Zheng Wu, Zhou Jianhong, Luan Yanan, Yang Jianglan, Ge Yuanyuan, Wang Muran, Wu Beibei, Wu Zhongnan, Chen Xingjun, Li Fei, Li Zhihui, Vakal Sergii, Guo Wei, Chen Jiang-Fan
Molecular Neuropharmacology Laboratory, School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, Wenzhou, China.
State Key Laboratory of Optometry & Vision Science, Wenzhou, China.
Front Mol Neurosci. 2018 Mar 28;11:95. doi: 10.3389/fnmol.2018.00095. eCollection 2018.
Despite the progress in deorphanization of G Protein-Coupled Receptors (GPCRs), ≈100 GPCRs are still classified as orphan receptors without identified endogenous ligands and with unknown physiological functions. The lack of endogenous ligands triggering GPCR signaling has hampered the study of orphan GPCR functions. Using GPR37 as an example, we provide here the first demonstration of the channelrhodopsin 2 (ChR2)-GPCR approach to bypass the endogenous ligand and selectively activate the orphan GPCR signal by optogenetics. Inspired by the opto-XR approach, we designed the ChR2-GPR37 chimera, in which the corresponding parts of GPR37 replaced the intracellular portions of ChR2. We showed that optogenetic activation of ChR2/opto-GPR37 elicited specific GPR37 signaling, as evidenced by reduced cAMP level, enhanced ERK phosphorylation and increased motor activity, confirming the specificity of opto-GPR37 signaling. Besides, optogenetic activation of opto-GPR37 uncovered novel aspects of GPR37 signaling (such as IP-3 signaling) and anxiety-related behavior. Optogenetic activation of opto-GPR37 permits the causal analysis of GPR37 activity in the defined cells and behavioral responses of freely moving animals. Importantly, given the evolutionarily conserved seven-helix transmembrane structures of ChR2 and orphan GPCRs, we propose that opto-GPR37 approach can be readily applied to other orphan GPCRs for their deorphanization in freely moving animals.
尽管在G蛋白偶联受体（GPCRs）的去孤儿化方面取得了进展，但仍有大约100种GPCR被归类为孤儿受体，它们尚未确定内源性配体，生理功能也未知。缺乏触发GPCR信号传导的内源性配体阻碍了孤儿GPCR功能的研究。以GPR37为例，我们在此首次展示了通过光遗传学方法利用通道视紫红质2（ChR2）-GPCR绕过内源性配体并选择性激活孤儿GPCR信号。受光遗传学-XR方法的启发，我们设计了ChR2-GPR37嵌合体，其中GPR37的相应部分取代了ChR2的细胞内部分。我们发现，ChR2/opto-GPR37的光遗传学激活引发了特定的GPR37信号传导，cAMP水平降低、ERK磷酸化增强和运动活性增加证明了这一点，证实了opto-GPR37信号传导的特异性。此外，opto-GPR37的光遗传学激活揭示了GPR37信号传导（如IP-3信号传导）和焦虑相关行为的新方面。opto-GPR37的光遗传学激活允许对定义细胞中GPR37活性以及自由活动动物的行为反应进行因果分析。重要的是，鉴于ChR2和孤儿GPCR在进化上保守的七螺旋跨膜结构，我们提出opto-GPR37方法可以很容易地应用于其他孤儿GPCR，以便在自由活动的动物中实现它们的去孤儿化。
