Elucidation of Activation Mechanism of Granulocyte Colony-Stimulating Factor Receptor

粒细胞集落刺激因子（GCSF）通过调节嗜中性粒细胞前体细胞的成熟、增殖和分化，成为治疗粒细胞减少症的重要细胞因子。GCSF与其受体的细胞外Ig样和CRH结构域（GCSF-R）的结合触发受体同源二聚化，导致JAK-STAT型信号级联的激活。GCSF/GCSF-R复合物的化学计量一直存在争议，有各种建议值（1：1，2：2和/或4：4）。本研究成功地将人GCSF-R（hGCSF-R）的Ig样结构域和CRH结构域与人GCSF（hGCSF-R）的2：2复合物结晶化，并用X射线衍射法测定了其三级结构。信号传导2：2复合物通过hGCSF-R的Ig样结构域和相邻的hGCSF之间的交叉相互作用形成，形成双重晶体对称轴。这种构象与异质mGCSF-R复合物的构象完全不同，并且更接近于人白细胞介素-6（IL-6）、人ILr 6 α-受体和人gp 130（其是几种细胞因子的共享信号转导受体）的2：2：2活性组装，以及病毒IL-6和人gp 130的2：2组装。GCSF-R激活所需的Ig样结构域交叉结构与先前报道的热力学和突变分析一致。

Granulocyte colony-stimulating factor (GCSF) has become an important cytokine for medical treatment of patients suffering from granulopoenia through regulating the maturation, proliferation, and differentiation of the precursor cells of neutrophilic granulocytes. Binding of GCSF to the extracellular Ig-like and CRH domain of its receptor (GCSF-R) triggers receptor homodimerization, resulting in activation of JAK-STAT type signaling cascades. The stoichiometry of the GCSF/GCSF-R complex has been a matter of some debate, with various proposed values (1:1, 2:2 and/or 4:4). Our previous report demonstrated that only the 2:2 stoichiometry is observed as a stable complex.In this research, we have succeeded in crystallization of 2:2 complex between human GCSF (hGCSF) and the Ig-like and CRH domains of human GCSF-R (hGCSF-R) and determined its tertiary structure by X-ray crystallography at 2.8 A resolution. The signaling 2:2 complex is formed via cross-over interactions between the Ig-like domain of hGCSF-R and the neighboring hGCSF, forming a two-fold axis of crystallographic symmetry. This conformation is quite different from that of the heterogeneous mGCSF-R complex, and more closely resembles the 2:2:2 active assembly of human interleukin-6 (IL-6), human ILr6 a-receptor and human gp 130 (which is a shared signal transducing receptor for several cytokines), and the 2:2 assembly of viral IL-6 and human gp 130. The Ig-like domain cross-over structure necessary for GCSF-R activation is consistent with previously reported thermodynamic and mutational analyses.