On the G protein-coupling selectivity of the native A_2B adenosine receptor

A_2B adenosine receptor (A_2BAR) activation induces Gs-dependent cyclic AMP accumulation. However, A_2BAR G protein-coupling to other signaling events, e.g. ERK1/2 and calcium, is not well documented. We explored Gi, Gq/11 and Gs coupling in 1321 N1 astrocytoma, HEK293, and T24 bladder cancer cells endogenously expressing human A_2BAR, using NECA or nonnucleoside BAY60-6583 as agonist, selective Gi, Gs and Gq/11 blockers, and CRISPR/Cas9-based Gq- and Gs-null HEK293 cells. In HEK293 cells, A_2BAR-mediated ERK1/2 activity occurred via both Gi and Gs, but not Gq/11. However, HEK293 cell calcium mobilization was completely blocked by Gq/11 inhibitor UBO-QIC and by Gq/11 knockout. In T24 cells, Gi was solely responsible for A_2BAR-mediated ERK1/2 stimulation, and Gs suppressed ERK1/2 activity. A_2BAR-mediated intracellular calcium mobilization in T24 cells was mainly via Gi, although Gs may also play a role, but Gq/11 is not involved. In 1321 N1 astrocytoma cells A_2BAR activation suppressed rather than stimulated ERK1/2 activity. The ERK1/2 activity decrease was reversed by Gs downregulation using cholera toxin, but potentiated by Gi inhibitor pertussis toxin, and UBO-QIC had no effect. EPACs played an important role in A_2BAR-mediated ERK1/2 signaling in all three cells. Thus, A_2BAR may: couple to the same downstream pathway via different G proteins in different cell types; activate different downstream events via different G proteins in the same cell type; activate Gi and Gs, which have opposing or synergistic roles in different cell types/signaling pathways. The findings, relevant to drug discovery, address some reported controversial roles of A_2BAR and could apply to signaling mechanisms in other GPCRs.