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 non-excitable human embryonic kidney (HEK)-293S cells. Down-regulation of Gs with sustained cholera toxin pre-treatment and the use Gs-null HEK293 (ΔGs-HEK293) cells generated using the 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 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 adenosine triphosphate (ATP) and pre-treatment 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 co-expressed in various tissues, this novel signalling 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 signalling.
- Gs family
- Gq/11 family
- Adenylyl cyclases
- G protein regulation
- The American Society for Pharmacology and Experimental Therapeutics