beta c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a beta-subunit that specifically binds IL-3 (beta(IL-3); present in mice but not humans). We recently identified two splice variants of the alpha-subunit of the IL-3 receptor (IL-3R alpha) that are relevant to hematopoietic progenitor cell differentiation or proliferation: the full length ("SP1" isoform) and a novel isoform (denoted "SP2") lacking the N-terminal Ig-like domain. Although our studies demonstrated that each mouse IL-3 (mIL-3) R alpha isoform can direct mIL-3 binding to two distinct sites on the beta(IL-3) subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of beta(IL-3) recognition and whether the human IL-3R alpha SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human beta c subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu(23), in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the beta(IL-3) and mIL-3R alpha SP2 subunits, whereas an overlapping cluster was required for binding and activation of beta(IL-3) in the presence of mIL-3R alpha SP1. Similarly, our studies of human IL-3 indicate that two different modes of beta c binding are utilized in the presence of the hIL-3R alpha SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.
Highlights
Interleukin-3 (IL-3)3 is a cytokine produced principally by activated T-cells during immune responses that is known to be a key regulator of inducible hematopoiesis and inflammation
Our studies demonstrated that each mouse IL-3 R␣ isoform can direct mIL-3 binding to two distinct sites on the IL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of IL-3 recognition and whether the human IL-3R␣ SP1 and SP2 orthologs instruct human IL-3 binding to two distinct sites on the human c subunit
Activation of Janus kinase 2 (Jak2) and Erk1/2 Signaling by Wild-type and E23A mIL-3—To examine the roles of the two distinct mIL-3 binding modes in governing the initiation of intracellular signaling, we investigated the capacity of wild-type and E23A mIL-3 to activate Jak2 and Erk1/2 in two mIL-3-dependent selfrenewal cell line models: Ba/F3, a pro-B cell line; and Stem cell factor (SCF) ERHoxb8, a neutrophil progenitor cell line prevented from undergoing differentiation when cultured in the presence of -estradiol, which is required for the activity of a retrovirally introduced Hoxb8 gene [26]
Summary
Cell Lines—The generation of CTLL2 cell lines stably expressing wild-type or mutant mIL-3R␣ SP1, mIL-3R␣ SP2, hIL-3R␣ SP1, or hIL-3R␣ SP2 and wild-type or mutant hc or IL-3 subunits was described previously [16]. Labeled wild-type mIL-3 or hIL-3, respectively, to saturate relevant receptors stably expressed in CTLL2 cells, cold competition binding assays were performed on CTLL2 cells stably transfected with relevant receptors. For the preparation of lysates of Ba/F3 and SCF ER-Hoxb neutrophil progenitors, Ba/F3 or SCF ER-Hoxb cells were washed with plain RPMI 1640 or OptiMEM, respectively, three times and starved in plain RPMI 1640 or OptiMEM, respectively, for 4 h before incubation with or without 1% (v/v) conditioned insect cell medium containing either wild-type or E23A mutant mIL-3 for 10 min. Total protein loading was controlled by gel staining with GelCode Blue (Pierce) and visualizing protein bands using a Fuji LAS-1000 Plus Imager (Fujifilm)
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