AbstractWe performed a series of experiments using alanine‐scanning mutagenesis to locate side chains within human granulocyte colony‐stimulating factor (G‐CSF) that are involved in human G‐CSF receptor binding. We constructed a panel of 28 alanine mutants that examined all surface exposed residues on helices A and D, as well as all charged residues on the surface of G‐CSF. The G‐CSF mutants were expressed in a transiently transfected mammalian cell line and quantitated by a sensitive biosensor method. We measured the activity of mutant proteins using an in vitro proliferation assay and an ELISA binding competition assay. These studies show that there is a region of five charged residues on helices A and C employed by G‐CSF in binding its receptor, with the most important residue in this binding patch being Glu 19. Both wild‐type G‐CSF and the E19A mutant were expressed in E. coli. The re‐folded proteins were found to have proliferative activities similar to the analagous proteins from mammalian cells: furthermore, biophysical analysis indicated that the E19A mutation does not cause gross structural perturbations in G‐CSF. Although G‐CSF is likely to signal through receptor homo‐dimerization, we found no compelling evidence for a second receptor binding region. We also found no evidence of self‐antagonism at high G‐CSF concentrations, suggesting that, in contrast to human growth hormone (hGH) and erythropoietin (EPO), G‐CSF probably does not signal via a pure 2.1 receptor:ligand complex. Thus, G‐CSF, while having a similar tertiary structure to hGH and EPO, uses different areas of the four helix bundle for high‐affinity interaction with its receptor.