Layton J E, Hall N E, Connell F, Venhorst J, Treutlein H R
Ludwig Institute for Cancer Research, Melbourne Tumour Biology Branch and the Cooperative Research Centre for Cellular Growth Factors, Parkville, Victoria 3050, Australia.
J Biol Chem. 2001 Sep 28;276(39):36779-87. doi: 10.1074/jbc.M104787200. Epub 2001 Jul 23.
The granulocyte colony-stimulating factor receptor (G-CSF-R) forms a tetrameric complex with G-CSF containing two ligand and two receptor molecules. The N-terminal Ig-like domain of the G-CSF-R is required for receptor dimerization, but it is not known whether it binds G-CSF or interacts elsewhere in the complex. Alanine scanning mutagenesis was used to show that residues in the Ig-like domain of the G-CSF-R (Phe(75), Gln(87), and Gln(91)) interact with G-CSF. This binding site for G-CSF overlapped with the binding site of a neutralizing anti-G-CSF-R antibody. A model of the Ig-like domain showed that the binding site is very similar to the viral interleukin-6 binding site (site III) on the Ig-like domain of gp130, a related receptor. To further characterize the G-CSF-R complex, exposed and inaccessible regions of monomeric and dimeric ligand-receptor complexes were mapped with monoclonal antibodies. The results showed that the E helix of G-CSF was inaccessible in the dimeric but exposed in the monomeric complex, suggesting that this region binds to the Ig-like domain of the G-CSF-R. In addition, the N terminus of G-CSF was exposed to antibody binding in both complexes. These data establish that the dimerization interface of the complete receptor complex is different from that in the x-ray structure of a partial complex. A model of the tetrameric G-CSF.G-CSF-R complex was prepared, based on the viral interleukin-6.gp130 complex, which explains these and previously published data.
粒细胞集落刺激因子受体（G-CSF-R）与粒细胞集落刺激因子（G-CSF）形成一种四聚体复合物，其中包含两个配体分子和两个受体分子。G-CSF-R的N端免疫球蛋白样结构域是受体二聚化所必需的，但尚不清楚它是否结合G-CSF或在复合物的其他部位相互作用。采用丙氨酸扫描诱变表明，G-CSF-R免疫球蛋白样结构域中的残基（苯丙氨酸75、谷氨酰胺87和谷氨酰胺91）与G-CSF相互作用。这个G-CSF结合位点与一种中和性抗G-CSF-R抗体的结合位点重叠。免疫球蛋白样结构域的模型显示，该结合位点与相关受体gp130免疫球蛋白样结构域上的病毒白细胞介素-6结合位点（位点III）非常相似。为了进一步表征G-CSF-R复合物，用单克隆抗体绘制了单体和二聚体配体-受体复合物的暴露区域和不可及区域。结果显示，G-CSF的E螺旋在二聚体复合物中不可及，但在单体复合物中暴露，这表明该区域与G-CSF-R的免疫球蛋白样结构域结合。此外，G-CSF的N端在两种复合物中均暴露于抗体结合。这些数据表明，完整受体复合物的二聚化界面与部分复合物的X射线结构中的不同。基于病毒白细胞介素-6.gp130复合物制备了四聚体G-CSF.G-CSF-R复合物的模型，该模型解释了这些数据以及先前发表的数据。
