LAUSR

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.