Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca(2+) in Nonexcitable Cells.
Department of Biochemistry (W.S., E.T.v.d.W., A.-M.S., B.P., M.B.) and Institute for Research in Immunology and Cancer (W.S., E.T.v.d.W., A.-M.S., B.P., M.H., V.L., C.L.G., M.B.), Université de Montréal, Montréal, QC, Canada; Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Miyagi, Japan (A.I., S.I., J.A.); Japan Science and Technology Agency, Precursory Research for Embryonic Science and Technology, Kawaguchi, Saitama, Japan (A.I.); and Japan Agency for Medical Research and Development, Core Research for Evolutional Science and Technology, Chiyoda-ku, Tokyo, Japan (J.A.).
The β2 adrenergic receptor (β2AR) increases intracellular Ca(2+) in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the β2AR promotes Ca(2+) 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 Ca(2+) following β2AR stimulation. The increase in cytoplasmic Ca(2+) 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 Ca(2+) 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 Ca(2+) 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.
Mol. Pharmacol. 2017;91(5):533-544.
Pubmed ID: 28280061