Identification of critical amino acid residues in human and mouse granulocyte-macrophage colony-stimulating factor and their involvement in species specificity.

Segments critical to the activity of human granulocyte-macrophage colony-stimulating factor (GM-CSF) were identified by scanning deletion analysis and compared with the critical regions previously identified in the homologous mouse GM-CSF protein. Three of the four critical regions thus identified are in equivalent positions in their respective polypeptides, while a fourth critical region of each is uniquely located. To investigate whether unique critical regions are responsible for the observed species specificity of human and mouse GM-CSF, all critical regions were substituted into their opposite homologue. This identified one specific, but different, critical region in each homologue that could not be replaced. Further characterization of the nature of the species specificity of these two proteins was accomplished by the generation of a series of human/mouse GM-CSF hybrids. Each hybrid protein was assayed for specific activity on human- and mouse GM-CSF-dependent cell lines. Significant differences in the specific activity of these hybrids was observed, suggesting that different segments of each molecule interact with their respective receptors. Based on these two approaches, individual amino acids were identified that could provide, at least in part, the interactions between these protein ligands and their respective receptors. These residues are Thr-78 and Met-80 in human GM-CSF and Asp-92, Thr-98, and Asp-102 in mouse GM-CSF.