title: Two contiguous residues in human interleukin-3, Asp21 and Glu22, selectively interact with the alpha- and beta-chains of its receptor and participate in function.
abstract: We have previously reported that the predicted first helix of human interleukin (IL)-3 contains a hydrophilic region encompassing residues Asp21, Glu22, and Thr25 that is crucial for biological activity and IL-3 receptor binding. Using single amino acid substitution mutagenesis, we have now determined that Asp21 and Glu22, but not Thr25, were crucial for full IL-3 activity. Mutant D21R was 30-fold less potent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the cloned high affinity IL-3 receptor complex (alpha- and beta-chains) or to the receptor alpha-chain alone, indicating that residue 21 is involved in contacts with the alpha-chain. Mutant E22R was approximately 20,000-fold less potent than wild type IL-3 in the stimulation of biological activity and in binding to the IL-3 receptor high affinity complex. However, the binding of E22R to the IL-3 receptor alpha-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor beta-chain. These results show that two contiguous residues in the N-terminal region of IL-3 mediate binding to the two different chains of the IL-3 receptor and emphasize the functional significance of the conserved Glu in the first helix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.
authors.affiliation: Hanson Centre for Cancer Research, Division of Human Immunology, Adelaide, South Australia.
authors.forename: S C
authors.initials: SC
authors.lastname: Barry
authors.forename: C J
authors.initials: CJ
authors.lastname: Bagley
authors.forename: J
authors.initials: J
authors.lastname: Phillips
authors.forename: M
authors.initials: M
authors.lastname: Dottore
authors.forename: B
authors.initials: B
authors.lastname: Cambareri
authors.forename: P
authors.initials: P
authors.lastname: Moretti
authors.forename: R
authors.initials: R
authors.lastname: D'Andrea
authors.forename: G J
authors.initials: GJ
authors.lastname: Goodall
authors.forename: M F
authors.initials: MF
authors.lastname: Shannon
authors.forename: M A
authors.initials: MA
authors.lastname: Vadas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D002460:Cell Line; D003001:Cloning, Molecular; D005971:Glutamates; D018698:Glutamic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry; D006801:Humans; D007377:Interleukin-3 / Q000737:chemistry* / Q000378:metabolism*; D015848:Interleukin-5 / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016185:Receptors, Interleukin-3 / Q000378:metabolism*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013912:Threonine; D014162:Transfection
chemical_list: D005971:Glutamates; D007377:Interleukin-3; D015848:Interleukin-5; D016185:Receptors, Interleukin-3; D013912:Threonine; D001224:Aspartic Acid; D018698:Glutamic Acid; D016178:Granulocyte-Macrophage Colony-Stimulating Factor