Interaction of granulocyte colony-stimulating factor (G-CSF) with its receptor -: evidence that Glu19 of G-CSF interacts with Arg288 of the receptor

Granulocyte colony-stimulating factor (G-CSF) forms a tetrameric complex with its receptor, comprising two G-CSF and two receptor molecules. The structure of the complex is unknown, and it is unclear whether there are one or two binding sites on G-CSF and the receptor. The immunoglobulin-like domain and the cytokine receptor homologous module of the receptor are involved in G-CSF binding, and Arg(288) in the cytokine receptor homologous module is particularly important. To identify residues in G-CSF that interact with Arg(288), selected charged residues in G-CSF were mutated to Ale. To clarify whether there are two binding sites, a chimeric receptor was created in which the Ig domain was replaced with that of the related receptor gp130, This chimera bound G-CSF but could not transduce a signal, consistent with failure of dimerization and loss of one binding site, The G-CSF mutants had reduced mitogenic activity on cells expressing wild-type receptor. When tested with the chimeric receptor, all G-CSF mutants except one (E46A) showed reduced binding, suggesting that Glu(46) is important for interaction with the Ig domain, On cells expressing R288A receptor, all the G-CSF mutants except E19A showed reduced mitogenic activity, indicating that Glu(19) of G-CSF interacts with Arg(288) of the receptor.