2 CONTIGUOUS RESIDUES IN HUMAN INTERLEUKIN-3, ASP(21) AND GLU(22), SELECTIVELY INTERACT WITH THE ALPHA-CHAIN AND BETA-CHAIN OF ITS RECEPTORAND PARTICIPATE IN FUNCTION

We have previously reported that the predicted first helix of human in terleukin (IL)-3 contains a hydrophilic region encompassing residues A sp(21), Glu(22), and Thr(25) that is crucial for biological activity a nd IL-3 receptor binding. Using single amino acid substitution mutagen esis, we have now determined that Asp(21) and Glu(22), but not Thr(25) , were crucial for full IL-3 activity. Mutant D21R was 30-fold less po tent than wild type IL-3 in the stimulation of biological activity. It also exhibited a similar reduction in its ability to bind to the clon ed 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 20,00 0-fold less potent than wild type IL-3 in the stimulation of biologica l 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 wa s similar to that of wild type IL-3, suggesting that this mutant was d efective in interactions with the receptor beta-chain. These results s how that two contiguous residues in the N-terminal region of IL-3 medi ate binding to the two different chains of the IL-3 receptor and empha size the functional significance of the conserved Glu in the first hel ix of the IL-3, granulocyte-macrophage colony-stimulating factor, and IL-5 cytokine subfamily.