Hong Ling-Juan, Jiang Quan, Long Sen, Wang Huan, Zhang Ling-di, Tian Yun, Wang Cheng-Kun, Cao Jing-Jing, Tao Rong-Rong, Huang Ji-Yun, Liao Mei-Hua, Lu Ying-Mei, Fukunaga Kohji, Zhou Nai-Ming, Han Feng
College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China.
Department of Pharmacy, Hangzhou No.7 People's Hospital, Hangzhou, China.
Mol Neurobiol. 2016 Mar;53(2):1237-1246. doi: 10.1007/s12035-014-9085-y. Epub 2015 Jan 24.
Valproate exposure is associated with increased risks of autism spectrum disorder. To date, the mechanistic details of disturbance of melatonin receptor subtype 1 (MTNR1A) internalization upon valproate exposure remain elusive. By expressing epitope-tagged receptors (MTNR1A-EGFP) in HEK-293 and Neuro-2a cells, we recorded the dynamic changes of MTNR1A intracellular trafficking after melatonin treatment. Using time-lapse confocal microscopy, we showed in living cells that valproic acid interfered with the internalization kinetics of MTNR1A in the presence of melatonin. This attenuating effect was associated with a decrease in the phosphorylation of PKA (Thr197) and ERK (Thr202/Tyr204). VPA treatment did not alter the whole-cell currents of cells with or without melatonin. Furthermore, fluorescence resonance energy transfer imaging data demonstrated that valproic acid reduced the melatonin-initiated association between YFP-labeled β-arrestin 2 and CFP-labeled MTNR1A. Together, we suggest that valproic acid influences MTNR1A intracellular trafficking and signaling in a β-arrestin 2-dependent manner.
丙戊酸盐暴露与自闭症谱系障碍风险增加有关。迄今为止，丙戊酸盐暴露后褪黑素受体亚型1（MTNR1A）内化紊乱的机制细节仍不清楚。通过在HEK-293和Neuro-2a细胞中表达表位标记的受体（MTNR1A-EGFP），我们记录了褪黑素处理后MTNR1A细胞内运输的动态变化。使用延时共聚焦显微镜，我们在活细胞中显示丙戊酸在褪黑素存在的情况下干扰了MTNR1A的内化动力学。这种减弱作用与PKA（Thr197）和ERK（Thr202/Tyr204）磷酸化的降低有关。丙戊酸处理并未改变有或没有褪黑素的细胞的全细胞电流。此外，荧光共振能量转移成像数据表明丙戊酸减少了褪黑素引发的YFP标记的β-抑制蛋白2和CFP标记的MTNR1A之间的结合。总之，我们认为丙戊酸以β-抑制蛋白2依赖性方式影响MTNR1A的细胞内运输和信号传导。
