Identification of residues in the first and fourth helices of human granulocyte-macrophage colony-stimulating factor involved in biologic activity and in binding to the α- and β-chains of its receptor

Residues within the first and fourth helices of human granulocyte-macrophage colony-stimulating factor (hGM-CSF) were analyzed for their role in biologic activity and interaction with the α- and β-chains of the hGM-CSF receptor. Within the first helix substitution of the surface residues Glu¹⁴, Asn¹⁷, Gln²⁰, Arg²³, Arg²⁴, and Asn²⁷ or the buried residues Ala¹⁸, Leu²⁵, and Leu²⁸ did not significantly impair bioactivity or receptor binding. Substitutions at the buried residues Ala²² and Leu²⁶ had intermediate bioactivity. However, substitutions of the surface residue Glu²¹ or the buried residue lle¹⁹ reduced the relative bioactivity of the analogues to as little as 0.45% and 0.3%, respectively. Substitution of the charged surface residues of the fourth helix showed that substitution at Glu¹⁰⁴, Lys¹⁰⁷, and Lys¹¹¹ had no significant effect on bioactivity, but substitution at Glu¹⁰⁸ and Asp¹¹² reduced the potency of the analogues to 34% and 7%, respectively. Receptor binding studies showed that, whereas Glu²¹ is the critical residue for binding to the hGM-CSF-receptor β-chain, Asp¹¹² is likely to be involved in binding to the GM-CSF-receptor α-chain. These results establish the relative contribution of residues in the first and fourth helices for GM-CSF bioactivity and receptor binding, and support a model where the fourth helix of GM-CSF interacts with the α-chain, and the first helix with the β-chain of the GM-CSF receptor.