5-HT4R,5-HT6R,and 5-HT7AR are three constitutively active Gs-coupled 5-HT receptors that have key roles in brain development,leaing,memory,cognition,and other physiological processes in the central nervous system.In addition to Gs signaling cascade mediated by these three 5-HT receptors,the ERK1/2 signaling which is dependent on cyclic adenosine monophosphate (cAMP) production and protein kinase A (PKA) activation downstream of Gs signaling has also been widely studied.In this study,we investigated these two signaling pathways originating from the three Gs-coupled 5-HT receptors in AD293 cells.We found that the phosphorylation and activation of ERK1/2 are ligand-induced,in contrast to the constitutively active Gs signaling.This indicates that Gs signaling alone is not sufficient for ERK1/2 activation in these three 5-HT receptors.In addition to Gs,we found that β-arrestin and Fyn are essential for the activation of ERK1/2.Together,these results put forth a novel mechanism for ERK1/2 activation involving the cooperative action of Gs,β-arrestin,and Fyn.