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

Abstract ID 90367 Poster Board 342 GPCRs are dynamic proteins that transduce extracellular stimuli into intracellular signaling pathways to ultimately induce a cellular response. Ca²⁺ mobilization is a well-known second messenger that can be induced by GPCR activation. The primary canonical pathway for GPCR-promoted Ca²⁺ mobilization involves Gaq-mediated activation of phospholipase Cb (PLCb), while Gbg subunits from activated Gi can also induce Ca²⁺ mobilization through PLCb. Since previous studies support a role for Gs in b₂-adrenergic receptor (b₂AR)-mediated activation of Ca²⁺ mobilization, here we evaluated whether different Gs-coupled receptors endogenously expressed in HEK293 cells utilized common pathways for mediating Ca²⁺ mobilization. For the b₂AR, we found an essential role for Gq in mediating agonist (isoproterenol or formoterol) promoted activation of Ca²⁺ mobilization while disrupting Gs using DGas cells or Gi using pertussis toxin (PTX) had no effect on this process. b-agonist promoted Ca²⁺ mobilization was also effectively blocked by the b₂AR-selective inverse agonist ICI 118,551 as well as the Gq-selective inhibitor YM-254892 (YM) and was not observed in DGaq/11 cells. BRET analysis also revealed agonist-dependent association of b₂AR-Rluc with Nes-Venus-mGq. We also evaluated Ca²⁺ mobilization through the endogenous prostaglandin E receptors EP2 and EP4 in HEK293 cells. EP2 has been reported to selectively couple to Gs while EP4 couples to Gs and Gi. When activated by their selective agonists, both EP2 and EP4 were able to induce Ca²⁺ mobilization in HEK293 cells. The EP2 response was pertussis toxin sensitive but YM insensitive, while EP4 was sensitive to both PTX and YM. Interestingly, both EP2 and EP4 were largely unable to induce Ca²⁺ mobilization in DGas cells supporting a strong dependency of these receptors on Gs signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are utilized to mediate Ca²⁺ mobilization in HEK293 cells. The b₂AR primarily utilizes Gq, EP2 uses Gs and Gi, and EP4 appears to utilize Gs, Gi and Gq. Further characterization in other cellular models will be important to address the contribution of system bias to GPCR signaling.