Identification of a Ligand-binding Site on the Granulocyte Colony-stimulating Factor Receptor by Molecular Modeling and Mutagenesis*
Identification of a Ligand-binding Site on the Granulocyte Colony-stimulating Factor Receptor by Molecular Modeling and Mutagenesis*
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通过分子建模和诱变鉴定粒细胞集落刺激因子受体上的配体结合位点*
DOI：
10.1074/jbc.272.47.29735
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
H. Treutlein
中科院分区：
文献类型：
--
作者：
J. Layton;J. Iaria;D. Smith;H. Treutlein
粒细胞集落刺激因子（G-CSF）通过诱导同型二聚体受体复合物的形成来启动其对中性粒细胞谱系细胞的作用。G-CSF受体的结构尚未确定，因此我们使用分子建模来鉴定可能参与配体结合的受体区域。将G-CSF受体序列与gp 130和生长激素受体家族的所有可用序列进行比对，并基于生长激素受体结构构建细胞因子受体同源结构域的模型。对预测结合配体的环和单个残基进行丙氨酸取代诱变。突变体受体在因子依赖性Ba/F3细胞中表达，并评估增殖反应和配体结合。鉴定出6个残基显著降低受体功能，其中F′-G′环中的Arg 288具有最大的影响。这些残基在受体模型上形成类似于生长激素受体位点的结合面，这表明该模型是合理的。然而，静电分析的模型提供了进一步的证据，受体二聚化的机制是不同的生长激素受体。
Granulocyte colony-stimulating factor (G-CSF) initiates its effects on cells of the neutrophil lineage by inducing formation of a homodimeric receptor complex. The structure of the G-CSF receptor has not yet been determined, therefore we used molecular modeling to identify regions of the receptor that were likely to be involved in ligand binding. The G-CSF receptor sequence was aligned with all the available sequences of the gp130 and growth hormone receptor families and a model of the cytokine receptor homologous domain was constructed, based on the growth hormone receptor structure. Alanine substitution mutagenesis was performed on loops and individual residues that were predicted to bind ligand. Mutant receptors were expressed in factor-dependent Ba/F3 cells and assessed for proliferation response and ligand binding. Six residues were identified that significantly reduced receptor function, with Arg288 in the F′-G′ loop having the greatest effect. These residues formed a binding face on the receptor model resembling the growth hormone receptor site, which suggests that the model is reasonable. However, electrostatic analysis of the model provided further evidence that the mechanism of receptor dimerization is different from that of the growth hormone receptor.