Two modes of β-receptor recognition are mediated by distinct epitopes on mouse and human interleukin-3

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 α-subunit and common β-subunit (βc; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a β-subunit that specifically binds IL-3 (β_IL-3; present in mice but not humans). We recently identified two splice variants of the α-subunit of the IL-3 receptor (IL-3Rα) 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) Rα isoform can direct mIL-3 binding to two distinct sites on the β_IL-3 subunit, it has remained unclear which residues in mIL-3 itself are critical to the two modes of β_IL-3 recognition and whether the human IL-3Rα SP1 and SP2 orthologs similarly instruct human IL-3 binding to two distinct sites on the human βc subunit. Herein, we describe the identification of residues clustering around the highly conserved A-helix residue, Glu²³, in the mIL-3 A- and C-helices as critical for receptor binding and growth stimulation via the β_IL-3 and mIL-3Rα SP2 subunits, whereas an overlapping cluster was required for binding and activation of β_IL-3 in the presence of mIL-3Rα SP1. Similarly, our studies of human IL-3 indicate that two different modes of βc binding are utilized in the presence of the hIL-3Rα SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.