Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells
Purinergic Receptor Transactivation by the β2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells
复制标题
DOI：
10.1124/mol.116.106419
复制
发表时间：
2017-05-01
影响因子：
3.6
通讯作者：
Bouvier, Michel
中科院分区：
文献类型：
--
作者：
Stallaert, Wayne;van der Westhuizen, Emma T.;Bouvier, Michel
β(2) 肾上腺素能受体 (β(2)AR) 可增加多种细胞类型的细胞内 Ca2+。通过结合药理学和遗传操作，我们揭示了一种新机制，通过该机制，β(2)AR 促进非兴奋性人胚肾 (HEK) 293S 细胞中 Ca2+$ 动员 (pEC(50) = 7.32 +/- 0.10)。通过持续霍乱毒素预处理和使用通过成簇规则间隔短回文重复相关蛋白9核酸酶（CRISPR/Cas9）系统生成的Gs无效HEK293（Delta Gs-HEK293）细胞下调Gs，结合cAMP形成的药理学调节，揭示了β（2）AR刺激后细胞内Ca2+的Gs依赖性但不依赖于cAMP的增加。细胞质 Ca2+ 的增加受到 P2Y 嘌呤能受体拮抗剂以及 Gq 显性失活突变形式（一种 Gq 选择性抑制剂）和肌醇 1,4,5-三磷酸 (IP3) 受体拮抗剂的抑制，表明该 Gq 偶联受体家族在 β(2)AR 激活下游发挥作用。与这一机制一致，β(2) AR 刺激促进了 ATP 的细胞外释放，而腺苷三磷酸双磷酸酶预处理抑制了 β(2) AR 促进的 Ca2+ 动员。总之，这些数据支持这样一个模型：β(2)AR 刺激 Gs 依赖性 ATP 释放，从而通过由内而外的机制反式激活 Gq 偶联的 P2Y 受体，导致 Gq 和 IP3 依赖性 Ca2+ 从细胞内储备中动员。鉴于 β(2)AR 和 P2Y 受体在多种组织中共表达，这种新颖的信号传导范式可能具有重要的生理学意义并具有治疗意义。此外，本研究报告了使用 CRISPR/Cas9 基因组编辑技术删除 Gs 的 HEK293 细胞的生成和验证，这无疑将成为研究 Gs 依赖性信号传导的有力工具。
The beta(2) adrenergic receptor (beta(2)AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the beta(2)AR promotes Ca2+$ mobilization (pEC(50) = 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 (Delta 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 beta(2)AR 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 beta(2)AR activation. Consistent with this mechanism, beta(2)AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the beta(2) AR-promoted Ca2+ mobilization. Together, these data support a model whereby the beta(2)AR 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 beta(2)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.