Purinergic Receptor Transactivation by theβ₂-Adrenergic Receptor Increases Intracellular Ca²⁺in Nonexcitable Cells

Abstract

The adrenergic receptor (AR) increases intracellular Ca in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the AR promotes Ca mobilization (pEC = 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 following AR stimulation. The increase in cytoplasmic Ca 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 (IP) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the AR activation. Consistent with this mechanism,AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the AR-promoted Ca mobilization. Together, these data support a model whereby the AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP-dependent Ca mobilization from intracellular stores. Given that AR 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.

Bioluminescence resonance energy transfer-based biosensors allow monitoring of ligand- and transducer-mediated GPCR conformational changes

“…To determine if this is the case or if ISO can promote a conformational change on its own, the conformational change was assessed in cells devoid of Gs or β-arrestin. For this purpose, we took advantage of the Gs and β-arrestin-deficient cell lines that were recently generated using CRISPR/Cas9 34,35 . The lack of Gs and β-arrestin was functionally confirmed by the absence of ISO-stimulated cAMP production and β2AR endocytosis in the Gs and β-arrestin-deficient cells, respectively, when compared with their parental cells (Supplementary Fig.…”

“…HEK293T cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% newborn calf serum at 37 °C with 5% CO 2 . HEK293-ΔGs and HEK293-Δβarr1/2 were generated by CRISPR/Cas9 gene editing as previously reported 34,35 . HEK293-ΔGs, HEK293-Δβarr1/2, and their respective parental cells were grown in DMEM supplemented with 10% fetal bovine serum at 37 °C with 5% CO 2 .…”

Genetic evidence that β-arrestins are dispensable for the initiation of β 2 -adrenergic receptor signaling to ERK

“…Gnas f/f MEFs were kindly provided by Jean Regard and generated as previously described (41). Gαs knockout HEK293 cells were prepared using CRISPR/Cas9 as previously reported (42). …”

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

“…Experiments were conducted in HEK293T cells stably expressing RXFP1 (HEK‐RXFP1), RXFP2 (HEK‐RXFP2), or RXFP3 (HEK‐RXFP3), THP‐1 cells endogenously expressing RXFP1, CHO‐K1 (FlpIn) cells stably expressing RXFP3 (CHO‐RXFP3), and CHO‐K1 cells stably expressing RXFP4 (CHO‐RXFP4) . Experiments were also conducted in HEK293A parental cells and HEK293A cells that do not express the Gα s subunit (ΔGα s ) …”