Diverse pathways in GPCR-mediated activation of Ca2+ mobilization in HEK293 cells.

G protein-coupled receptors transduce extracellular stimuli into intracellular signaling. Ca2+ is a well-known second messenger that can be induced by G protein-coupled receptor activation through the primary canonical pathways involving Gαq- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether Gs-coupled receptors including the β2-adrenergic receptor (β2AR) and the prostaglandin EP2 and EP4 receptors (EP2R and EP4R) that are endogenously expressed in human embryonic kidney 293 (HEK293) cells utilize common pathways for mediating Ca2+ mobilization. For the β2AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. β-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in ΔGαq/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β2AR with Gq. For the EP2R, which couples to Gs, agonist treatment induced Ca2+ mobilization in a pertussis toxin-sensitive but YM-254890-insensitive manner. In contrast, EP4R, which couples to Gs and Gi, exhibited Ca2+ mobilization that was sensitive to both pertussis toxin and YM-254890. Interestingly, both EP2R and EP4R were largely unable to induce Ca2+ mobilization in ΔGαs or ΔPLCβ cells, supporting a strong dependency on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are used to mediate Ca2+ mobilization in HEK293 cells where the β2AR primarily uses Gq, EP2R uses Gs and Gi, and EP4R uses Gs, Gi, and Gq.