Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5203, Institut de Génomique Fonctionnelle, Université Montpellier 1 and 2, Montpellier, France.
Mol Pharmacol. 2010 Nov;78(5):818-26. doi: 10.1124/mol.110.066035. Epub 2010 Aug 10.
Serotonin (5-HT)(2C) receptor is a G(q)-coupled receptor exhibiting a high degree of constitutive activity toward phospholipase C effector pathway, a process regulated by receptor mRNA editing. In addition to G protein-dependent signaling, 5-HT(2C) receptors also activate the extracellular signal-regulated kinase (ERK) 1/2 pathway independently of receptor coupling to G proteins. Constitutive activity at ERK signaling has not yet been explored. Transient expression of unedited 5-HT(2C-INI) receptors in human embryonic kidney (HEK) 293 cells resulted in a marked increase in ERK1/2 phosphorylation compared with nontransfected cells. No increase in ERK1/2 phosphorylation was measured in cells expressing fully edited (5-HT(2C-VGV)) receptors. Basal ERK1/2 phosphorylation in 5-HT(2C-INI) receptor-expressing cells was abolished by 5-methyl-1-(3-pyridylcarbamoyl)-1,2,3,5-tetrahydropyrrolo[2,3-f]indole (SB206,553), a 5-HT(2C) inverse agonist toward phospholipase C. This effect was prevented by the neutral antagonist 6-chloro-5-methyl-1-[6-(2-methylpyridin-3-yloxy)pyridin-3-ylcarbamoyl]indoline (SB242,084), which alone did not alter basal activity. Similar observations were made in vivo in mouse choroid plexus, a structure rich in constitutively active 5-HT(2C) receptors. Reminiscent of agonist-induced ERK1/2 phosphorylation, basal activity in HEK 293 cells was unaffected by cellular depletion of Gα(q/11) and Gα(13) proteins but strongly reduced in cells expressing a dominant-negative β-arrestin or depleted of β-arrestin by RNA interference and in cells expressing a dominant-negative calmodulin or a 5-HT(2C-INI) receptor mutant not capable of interacting with calmodulin. The tetracyclic antidepressants mirtazapine and mianserin likewise reduced basal ERK activation. On the other hand, the m-chlorophenylpiperazine derivative trazodone and the selective serotonin reuptake inhibitor fluoxetine were inactive alone but blocked 5-HT-induced ERK1/2 phosphorylation. Together, these data provide the first evidence of constitutive activity of a G protein-coupled receptor toward G-independent, β-arrestin-dependent, receptor signaling.
血清素（5-HT）（2C）受体是一种 G(q)偶联受体，对磷脂酶 C 效应途径表现出高度的组成型活性，该过程受受体 mRNA 编辑调节。除了 G 蛋白依赖的信号转导外，5-HT（2C）受体还可以独立于 G 蛋白与受体的偶联激活细胞外信号调节激酶（ERK）1/2 途径。ERK 信号的组成型活性尚未得到探索。在人胚肾（HEK）293 细胞中转染未经编辑的 5-HT（2C-INI）受体可导致 ERK1/2 磷酸化明显增加，而非转染细胞则无此变化。在表达完全编辑（5-HT（2C-VGV））受体的细胞中未测量到 ERK1/2 磷酸化的增加。5-HT（2C-INI）受体表达细胞中基础 ERK1/2 磷酸化被 5-甲基-1-（3-吡啶基羰基）-1,2,3,5-四氢吡咯并[2,3-f]吲哚（SB206,553），一种针对磷脂酶 C 的 5-HT（2C）反向激动剂所消除。这种作用被中性拮抗剂 6-氯-5-甲基-1-[6-（2-甲基-3-吡啶基氧基）吡啶-3-基甲酰胺基]吲哚啉（SB242,084）所阻止，后者单独使用不会改变基础活性。在富含组成型激活的 5-HT（2C）受体的小鼠脉络丛中也进行了类似的体内观察。类似于激动剂诱导的 ERK1/2 磷酸化，HEK 293 细胞中的基础活性不受细胞内 Gα（q/11）和 Gα（13）蛋白耗竭的影响，但在表达显性负性β-arrestin 或通过 RNA 干扰耗竭β-arrestin 的细胞中以及在表达不能与钙调蛋白相互作用的显性负性钙调蛋白或 5-HT（2C-INI）受体突变体的细胞中，基础活性受到强烈抑制。四环抗抑郁药米氮平和米安色林同样降低了基础 ERK 激活。另一方面，m-氯苯基哌嗪衍生物曲唑酮和选择性 5-羟色胺再摄取抑制剂氟西汀单独使用时无活性，但可阻断 5-HT 诱导的 ERK1/2 磷酸化。综上所述，这些数据首次提供了 G 蛋白偶联受体对 G 蛋白非依赖性、β-arrestin 依赖性、受体信号的组成型活性的证据。
