G protein-coupled receptors (GPCRs) transmit extracellular signals into cells by activating G protein- and β-arrestin-dependent pathways. Extracellular signal-regulated kinases (ERKs) play a central role in integrating these different linear inputs coming from a variety of GPCRs to regulate cellular functions. Here, we investigated human melatonin MT1 and MT2 receptors signaling through the ERK1/2 cascade by employing different biochemical techniques together with pharmacological inhibitors and siRNA molecules. We show that ERK1/2 activation by both receptors is exclusively G protein-dependent, without any participation of β-arrestin1/2 in HEK293 cells. ERK1/2 activation by MT1 is only mediated though Gi/o proteins, while MT2 is dependent on the cooperative activation of Gi/o and Gq/11 proteins. In the absence of Gq/11 proteins, however, MT2 -induced ERK1/2 activation switches to a β-arrestin1/2-dependent mode. The signaling cascade downstream of G proteins is the same for both receptors and involves activation of the PI3K/PKCζ/c-Raf/MEK/ERK cascade. The differential G protein dependency of MT1 - and MT2 -mediated ERK activation was confirmed at the level of EGR1 and FOS gene expression, two ERK1/2 target genes. Gi/o /Gq/11 cooperativity was also observed in Neuroscreen-1 cells expressing endogenous MT2 , whereas in the mouse retina, where MT2 is engaged into MT1 /MT2 heterodimers, ERK1/2 signaling is exclusively Gi/o -dependent. Collectively, our data reveal differential signaling modes of MT1 and MT2 in terms of ERK1/2 activation, with an unexpected Gi/o /Gq/11 cooperativity exclusively for MT2 . The plasticity of ERK activation by MT2 is highlighted by the switch to a β-arrestin1/2-dependent mode in the absence of Gq/11 proteins and by the switch to a Gi/o mode when engaged into MT1 /MT2 heterodimers, revealing a new mechanism underlying tissue-specific responses to melatonin.

中文翻译：

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褪黑素MT1和MT2受体ERK信号传导差异地依赖于Gi / o和Gq / 11蛋白。

G蛋白偶联受体（GPCR）通过激活G蛋白和β-arrestin依赖性途径将细胞外信号传递到细胞中。细胞外信号调节激酶（ERK）在整合来自各种GPCR的这些不同的线性输入以调节细胞功能中起着核心作用。在这里，我们通过使用不同的生化技术以及药理抑制剂和siRNA分子研究了人类褪黑激素MT1和MT2受体通过ERK1 / 2级联的信号传导。我们显示，这两个受体的ERK1 / 2激活完全是G蛋白依赖性的，而HEK293细胞中没有任何β-arrestin1/ 2的参与。MT1激活ERK1 / 2仅通过Gi / o蛋白介导，而MT2依赖于Gi / o和Gq / 11蛋白的协同激活。但是，在没有Gq / 11蛋白的情况下，MT2诱导的ERK1 / 2激活切换至β-arrestin1/ 2依赖性模式。G蛋白下游的信号级联对于两个受体都是相同的，并且涉及PI3K /PKCζ/ c-Raf / MEK / ERK级联的激活。在两个ERK1 / 2靶基因EGR1和FOS基因表达水平，证实了MT1和MT2介导的ERK活化对G蛋白的依赖性不同。在表达内源性MT2的Neuroscreen-1细胞中也观察到Gi / o / Gq / 11协同作用，而在小鼠视网膜中，MT2参与MT1 / MT2异二聚体，ERK1 / 2信号仅依赖Gi / o依赖性。总的来说，我们的数据揭示了在ERK1 / 2激活方面MT1和MT2的差分信号模式，以及专门针对MT2的出乎意料的Gi / o / Gq / 11合作性。