Identification of ligand-binding site III on the immunoglobulin-like domain of the granulocyte colony-stimulating factor receptor

The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetram eric complex with G-CSF containing two ligand and two receptor molecules. T he N-terminal Ig-like domain of the G-CSF-R is required for receptor dimeri zation, but it is not known whether it binds G-CSF or interacts elsewhere i n the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe(75), Gln(87), and Gln(91)) intera ct with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain sho wed that the binding site is very similar to the viral interleukin-6 bindin g site (site III) on the Ig-like domain of gp130, a related receptor. To fu rther characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclona l antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N termi nus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF.G-CSF-R complex was prepared, based on the viral i nterleukin-6.gp130 complex, which explains these and previously published d ata.