Purinergic Receptor Transactivation by the β₂-Adrenergic Receptor Increases Intracellular Ca²⁺ in Nonexcitable Cells

The β₂ adrenergic receptor (β₂AR) increases intracellular Ca²⁺ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β₂AR promotes Ca²⁺ mobilization (pEC₅₀ = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca²⁺ following β₂AR stimulation. The increase in cytoplasmic Ca²⁺ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP₃) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the β₂AR activation. Consistent with this mechanism, β₂AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the β₂AR-promoted Ca²⁺ mobilization. Together, these data support a model whereby the β₂AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP₃-dependent Ca²⁺ mobilization from intracellular stores. Given that β₂AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.