μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis
μ-Opioid Receptor Forms a Functional Heterodimer With Cannabinoid CB1 Receptor: Electrophysiological and FRET Assay Analysis
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DOI：
10.1254/jphs.08244fp
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发表时间：
2008-11-01
影响因子：
3.5
通讯作者：
Uezono, Yasuhito
中科院分区：
文献类型：
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作者：
Hojo, Minoru;Sudo, Yuka;Uezono, Yasuhito
采用形态学和电生理方法研究了mu-阿片受体（mu OR）与大麻素CB1受体（CB1R）的相互作用。在共表达mu OR与黄色荧光蛋白Venus融合和CB1R与青色荧光蛋白Cerulean融合的幼鼠肾（BHK）细胞中，细胞表面检测到两种颜色；荧光共振能量转移（FRET）分析显示，mu OR与CB1R形成异源二聚体。共免疫沉淀和Western blotting分析也证实了mu OR和CB1R的异源二聚体。[D-Ala(2),N-Me-Phe(4),Gly(5)-ol]脑啡肽（DAMGO）或CP55,940分别在表达mu or或CB1R以及G蛋白激活的内向纠偏K+通道（GIRKs）的爪蟾卵母细胞中引发K+电流。在共表达这两种受体的卵母细胞中，其中一种受体与激活磷脂酶C通路的嵌合G α蛋白G（qi5）融合，DAMGO和CP55,940都引发Ca2+激活的Cl-电流，表明每种激动剂都可以通过Gqi5与自身受体或其他受体融合诱导反应。内源性G（i/o）实验。百日毒（PTX）的蛋白失活通过PTX不敏感的G（qi5(m)）融合到每个受体上，支持mu OR/CB1R的功能性异二聚化。因此，mu OR和CB1R形成异源二聚体，并通过共同的G蛋白传递信号。我们的电生理方法可用于测定异源二聚G蛋白偶联受体介导的信号。
Interactions between mu-opioid receptor (mu OR) and cannabinoid CB1 receptor (CB1R) were examined by morphological and electrophysiological methods. In baby hamster kidney (BHK) cells coexpressing mu OR fused to the yellow fluorescent protein Venus and CB1R fused to the cyan fluorescent protein Cerulean, both colors were detected on the cell surface; and fluorescence resonance energy transfer (FRET) analysis revealed that mu OR and CB1R formed a heterodimer. Coimmunoprecipitation and Western blotting analyses also confirmed the heterodimers of mu OR and CB1R. [D-Ala(2),N-Me-Phe(4),Gly(5)-ol]enkephalin (DAMGO) or CP55,940 elicited K+ currents in Xenopus oocytes expressing mu OR or CB1R together with G protein activated-inwardly rectifying K+ channels (GIRKs), respectively. In oocytes coexpressing both receptors, either of which was fused to the chimeric G alpha protein G(qi5) that activates the phospholipase C pathway, both DAMGO and CP55,940 elicited Ca2+-activated Cl- currents, indicating that each agonist can induce responses through Gqi5 fused to either its own receptor or the other. Experiments with endogenous G(i/o). protein inactivation by pertussis toxin (PTX) supported the functional heterodimerization of mu OR/CB1R through PTX-insensitive G(qi5(m)) fused to each receptor. Thus, mu OR and CB1R form a heterodimer and transmit a signal through a common G protein. Our electrophysiological method could be useful for determination of signals mediated through heterodimerized G protein-coupled receptors.