Interleukin-3 Binding to the Murine βIL-3 and Human βc Receptors Involves Functional Epitopes Formed by Domains 1 and 4 of Different Protein Chains

Abstract

Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broadrange of hematopoietic cells and in the nervous system and appears to be important in several chronic inflammatory diseases. In this study, alanine substitutions were used to investigate the role of residues of the human ␤-common (h␤c) receptor and the murine IL-3-specific (␤ IL-3 ) receptor in IL-3 binding. We show that the domain 1 residues, Tyr 15 and Phe 79 , of the h␤c receptor are important for high affinity IL-3 binding and receptor activation as shown previously for the related cytokines, interleukin-5 and granulocyte-macrophage colony-stimulating factor, which also signal through this receptor subunit. From the x-ray structure of h␤c, it is clear that the domain 1 residues cooperate with domain 4 residues to form a novel ligand-binding interface involving the two protein chains of the intertwined homodimer receptor. We demonstrate by ultracentrifugation that the ␤ IL-3 receptor is also a homodimer. Its high sequence homology with h␤c suggests that their structures are homologous, and we identified an analogous binding interface in ␤ Interleukin-3 (IL-3) 1 is a cytokine produced by activated T-cells and mast cells that has been shown to stimulate renewal of pluripotent hematopoietic stem cells and to be a potent regulator of many hematopoietic cell lineages (1-3). Its role appears to be in stimulating inducible hematopoiesis in response to parasite infections (4), and it has also been implicated in the pathogenesis of several chronic inflammatory diseases, including asthma (1), and neurodegenerative disorders, such as multiple sclerosis (5). The effect of IL-3 on human cells is mediated by a receptor system composed of a ligand-specific ␣ subunit and a ␤ subunit (denoted ␤c) that is also part of the receptor systems for the related cytokines, interleukin-5 (IL-5) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (6 -9). Signaling through the ␤c receptor requires the formation of a high affinity complex involving each cytokine and its respective ␣ subunit (7-10). Whereas the ␣ subunits bind their ligands with low affinity, ␤c does not measurably bind any of the ligands alone. Upon receptor activation, the cytoplasmic portion of the ␤c subunit, which lacks any intrinsic kinase activity (11), initiates a number of signaling pathways including the Janus kinase 2/signal transducers and activators of transcription, phosphatidylinositol 3-kinase, and Ras/mitogen-activated protein kinase pathways (reviewed in Ref. 12).Mice also possess a ␤c subunit (m␤c) but have an additional IL-3-specific ␤ receptor (␤ IL-3 ). ␤ IL-3 differs from the m␤c subunit in its ability to bind murine IL-3 (mIL-3) directly (13), although the presence of the mIL-3 ␣ subunit is absolutely required for signaling (14). The properties of mIL-3-responsive precursor cells from gene knock-out mice lacking expression of the ␤ IL-3 subunit indicate that this subunit plays an important role in the response to mIL-3 stimulation (15)....

Interleukin-5 Receptor Subunit Oligomerization and Rearrangement Revealed by Fluorescence Resonance Energy Transfer Imaging

“…Previous high resolution structure analyses have shown that the extracellular region of ␤c can exist as a dimer (44,48). In this structure, the ␤c is a homodimer stabilized by domain swapping between domain 1 (D1) of one chain and domain 3 (D3) of the symmetry-related chain (Fig.…”

Slow-dissociation effect of common signaling subunit β on IL5 and GM-CSF receptor assembly

“…4A). Structural integrity of these mutants was previously verified by measuring their cell surface expression with flow cytometry [23] [24]. The two alanine-mutational variants, sβc(Y18A) and sβc(Y344A), were successfully expressed in Drosophila S2 cells and secreted into the cell culture in amounts roughly equal to that achieved for wild type sβc protein.…”

“…Tyr18 and Tyr344 of βc (corresponding to Tyr15 and Tyr347 in the crystal structure [25]) have been found to be critical for IL5, IL3 and GM-CSF association into their respective trimolecular complexes [21] [22] [23] [24]. These residues are located in a hypothetical ligand-binding interface of D1 and D4 domains (Fig.…”

“…Although βc itself has no measurable affinity for cytokine in a bimolecular (cytokine·βc) complex [9] [10] [11] [19] [20], it is conceivable that IL5, IL3 and GM-CSF make direct contacts with βc in the high affinity trimolecular (cytokine·α·βc) complex. The ligand-binding epitope in βc appears to be composed of mostly aromatic residues from D1 (Tyr18, Phe82) and D4 (Tyr344, Tyr400) domains [21] [22] [23] [24]. Crystallographic analysis has shown that the extracellular regions of βc exist as a domain-swapped dimer composed of two identical polypeptide chains [25], and that these binding residues are clustered in the interface of D1 and D4, domains from each polypeptide chain, that constitute a CRM in the three dimensional structure [23] [24].…”

“…The ligand-binding epitope in βc appears to be composed of mostly aromatic residues from D1 (Tyr18, Phe82) and D4 (Tyr344, Tyr400) domains [21] [22] [23] [24]. Crystallographic analysis has shown that the extracellular regions of βc exist as a domain-swapped dimer composed of two identical polypeptide chains [25], and that these binding residues are clustered in the interface of D1 and D4, domains from each polypeptide chain, that constitute a CRM in the three dimensional structure [23] [24].…”