Wang Yan, Ge Yan-Hui, Wang Yan-Xia, Liu Ting, Law Ping-Yee, Loh Horace H, Chen Hong-Zhuan, Qiu Yu
Department of Pharmacology, Institute of Medical Sciences, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
CNS Neurosci Ther. 2015 Jul;21(7):591-8. doi: 10.1111/cns.12409.
Mechanistic/mammalian target of rapamycin (mTOR) activation by μ-opioid receptor (OPRM1) participates in antinociceptive tolerance, hyperalgesia, and physical dependence. Our previous study also showed that mTOR activation by OPRM1 could attenuate β amyloid oligomers-induced neurotoxicity. OPRM1 is demonstrated to interact with FK506-binding protein 12 (FKBP12). It is our great interest to investigate whether OPRM1-mediated mTOR signaling is related to receptor-FKBP12 association.
The activities of mTOR and its downstream effector p70 S6K were measured by immunoblotting their phosphorylation status. The interaction of receptor with mTOR was detected by co-immunoprecipitation and immunofluorescence.
OPRM1 activation by morphine-induced time-dependent mTOR activation. PI3K-specific inhibitor LY294002 only blocked the late phase of mTOR activation. However, morphine-induced mTOR activation was totally blocked at all time points in cells expressing FKBP12 association-deficient mutant receptor. FKBP12 knockdown also blocked morphine-induced mTOR activation. Further analysis demonstrated that morphine treatment enhanced the association of receptor with phosphorylated mTOR, whereas decreased association was observed after FKBP12 knockdown, mTOR inhibition or in cells expressing FKBP12 association-deficient mutant.
OPRM1-FKBP12 association played a key role in OPRM1-mediated mTOR activation, which could underlie the mechanisms of multiple physiological and pathological processes. Thus, our findings provide new avenue to modulating these processes.
μ-阿片受体（OPRM1）激活雷帕霉素作用机制/哺乳动物雷帕霉素靶蛋白（mTOR）参与抗伤害感受性耐受、痛觉过敏和身体依赖性。我们之前的研究还表明，OPRM1激活mTOR可减轻β淀粉样寡聚体诱导的神经毒性。已证实OPRM1与FK506结合蛋白12（FKBP12）相互作用。我们非常感兴趣的是研究OPRM1介导的mTOR信号传导是否与受体-FKBP12结合有关。
通过免疫印迹法检测mTOR及其下游效应因子p70 S6K的磷酸化状态来测定其活性。通过免疫共沉淀和免疫荧光检测受体与mTOR的相互作用。
吗啡诱导的OPRM1激活导致mTOR呈时间依赖性激活。PI3K特异性抑制剂LY294002仅阻断mTOR激活的后期阶段。然而，在表达FKBP12结合缺陷型突变受体的细胞中，吗啡诱导的mTOR激活在所有时间点均被完全阻断。敲低FKBP12也阻断了吗啡诱导的mTOR激活。进一步分析表明，吗啡处理增强了受体与磷酸化mTOR的结合，而在敲低FKBP12、抑制mTOR或表达FKBP12结合缺陷型突变体的细胞中观察到结合减少。
OPRM1-FKBP12结合在OPRM1介导的mTOR激活中起关键作用，这可能是多种生理和病理过程机制的基础。因此，我们的研究结果为调节这些过程提供了新途径。
