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

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 Nterminal 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-CSF⅐G-CSF-R complex was prepared, based on the viral interleukin-6⅐gp130 complex, which explains these and previously published data. The granulocyte colony-stimulating factor receptor (G-CSF-R)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 function (1, 2). The extracellular region comprises six structural domains: an N-terminal Ig-like domain followed by five fibronectin type III (FNIII) domains (3) (Fig. 1A). The first two of the FNIII domains (called D2 and D3) form the cytokine receptor homology (CRH) module, which contains four cysteine residues in D2 and a WSXWS motif in D3. These features of the CRH module are conserved in members of the class 1 cytokine receptor family (4). This domain structure is also found in the closest homologue of the G-CSF-R, gp130, which is the shared signal transducing receptor chain of the interleukin (IL)-6 family of cytokines (5). The G-CSF-R and gp130 share 46% sequence similarity in the extracellular region (6). The ligands for the class 1 cytokine receptors have a conserved 4-␣-helical bundle structure but little sequence similarity (7).In the gp130 family of ligands and receptors, three binding interfaces have been identified (reviewed in Ref. 8). For example, IL-6 interacts with the IL-6 receptor at site I and gp130 at site II. A second gp130 receptor interacts at site III, resulting in a hexameric complex of two components each of IL-6, IL-6 receptor, and gp130 (9). On the IL-6 family of ligands, site I is located at the end of the D helix, site II on the A and C helices, and site III at the N...

GPL, a Novel Cytokine Receptor Related to GP130 and Leukemia Inhibitory Factor Receptor

“…Then, the CBD of some large-size cytokine receptors (gp130, IL-12R␤1, and granulocyte-colony stimulating factor) interact with the cytokine(s) through site II (57,(61)(62)(63)(64). Recruitment of an additional site of interaction (site III) located on the Ig-like domain of a second large-size receptor leads to oligomerization and signaling of the receptor complex (65)(66)(67)(68)(69). Because no Ig-like domain is present in GPL, we can hypothesize that GPL should interact with its potential ligand(s) through a site II or a site I. GPL also likely implicates a neighbor receptor holding an Ig-like module to define the site III and to allow the dimerization process of large-size signaling subunits and subsequent signaling events.…”

Leukemia Inhibitory Factor (LIF), Cardiotrophin-1, and Oncostatin M Share Structural Binding Determinants in the Immunoglobulin-like Domain of LIF Receptor

“…CNTF, CLC, IL-6, and IL-11) binds to this co-receptor (CNTFR, IL-6R, and IL-11R) through binding site I (C-terminal parts of the AB loop and of helix D (8,20,26,(33)(34)(35)(36)). The glycoprotein gp130 interacts through binding site II, located on the solvent-exposed faces of helices A and C (3,37,38). These sites are similar to binding sites I and II of the growth hormone (32).…”

“…This is consistent with the much smaller free energy of binding observed for the second interacting receptor in the high affinity complex (LIFR binding to the OSM⅐gp130 complex) than for the first interacting receptor (LIFR binding to LIF or CT-1). In a study aimed to find the binding epitope of the Ig-like domain of G-CSF receptor, Layton et al (37) observed that single mutations in the Ig-like domain of the granulocyte-colony stimulating factor receptor did not impair binding or biological activity but that double mutations were required to observe an effect. They explained this behavior by the low additional change in free energy upon the second receptor binding, which should be shared by several residues at the interface, corresponding to weak interactions.…”

Homodimeric cross-over structure of the human granulocyte colony-stimulating factor (GCSF) receptor signaling complex

“…The aromatic ring of Tyr-39 in hGCSF exhibits a stacking interaction parallel to that of Phe-75 in hGCSF-R. Five hydrogen-bonding interactions also contribute to the site III interaction as shown in Table 1. In these residues, Glu-46 and Val-48 in hGCSF and Phe-75, Gln-87, and Gln-91 in hGCSF-R are reported to be important for ligand binding and biological function (16)(17)(18)20).…”