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 (∼55 Å). 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 A2)] 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 Å) between two CRH domains in the 2:2 complex is much shorter than in our previous model (∼90 Å) predicted from the same crystal structure of the GCSF-CRH complex.
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