Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding

Abstract

The granulocyte colony-stimulating factor receptor (GCSFR), containing the Ig-like domain (Ig) and cytokine receptor homologous region (CRH), was prepared as a preformed dimer (Ig-CRH-Fc)(2) after fusion to the mouse Fc region via an eight-residue linker (approximately 55 A). Monomer Ig-CRH was also prepared after the Fc region was removed from (Ig-CRH-Fc)(2). GCSF binding to Ig-CRH and (Ig-CRH-Fc)(2) was investigated using light scattering and isothermal titration calorimetry. The average molecular mass determined by light scattering showed that both Ig-CRH and (Ig-CRH-Fc)(2) formed a 2:2 dimer with GCSF. Moreover, isothermal titration calorimetry showed that the thermodynamic parameters upon binding of GCSF to Ig-CRH and (Ig-CRH-Fc)(2) were comparable, suggesting a similar binding stoichiometry and interface [including similar buried surface area (5700-6000 A(2))] despite the presence of the eight-residue linker. The buried surface area is much larger than that calculated from our previous report of the crystal structure of the GCSF-CRH complex [Aritomi, M., et al. (1999) Nature 401, 713-717], suggesting a substantial contribution of the Ig domain to GCSF binding. The data also indicate that the distance (55 A) between two CRH domains in the 2:2 complex is much shorter than in our previous model (approximately 90 A) predicted from the same crystal structure of the GCSF-CRH complex.

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

“…The stoichiometry of the GCSF͞GCSF-R complex has been a matter of some debate, with various proposed values (1:1, 2:2, and͞or 4:4) (7)(8)(9). Our recent report demonstrated that only the 2:2 stoichiometry is observed as a stable complex (10).…”

A hingeless Fc fusion system for site-specific cleavage by IdeS

“…We initially evaluated thrombin for this purpose, by inserting a thrombin recognition site between the TRAILR2 partner protein and Fc region as described by others. 8,15 However, in addition to cleavage at this designed site, we unexpectedly found that thrombin also cleaved TRAILR2 at an internal arginine residue within the motif -PQQR^AA-(data not shown). IdeS has been shown to cleave some Fc-fusion proteins, 12-14 so we decided to investigate this in the context of our protein of interest.…”

Crystallization of a 2:2 complex of granulocyte-colony stimulating factor (GCSF) with the ligand-binding region of the GCSF receptor

“…Recombinant human GCSF expressed in Escherichia coli was obtained from the pharmaceutical division of Kirin Brewery Co. Ltd (Tokyo, Japan). The receptor-binding region (308 amino acids) of human GCSF receptor (GCSFR) comprised of an Ig-like domain (Ig) and a cytokine-receptor homologous region (CRH) was prepared as a fusion protein with the Fc region of immunoglobulin (Mine et al, 2004). Three cysteine mutations (Cys79!Ser, Cys164!Ser and Cys229!Ser) were incorporated into Ig-CRH to reduce cysteinemediated aggregation (Mine et al, 2004).…”

“…Recently, not only site II but also site III interactions were elucidated by the structural analysis of a complex of the Ig-CRH domains of human gp130 with viral IL-6 (vIL-6;Chow et al, 2001) and with human IL-6 (Boulanger et al, 2003). The extracellular region of GCSFR shares 46% sequence similarity with that of gp130 (Hammacher et al, 1998); additionally, the Ig-CRH domain of GCSFR forms a 2:2 stoichiometric complex with GCSF (Mine et al, 2004), suggesting a receptor-activation mechanism similar to that of the vIL-6-gp130 complex (Layton et al, 2001). A model of GCSF-Ig-CRH based on the structure of the vIL6-gp130 complex is plausible since it would explain previous data obtained using a chimeric receptor (Layton et al, 1999) and neutralizing mAbs (Layton et al, 1997); however, there has been no structural evidence for the ligandrecognition scheme in GCSF.…”