Abstract
Interleukin-3 (IL-3) is a cytokine produced by activated T-cells and mast cells that is active on a broad range 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 beta-common (hbetac) receptor and the murine IL-3-specific (beta(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr(15) and Phe(79), of the hbetac 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 hbetac, 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 beta(IL-3) receptor is also a homodimer. Its high sequence homology with hbetac suggests that their structures are homologous, and we identified an analogous binding interface in beta(IL-3) for direct IL-3 binding to the high affinity binding site in hbetac. Tyr(21) (A-B loop), Phe(85), and Asn(87) (E-F loop) of domain 1; Ile(320) of the interdomain loop; and Tyr(348) (B'-C' loop) and Tyr(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg(84) and Tyr(317) major secondary roles in direct murine IL-3 binding to the beta(IL-3)receptor. Most surprising, none of the key residues for direct IL-3 binding were critical for high affinity binding in the presence of the murine IL-3 alpha receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hbetac.
Highlights
Newal of pluripotent hematopoietic stem cells and to be a potent regulator of many hematopoietic cell lineages [1,2,3]
The participation of domain 1 in ligand binding has been supported by recent mutagenesis studies that have shown that residues in the A–B and E–F loops of domain 1 of hc are required for the formation of the high affinity human GM-CSF complex and for receptor activation by hGM-CSF and human IL-5 [31]
Residues are contributed from a combination of the A–B and E–F loops of the membrane-distal domain and the BЈ–CЈ and FЈ–GЈ loops of the membrane-proximal domain to form a cluster of residues critical for ligand binding
Summary
Newal of pluripotent hematopoietic stem cells and to be a potent regulator of many hematopoietic cell lineages [1,2,3]. The human c (hc), mc, and IL-3 receptors are closely homologous in sequence and belong to the hematopoietin (or class I cytokine) receptor superfamily Members of this family have extracellular domains containing the cytokine-receptor homology module composed of two fibronectin III domains that have a number of conserved sequence elements [16, 17]. Mutagenesis studies of these receptors have shown that within the structural epitopes only a relatively small number of residues, which compose the functional epitope, are critical determinants of ligand binding [21,22,23,24,25] These residues are contributed by a combination of the A–B and E–F loops of the membrane-distal fibronectin III domain and the BЈ–CЈ and FЈ–GЈ loops of the membrane-proximal domain. A detailed analysis of the residues involved in direct and high affinity IL-3 binding has not been carried out previously nor has the structure of the IL-3 subunit extracellular domain been determined
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