Requirement for the Immunoglobulin-like Domain of Granulocyte Colony-stimulating Factor Receptor in Formation of a 2:1 Receptor-Ligand Complex

Abstract

The extracellular portion of the granulocyte colonystimulating factor (G-CSF) receptor has a mosaic structure of six domains (each approximately 100 amino acid residues) consisting of an immunoglobulin-like (Ig) domain, a cytokine receptor homologous region subdivided into amino-terminal (BN) and carboxyl-terminal (BC) domains, and three fibronectin type III repeats. In the present study, we expressed the Ig-BN and the BN-BC regions and purified them to homogeneity as monomers using G-CSF affinity column chromatography. Using gel filtration high performance liquid chromatography, we investigated the molecular composition of receptor-ligand complexes formed between G-CSF and purified BN-BC or Ig-BN domains. In contrast to the well characterized example of the human growth hormone (

Interaction of Granulocyte Colony-stimulating Factor (G-CSF) with Its Receptor

“…However, it is not yet clear whether there are one or two ligand-binding sites on the G-CSF-R. Horan et al (14,15) concluded that there is probably one ligand-binding site with dimerization caused by receptor-receptor interaction. On the other hand, studies on the complexes formed with G-CSF and two soluble receptor fragments (Ig-BN and the CRHM) indicated that each G-CSF-R in the complex has two binding sites for G-CSF, one in the CRHM and one requiring the Ig domain (19). In addition, this study established that the Ig domain of the G-CSF-R is required for receptor dimerization (19).…”

“…In both these studies, Glu 19 in the A helix of G-CSF was identified as particularly important and appeared to be a possible candidate for interaction with Arg 288 . In the present study, to identify which G-CSF residues interact with Arg 288 in the receptor, we have mutated Glu 19 , Lys 23 , Glu 46 , and Asp 112 and compared the activity of these G-CSF mutants on cells expressing wildtype (WT) and mutant (R288A) receptors. In addition, to clarify further whether there are one or two binding sites on G-CSF, a chimeric receptor was constructed in which the Ig domain of gp130 replaced the G-CSF-R Ig domain.…”

“…On the other hand, studies on the complexes formed with G-CSF and two soluble receptor fragments (Ig-BN and the CRHM) indicated that each G-CSF-R in the complex has two binding sites for G-CSF, one in the CRHM and one requiring the Ig domain (19). In addition, this study established that the Ig domain of the G-CSF-R is required for receptor dimerization (19). Similarly, the Ig domain of gp130 is required for receptor complex formation and signaling by interleukin-6 (IL-6) (12).…”

Interleukin‐6: Structure‐function relationships

“…We have taken into account that the extracellular region of gp130 has the same modular structure as the G-CSFR , Hibi et al, 1990Larsen et al, 1990), including an Ig-like domain that is lacking in the GHR (Leung et al, 1987). Both the Ig-like domain and the CBD of the G-CSFR are required for ternary complex formation with G-CSF (Hiraoka et al, 1995). In agreement with our model, we have recently identified neutralizing mAbs that recognize the Ig-like domain of gp130 (A. Hammacher & R.J. Simpson, in preparation) and interfere with the biological activity of IL-6 Wijdenes et al, 1995;Chevalier et al, 1996).…”

Determination of the Disulfide Structure andN-Glycosylation Sites of the Extracellular Domain of the Human Signal Transducer gp130

“…The Ig-like module of gp130 (domain 1) has been shown to be involved in the interaction of gp130 with ligands that induce homodimerization of gp130 (IL-6 (35,36) and IL-11 (37)) and essential for the formation of high affinity hexameric complexes (15,35). This domain is thought to bind site III in IL-6 and IL-11 (35), and Ig-like modules in the GCSFR and LIF receptor are also thought to make contact with their cognate ligands (38,39). The three membrane-proximal FN III modules of gp130, although not directly involved in ligand binding, may be required for gp130 dimerization by ligands such as IL-6 (40) and IL-11 (41) or transmembrane signaling events such as stabilization and/or orientation of transmembrane receptor dimers (5).…”