Two contiguous residues in human interleukin-3, Asp²¹ and Glu²², selectively interact with the α- and β-chains of its receptor and participate in function

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 (α- and β-chains) or to the receptor α-chain alone, indicating that residue 21 is involved in contacts with the α-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 α-chain alone was similar to that of wild type IL-3, suggesting that this mutant was defective in interactions with the receptor β-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.