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 Gα_q- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some G_s-coupled receptors are shown to trigger Ca²⁺ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether G_s-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 G_q in agonist-promoted Ca²⁺ mobilization while genetic or pharmacological inhibition of G_s or G_i had minimal effect. β-agonist-promoted Ca²⁺ mobilization was effectively blocked by the G_q-selective inhibitor YM-254890 and was not observed in ΔGα_q/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β₂AR with G_q. For the EP₂R, which couples to G_s, agonist treatment induced Ca²⁺ mobilization in a pertussis toxin-sensitive but YM-254890-insensitive manner. In contrast, EP₄R, which couples to G_s and G_i, 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 ΔGα_s or ΔPLCβ cells, supporting a strong dependency on G_s 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 G_q, EP₂R uses G_s and G_i, and EP₄R uses G_s, G_i, and G_q.