title: Interleukin-3 binding to the murine betaIL-3 and human betac 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 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.
authors.affiliation: Division of Molecular Bioscience, John Curtin School of Medical Research and Research School of Chemistry, Australian National University, Canberra, ACT 0200, Australia.
authors.forename: James M
authors.initials: JM
authors.lastname: Murphy
authors.forename: Sally C
authors.initials: SC
authors.lastname: Ford
authors.forename: Jane E
authors.initials: JE
authors.lastname: Olsen
authors.forename: Sonja E
authors.initials: SE
authors.lastname: Gustin
authors.forename: Peter D
authors.initials: PD
authors.lastname: Jeffrey
authors.forename: David L
authors.initials: DL
authors.lastname: Ollis
authors.forename: Ian G
authors.initials: IG
authors.lastname: Young
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002455:Cell Division; D003432:Cross-Linking Reagents / Q000494:pharmacology; D018360:Crystallography, X-Ray; D053648:Cytokine Receptor Common beta Subunit; D016207:Cytokines / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes; D005434:Flow Cytometry; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D006801:Humans; D007377:Interleukin-3 / Q000378:metabolism*; D015848:Interleukin-5 / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000737:chemistry* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014461:Ultracentrifugation
chemical_list: C115876:CSF2RB protein, human; D003432:Cross-Linking Reagents; D053648:Cytokine Receptor Common beta Subunit; D016207:Cytokines; D018076:DNA, Complementary; D000939:Epitopes; D007377:Interleukin-3; D015848:Interleukin-5; D011956:Receptors, Cell Surface; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000409:Alanine