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

Shouhei Mine, Takumi Koshiba, Eijiro Honjo, Tomoyuki Okamoto, Taro Tamada, Yoshitake Maeda, Yasuko Matsukura, Akane Horie, Matsujiro Ishibashi, Miharu Sato, Mizue Azuma, Masao Tokunaga, Katsutoshi Nitta, Ryota Kuroki

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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 (∼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.

Original languageEnglish
Pages (from-to)2458-2464
Number of pages7
JournalBiochemistry
Volume43
Issue number9
DOIs
Publication statusPublished - Mar 9 2004

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All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Mine, S., Koshiba, T., Honjo, E., Okamoto, T., Tamada, T., Maeda, Y., ... Kuroki, R. (2004). Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding. Biochemistry, 43(9), 2458-2464. https://doi.org/10.1021/bi0356855