Identification of Ligand-binding Site III on the Immunoglobulin-like Domain of the Granulocyte Colony-stimulating Factor Receptor

Judith E Layton,Nathan E Hall,Fiona Connell,Jennifer Venhorst,Herbert R Treutlein

doi:10.1074/jbc.m104787200

Abstract

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.

Highlights

The granulocyte colony-stimulating factor receptor (G-CSFR)1 is a transmembrane protein that is expressed predominantly on cells of the neutrophil lineage and is important in transmitting signals for their proliferation, differentiation, and ʈ Present address: Div. of Molecular Toxicology, Dept. of Pharmacochemistry, Faculty of Exact Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
We have proposed that the granulocyte colony-stimulating factor receptor (G-CSF-R) complex is similar to the IL-6 receptor complex but without the site I interaction, because there is no equivalent protein to the IL-6 receptor in the G-CSF-R complex [12] (Fig. 1B)
The mutated full-length receptor cDNAs were expressed in the murine pro-B cell line, Ba/F3, to enable measurement of the proliferative response to G-CSF and binding affinity for G-CSF, in comparison with the Wild type (WT) G-CSF-R

Summary

Introduction

The granulocyte colony-stimulating factor receptor (G-CSFR) is a transmembrane protein that is expressed predominantly on cells of the neutrophil lineage and is important in transmitting signals for their proliferation, differentiation, and ʈ Present address: Div. of Molecular Toxicology, Dept. of Pharmacochemistry, Faculty of Exact Sciences, Vrije Universiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands. The main receptor-binding site on G-CSF has been shown by mutagenesis to include residues on the A and C helices [13, 14] and is equivalent to site II of IL-6 (Fig. 1D) This site interacts with the “elbow” formed by D2 and D3 of the CRH module (G-CSF-R site II), shown in the crystal structure of G-CSF in complex with the CRH module [15]. On G-CSF, mutagenesis has identified some residues that appear to form a second binding site near the N-terminal end of the D helix [13], and we previously showed that Glu of G-CSF in the E helix (Fig. 1D) probably interacted with the Ig-like domain [12]. The recently described crystal structure of a 2:2 complex of the CRH module of G-CSF-R (with no Ig-like domain) and G-CSF (shown schematically in Fig. 1C) shows the site II interaction but provides no evidence for a site III interaction [15]. The putative site III of G-CSF (Fig. 1D) is exposed in the crystal complex, suggesting that the observed dimerization interface is different from that formed by the complete receptor and may represent an intermediate in complex formation [15]

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