Interleukin-3 binding to the murine β_IL-3 and human βc receptors involves functional epitopes formed by domains 1 and 4 of different protein chains

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 β-common (hβc) receptor and the murine IL-3-specific (β_IL-3) receptor in IL-3 binding. We show that the domain 1 residues, Tyr¹⁵ and Phe⁷⁹, of the hβc 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 hβc, 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 β_IL-3 receptor is also a homodimer. Its high sequence homology with hβc suggests that their structures are homologous, and we identified an analogous binding interface in β_IL-3 for direct IL-3 binding to the high affinity binding site in hβc. Tyr21 (A-B loop), Phe⁸⁵, and Asn⁸⁷ (E-F loop) of domain 1; Ile³²⁰ of the interdomain loop; and Tyr³⁴⁸ (B′-C′ loop) and Tyr⁴⁰¹ (F′-G′ loop) of domain 4 were shown to have critical individual roles and Arg⁸⁴ and Tyr³¹⁷ major secondary roles in direct murine IL-3 binding to the β_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 α receptor, indicating a fundamentally different mechanism of high affinity binding to that used by hβc.