Two Modes of β-Receptor Recognition Are Mediated by Distinct Epitopes on Mouse and Human Interleukin-3

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 ␣-subunit and common ␤-subunit (␤c; shared with IL-5 and granulocyte-macrophage colony-stimulating factor) or a ␤-subunit that specifically binds IL-3 (␤ IL-3 ; present in mice but not humans). We recently identified two splice variants of the ␣-subunit of the IL-3 receptor (IL-3R␣) 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␣ 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 similarly instruct human IL-3 binding to two distinct sites on the human ␤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 ␤ IL-3 and mIL-3R␣ SP2 subunits, whereas an overlapping cluster was required for binding and activation of ␤ IL-3 in the presence of mIL-3R␣ SP1. Similarly, our studies of human IL-3 indicate that two different modes of ␤c binding are utilized in the presence of the hIL-3R␣ SP1 or SP2 isoforms, suggesting a possible conserved mechanism by which the relative orientations of receptor subunits are modulated to achieve distinct signaling outcomes.

The Role of Interchain Heterodisulfide Formation in Activation of the Human Common β and Mouse βIL-3 Receptors

“…The differential effects observed for ligand binding by h␤c and ␤ IL-3 when co-expressed with the full-length IL-3␣ (SP1) or the splice variant lacking the N-terminal Ig-like domain (SP2) are consistent with our prior work suggesting that the two IL-3␣ isoforms dictate which of two distinct IL-3 binding interfaces on the ␤ receptors are used (25,31,47). It is clear that the ligand-binding epitopes on h␤c and ␤ IL-3 utilized by the IL-3⅐IL-3R␣ SP2 complex are the most affected by disruption of the domain 1 D-E loop disulfide.…”

The GM–CSF/IL‐3/IL‐5 cytokine receptor family: from ligand recognition to initiation of signaling

“…Although the location of these additional βc‐contact residues in human IL‐3 remains unknown, extensive mutagenesis studies of residues in helix C indicate that this region is not essential for IL‐3 function. The discovery of splice isoforms of the human and murine IL3Rα subunits lacking the NTD has revealed an interesting variation in IL‐3 interaction with the β‐subunit provided by the two isoforms . Thus, hIL‐3 E22A is a weak agonist through the full‐length IL3Rα isoform (SP1), but is completely devoid of agonist activity in the context of the IL3Rα isoform (SP2) lacking the NTD.…”

“…Thus, hIL‐3 E22A is a weak agonist through the full‐length IL3Rα isoform (SP1), but is completely devoid of agonist activity in the context of the IL3Rα isoform (SP2) lacking the NTD. Murine IL‐3 mutated at the critical conserved glutamate, E23A, is a full agonist on cells expressing murine IL3Rα SP1 and βIL‐3, but is completely devoid of agonist activity on cells expressing murine IL3Rα SP2 and βIL‐3 as well as being unable to directly bind βIL‐3 . Additional residues in mIL‐3 have also been identified as functionally important βIL‐3 contact residues including K27 in helix A and E65, V69, and N73 in helix C although, apart from E65, the importance of these additional contacts is largely revealed only in the presence of IL3Rα SP2 .…”

“…Murine IL‐3 mutated at the critical conserved glutamate, E23A, is a full agonist on cells expressing murine IL3Rα SP1 and βIL‐3, but is completely devoid of agonist activity on cells expressing murine IL3Rα SP2 and βIL‐3 as well as being unable to directly bind βIL‐3 . Additional residues in mIL‐3 have also been identified as functionally important βIL‐3 contact residues including K27 in helix A and E65, V69, and N73 in helix C although, apart from E65, the importance of these additional contacts is largely revealed only in the presence of IL3Rα SP2 . Not only do these results emphasize the critical role played by the conserved glutamate in βc binding by IL‐3 but they also reveal that alternative modes of IL‐3 presentation to βc can utilize secondary or peripheral contact residues.…”

Interleukin-5 and IL-5 receptor in health and diseases

“…The X-ray structure of the hβc is an intertwined homodimer in which each chain contains four domains with approximate fibronectin type-III topology. 80 ) By fluorescence resonance energy transfer imaging, βc subunit is demonstrated to exist as preformed homo-oligomers and the IL-5 stimulation induces βc assembly in the presence of IL-5Rα. 81 )…”