Katada Sayako, Touhara Kazushige
Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.
Nihon Yakurigaku Zasshi. 2004 Oct;124(4):201-9. doi: 10.1254/fpj.124.201.
Olfactory receptors (ORs) comprise the largest super-family of rhodopsin-like G-protein coupled receptors (GPCR) that involve the recognition and discrimination of thousands of odorants. We recently succeeded in functional reconstitution of mouse ORs in mammalian cell lines and provided molecular evidence that structurally-related ORs recognized overlapping sets of odorants with distinct ligand specificities. Here we show that mOR-EG, a mouse olfactory receptor that was isolated from a eugenol-responsive cell, recognizes 22 different odorants with EC50 values ranging from a few microM to several hundred microM. We constructed a molecular model of mOR-EG using the recent atomic-level structure of bovine rhodopsin. Site-directed mutations were introduced in a potential ligand-binding pocket based on computational ligand-docking simulation. Mutations of some amino acid residues in TM3, TM5, and TM6 dramatically affected the EC50 value of eugenol in Ca2+ imaging. Finally, we succeeded in rational receptor design with predicted ligand specificity by introducing point mutations in the binding site, confirming the accuracy of the binding site mapping. The current studies also help understand mechanisms underlying molecular recognition by GPCRs, with implications for therapeutic application.
嗅觉受体（ORs）构成了视紫红质样G蛋白偶联受体（GPCR）中最大的超家族，涉及对数千种气味剂的识别和区分。我们最近成功地在哺乳动物细胞系中对小鼠ORs进行了功能重建，并提供了分子证据，表明结构相关的ORs识别具有不同配体特异性的重叠气味剂集合。在此我们表明，从丁香酚反应性细胞中分离出的小鼠嗅觉受体mOR-EG识别22种不同的气味剂，其半数有效浓度（EC50）值范围从几微摩尔到几百微摩尔。我们利用最近获得的牛视紫红质原子水平结构构建了mOR-EG的分子模型。基于计算配体对接模拟，在潜在的配体结合口袋中引入了定点突变。跨膜螺旋3（TM3）、跨膜螺旋5（TM5）和跨膜螺旋6（TM6）中一些氨基酸残基的突变显著影响了Ca2+成像中丁香酚的EC50值。最后，我们通过在结合位点引入点突变成功地进行了具有预测配体特异性的合理受体设计，证实了结合位点映射的准确性。目前的研究也有助于理解GPCRs分子识别的潜在机制，对治疗应用具有启示意义。
