Kubota Ryou, Nomura Wataru, Iwasaka Takuma, Ojima Kento, Kiyonaka Shigeki, Hamachi Itaru
Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan.
Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan.
ACS Cent Sci. 2018 Sep 26;4(9):1211-1221. doi: 10.1021/acscentsci.8b00390. Epub 2018 Aug 24.
Investigating individual G-protein-coupled receptors (GPCRs) involved in various signaling cascades can unlock a myriad of invaluable physiological findings. One of the promising strategies for addressing the activity of each subtype of receptor is to design chemical turn-on switches on the target receptors. However, valid methods to selectively control class A GPCRs, the largest receptor family encoded in the human genome, remain limited. Here, we describe a novel approach to chemogenetically manipulate activity of engineered class A GPCRs carrying a His tag, using metal complex-agonist conjugates (MACs). This manipulation is termed coordination tethering. With the assistance of coordination bonds, MACs showed 10-100-fold lower EC values in the engineered receptors, compared with wild-type receptors. Such coordination tethering enabled selective activation of β-adrenoceptors and muscarinic acetylcholine receptors, without loss of natural receptor responses, in living mammalian cells, including primary cultured astrocytes. Our generalized, modular chemogenetic approach should facilitate more precise control and deeper understanding of individual GPCR signaling pathways in living systems.
研究参与各种信号级联反应的单个G蛋白偶联受体（GPCR），可以揭示无数宝贵的生理学发现。解决每种受体亚型活性的一种有前景的策略是在目标受体上设计化学开启开关。然而，选择性控制A类GPCR（人类基因组中编码的最大受体家族）活性的有效方法仍然有限。在此，我们描述了一种新方法，使用金属络合物-激动剂缀合物（MAC）对携带组氨酸标签的工程化A类GPCR的活性进行化学遗传操纵。这种操纵被称为配位连接。在配位键的帮助下，与野生型受体相比，MAC在工程化受体中的EC值低10-100倍。这种配位连接能够在包括原代培养星形胶质细胞在内的活哺乳动物细胞中选择性激活β-肾上腺素能受体和毒蕈碱型乙酰胆碱受体，而不会丧失天然受体反应。我们通用的模块化化学遗传方法应有助于更精确地控制和更深入地理解活系统中单个GPCR信号通路。
