A2Badenosine receptor-mediated intracellular calcium mobilization: Involvement of Gi, Gq, Gs, β-arrestins, and protein kinase C

Abstract ID 96308 Poster Board 554 Ca2+, a divalent cation, affects almost all aspects of cell life and death, thus understanding the basic principles controlling its cytosolic fluctuation is vital to the development of drugs targeting specific Ca2+ release mechanisms. Activation of PLCβ enzymes by GPCRs, the largest membrane protein family in the human genome and the targets of 30% of today's prescription medicines, is a common mechanism to trigger the cytosolic Ca2+ release in human cells. Both Giβγ and Gαq, activated by Gi-coupled and Gq-coupled receptors, respectively, can trigger intracellular calcium mobilization. We found previously that the recently available Gq inhibitor FR900358 (UBO) can inhibit both Giβγ and Gαq-mediated Ca2+ increase 1. Pfeil et al. (2020) confirmed our finding and further explored the underlying mechanisms 2. They found that "the G αi-Gβγ-PLCβ-Ca2+ signaling module is entirely dependent on the presence of active Gαq. If Gαq is pharmacologically inhibited or genetically ablated, Gαq can bind to PLCβ but does not elicit Ca2+ signals." The finding related to Gq/11 control of Giβγ-mediated Ca2+ signaling is an important discovery. However, the conclusion that Gαqcontrols Giβγ-mediated Ca2+ increases was drawn mostly from the study of HEK293 cells. In the present study, we further explored whether this is the case in other types of cells. Indeed, both Gq/11 knockout and Gq/11 inhibitors completely blocked P2Y1-, A1AR- and A2BAR-mediated calcium increase in HEK293 cells. However, in T24 bladder cancer cells, the Gi inhibitor PTX and Gs inactivator CTX, but not the Gq inhibitors FR900358 and YM254890 or Gq/11 siRNA, inhibited A2BAR-mediated intracellular Ca2+ mobilization. Simultaneous inactivation of both Gi and Gs completely blocked A2BAR-triggered Ca2+ increase. Both PKA inhibitor H89 and EPAC inhibitor ESI-09 partially diminished the Ca2+ increase in T24 cells, but the combination of two inhibitors did not produce an effect. Neither β-arrestin1 not β-arrestin2 siRNA affected the Ca2+ increase. PKC activator PMA only partially diminished A2BAR-mediated, but completely blocked β2-adrenergic receptor-mediated Ca2+ increase. The Gq inhibitor YM254890 partially inhibited β2-adrenergic receptor-induced Ca2+ increase, although without effect on A2BAR-triggered Ca2+ increase. Thus, we confirmed that Gαq is indeed vital for Giβγ-increased Ca2+ in HEK293 cells, but Gαq is not involved in Giβγ-triggered Ca2+ release in T24 bladder cancer cells upon the activation of the native A2BAR. It seems that Gαq/11 may or may not participate in Giβγ-mediated Ca2+ signaling depending on type of cells and receptors. Gao ZG, Jacobson KA. On the selectivity of the G αq inhibitor UBO-QIC: A comparison with the G αi inhibitor pertussis toxin. Biochem. Pharmacol. 2016;107:59–66. Pfeil EM et al. Heterotrimeric G protein subunit G αq Is a master switch for Gβγ-mediated calcium mobilization by Gi-coupled GPCRs. Mol. Cell. 2020;80:940–954.