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

• Published in: The Journal of biological chemistry Vol. 269; no. 11; pp. 8488 - 8492
• Main Authors: BARRY, S. C, BAGLEY, C. J, LOPEZ, A. F, PHILLIPS, J, DOTTORE, M, CAMBARERI, B, MORETTI, P, D'ANDREA, R, GOODALL, G. J, SHANNON, M. F, VADAS, M. A
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 18.03.1994
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Aspartic Acid; Biological and medical sciences; Cell Line; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; Glutamates; Glutamic Acid; Granulocyte-Macrophage Colony-Stimulating Factor - chemistry; Humans; Interleukin-3 - chemistry; Interleukin-3 - metabolism; Interleukin-5 - chemistry; Kinetics; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein hormones. Growth factors. Cytokines; Protein Structure, Secondary; Proteins; Receptors, Interleukin-3 - metabolism; Restriction Mapping; Sequence Homology, Amino Acid; Threonine; Transfection; Human; Site specificity; Membrane receptor; Structure activity relation; Interleukin 3; Cytokine; Site directed mutagenesis; Molecular interaction; Scatchard plot
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)37220-4

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.