Interleukin-3 Binding to the Murine beta sub(IL-3) and Human beta c 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 beta -common (h beta c) receptor and the murine IL-3-specific ( beta sub(IL-3)) receptor in IL-3 binding. We show that the domain 1 residues, Tyr super(15) and Phe super(79), of the h beta 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 beta 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 beta sub(IL-3) receptor is also a homodimer. Its high sequence homology with h beta c suggests that their structures are homologous, and we identified an analogous binding interface in beta sub(IL-3) for direct IL-3 binding to the high affinity binding site in h beta c. Tyr super(21) (A-B loop), Phe super(85), and Asn super(87) (E-F loop) of domain 1; Ile super(320) of the interdomain loop; and Tyr super(348) (B'-C' loop) and Tyr super(401) (F'-G' loop) of domain 4 were shown to have critical individual roles and Arg super(84) and Tyr super(317) major secondary roles in direct murine IL-3 binding to the beta sub(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 h beta c.