Thermodynamic Analysis of the Activation Mechanism of the GCSF Receptor Induced by Ligand Binding
2004; American Chemical Society; Volume: 43; Issue: 9 Linguagem: Inglês
10.1021/bi0356855
ISSN1943-295X
AutoresShouhei 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,
Tópico(s)T-cell and B-cell Immunology
ResumoThe 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.
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