A2B adenosine receptor-triggered intracellular calcium mobilization: Cell type-dependent involvement of Gi, Gq, Gs proteins and protein kinase C.

Abstract

Activation of PLCβ enzymes by G_iβγ and G_αq/11 proteins is a common mechanism to trigger cytosolic Ca²⁺ increase. We and others reported that G_αq/11 inhibitor FR900359 (FR) can inhibit both G_αq- and, surprisingly, G_iβγ-mediated intracellular Ca²⁺ mobilization. Thus, the G_αi-G_βγ-PLCβ-Ca²⁺ signaling axis depends entirely on the presence of active G_αq, which reasonably explained FR-inhibited G_iβγ-induced Ca²⁺ release. However, the conclusion that G_iβγ signaling is controlled by G_αq derives mostly from HEK293 cells. Here we show that indeed in HEK293 cells both G_αq/11 siRNA and G_αq/11 inhibitors diminished Ca²⁺ increase triggered by native G_q-coupled P2Y₁ receptors, or by transfected G_i-coupled A₁- or G_s-coupled A_2B adenosine receptors (ARs). However, in T24 bladder cancer cells, G_i inhibitor PTX, but not G_αq/11 inhibitors, FR, YM254890 (YM) or G_q/11 siRNA, inhibited Ca²⁺ increase triggered by native A_2BAR activation. Simultaneous inactivation of G_i and G_s further suppressed A_2BAR-triggered Ca²⁺ increase in T24 cells. The G_αq/11 inhibitor YM fully and partially inhibited endogenous P2Y₁- and β₂-adrenergic receptor-induced Ca²⁺ increase in T24 cells, respectively. PKC activator PMA partially diminished A_2BAR-triggered but completely diminished β₂-adrenergic receptor-triggered Ca²⁺ increase in T24 cells. Neither β-arrestin1 nor β-arrestin2 siRNA affected A_2BAR-mediated Ca²⁺ increase. Unlike in T24 cells, YM inhibited native A_2BAR-triggered calcium mobilization in MDA-MB-231 breast cancer cells. Thus, G_αq/11 is vital for Ca²⁺ increase in some cell types, but G_iβγ-mediated Ca²⁺ signaling can be Gα_q/11-dependent or independent based on cell type and receptor activated. Besides G proteins, PKC also modulates cytosolic Ca²⁺ increase depending on cell type and receptor.