基于SILAC和生物素标记蛋白复合物技术解析μ阿片类受体6次跨膜剪切异构体镇痛的分子机制

Opioid receptor is G protein-coupled receptors including μ, δ, κ three subtypes which play an important role in many physiological activities, especially in the pain management. It can cause dependence, addiction, respiratory depression and gastrointestinal motility inhibition if it is used long term, which has inhibited the clinical application of these drugs. μ opioid receptor gene has different splice variants, the proteins translated from there variants can be divided into two categories according to their structure and function: one is 7 transmembrane protein structure, another is 6 transmembrane protein lacking of extracellular domain and extracellular amino-terminal transmembrane protein structure. From the μ-opioid receptor 6 transmembrane protein and 7 transmembrane protein knockout mice show that the drug targeting 6 transmembrane receptor has a completely different analgesic pharmacology compared to 7-transmembrane receptors, and the drug targeting 6 transmembrane receptor has no such side effects like the drugs of 7 transmembrane receptor. In vitro studies have shown that the 6 transmembrane proteins in nerve cells must depend on combination with other molecules to perform the analgesia function. This aims of the study is to screen the 6-transmembrane protein chaperone, and reveal the molecular mechanism of its analgesic effect using stable isotope labeling in cell culture technology and biotin-labeled protein complexes methods.

阿片类受体是一种G蛋白偶联受体, 主要包括μ、δ、κ三种亚型，该受体在许多生理活动尤其是镇痛中发挥重要功能，但长期使用该类受体药物会引起依赖性、成瘾性、呼吸抑制和胃肠蠕动抑制等副作用，进而严重影响该类药物的临床应用。μ阿片类受体基因在体内有多种剪切形式而产生不同转录本，该转录本翻译产生的蛋白质从结构功能上主要分为两大类：一类为传统的7次跨膜蛋白；一类为细胞外氨基端缺乏膜外结构域而形成6次跨膜蛋白。从μ阿片类受体6次和7次跨膜蛋白基因敲除小鼠显示该6次跨膜结构受体与经典的7次跨膜结构受体具有完全不同的镇痛药理，且6次跨膜结构受体药物并无上述副作用；而体外研究显示该6次跨膜蛋白在神经细胞中必须与其它分子结合才可以形成其药物的结合位点。本研究旨在利用细胞培养稳定同位素标记技术和生物素标记蛋白复合物技术筛选该6次跨膜结构蛋白的分子伴侣，进而揭示其发挥镇痛作用的分子机制。

μ阿片类受体基因，OPRM1，在体内有多种剪切形式而产生不同转录本，该转录本翻译产生的蛋白质从结构功能上主要分为两大类：一类为传统的7次跨膜蛋白(7TM)；一类为细胞外氨基端缺乏膜外结构域而形成6次跨膜蛋白(6TM)。这些6TM变体是一种新型镇痛药物作用所必需的，包括3-碘苯甲酰-6β-纳曲沙酰胺(IBNtxA)，它可以有效对抗一系列疼痛模型，而不会表现出传统阿片类药物的副作用。在本研究中，我们证实了mMOR1G与增强全长7TMμ阿片受体mMOR1的不同药理功能。从μ阿片类受体6次和7次跨膜蛋白基因敲除小鼠显示该6次跨膜结构受体与经典的7次跨膜结构受体具有完全不同的镇痛药理，且6次跨膜结构受体药物并无上述副作用；而体外研究显示该6次跨膜蛋白在神经细胞中必须与其它分子结合才可以形成其药物的结合位点。在小鼠的5种6TM剪切异构体中，从啮齿动物到人类，mMOR1G数量丰富且保守。在此基础上利用原代神经元细胞培养及生物素标记，采用APEX2技术解析标记蛋白复合物，筛选该6次跨膜结构蛋白的分子伴侣，进而揭示其发挥镇痛作用的分子机制。