Department of Biomedical Sciences, University of Cagliari, 09042, Monserrato, Italy; Department of Biomedicine, University of Basel, Klingelbergstrasse 50-70, CH-4056, Basel, Switzerland.
Department of Biomedical Sciences, University of Cagliari, 09042, Monserrato, Italy.
Neuropharmacology. 2018 May 1;133:107-120. doi: 10.1016/j.neuropharm.2018.01.024.
Rimonabant is a potent and selective cannabinoid CB1 receptor antagonist widely used in animal and clinical studies. Besides its antagonistic properties, numerous studies have shown that, at micromolar concentrations rimonabant behaves as an inverse agonist at CB1 receptors. The mechanism underpinning this activity is unclear. Here we show that micromolar concentrations of rimonabant inhibited Gα-type G proteins, resulting in a receptor-independent block of G protein signaling. Accordingly, rimonabant decreased basal and agonist stimulated [S]GTPγS binding to cortical membranes of CB1- and GABA-receptor KO mice and Chinese Hamster Ovary (CHO) cell membranes stably transfected with GABA or D2 dopamine receptors. The structural analog of rimonabant, AM251, decreased basal and baclofen-stimulated GTPγS binding to rat cortical and CHO cell membranes expressing GABA receptors. Rimonabant prevented G protein-mediated GABA and D2 dopamine receptor signaling to adenylyl cyclase in Human Embryonic Kidney 293 cells and to G protein-coupled inwardly rectifying K channels (GIRK) in midbrain dopamine neurons of CB1 KO mice. Rimonabant suppressed GIRK gating induced by GTPγS in CHO cells transfected with GIRK, consistent with a receptor-independent action. Bioluminescent resonance energy transfer (BRET) measurements in living CHO cells showed that, in presence or absence of co-expressed GABA receptors, rimonabant stabilized the heterotrimeric Gαi/o-protein complex and prevented conformational rearrangements induced by GABA receptor activation. Rimonabant failed to inhibit Gαs-mediated signaling, supporting its specificity for Gα-type G proteins. The inhibition of Gα protein provides a new site of rimonabant action that may help to understand its pharmacological and toxicological effects occurring at high concentrations.
利莫那班是一种有效的、选择性的大麻素 CB1 受体拮抗剂，广泛应用于动物和临床研究。除了其拮抗特性外，许多研究表明，在微摩尔浓度下，利莫那班作为 CB1 受体的反向激动剂起作用。这种活性的机制尚不清楚。在这里，我们表明，微摩尔浓度的利莫那班抑制了 Gα 型 G 蛋白，导致 G 蛋白信号的受体非依赖性阻断。相应地，利莫那班降低了 CB1-和 GABA 受体 KO 小鼠皮质膜和稳定转染 GABA 或 D2 多巴胺受体的中国仓鼠卵巢 (CHO) 细胞膜中基础和激动剂刺激的[S]GTPγS 结合。利莫那班的结构类似物 AM251 降低了表达 GABA 受体的大鼠皮质和 CHO 细胞膜中基础和巴氯芬刺激的 GTPγS 结合。利莫那班防止 G 蛋白介导的 GABA 和 D2 多巴胺受体信号转导至人胚肾 293 细胞中的腺苷酸环化酶和 CB1 KO 小鼠中脑多巴胺神经元中的 G 蛋白偶联内向整流钾通道 (GIRK)。利莫那班抑制了转染 GIRK 的 CHO 细胞中由 GTPγS 诱导的 GIRK 门控，这与受体非依赖性作用一致。活 CHO 细胞中的生物发光共振能量转移 (BRET) 测量显示，在存在或不存在共表达的 GABA 受体的情况下，利莫那班稳定了异三聚体 Gαi/o-蛋白复合物并阻止了由 GABA 受体激活诱导的构象重排。利莫那班未能抑制 Gαs 介导的信号转导，支持其对 Gα 型 G 蛋白的特异性。Gα 蛋白的抑制提供了利莫那班作用的新位点，这可能有助于理解其在高浓度下发生的药理学和毒理学效应。
