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 tetrameric complex with G-CSF containing two ligand and two receptor molecules. The N-terminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in 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)) interact 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 showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal 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 terminus 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 interleukin-6.gp130 complex, which explains these and previously published data.
Layton
J E
JE
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch and the Cooperative Research Centre for Cellular Growth Factors, Parkville, Victoria 3050, Australia. Judy.Layton@ludwig.edu.au
Hall
N E
NE
Connell
F
F
Venhorst
J
J
Treutlein
H R
HR
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001665:Binding Sites; D002455:Cell Division; D002460:Cell Line; D019281:Dimerization; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry*; D015850:Interleukin-6 / Q000378:metabolism; D007700:Kinetics; D008024:Ligands; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016188:Receptors, Granulocyte Colony-Stimulating Factor / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid
D000911:Antibodies, Monoclonal; D007136:Immunoglobulins; D015850:Interleukin-6; D008024:Ligands; D016188:Receptors, Granulocyte Colony-Stimulating Factor