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 Gaq- and Gbg-mediated activation of phospholipase C-b (PLCb). While some Gs-coupled receptors are shown to trigger Ca2+ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether Gs-coupled receptors including the b 2-adrenergic receptor (b2AR) 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 b 2 AR, we found an essential role for Gq in agonist-promoted Ca2+ mobilization while genetic or pharmacological inhibition of Gs or Gi had minimal effect. b-agonist-promoted Ca2+ mobilization was effectively blocked by the Gq-selective inhibitor YM-254890 and was not observed in D G a q/11 or D PLC b cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the b 2 AR 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 D G a s or D PLC b 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 b 2 AR primarily uses Gq, EP2R uses Gs and Gi, and EP4R uses Gs, Gi, and Gq.