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临床常用β-肾上腺素受体激动剂和拮抗剂对β-arrestin2招募作用的研究

王怡然¹, 程德琴², 马兰¹, 刘星^1,*

¹复旦大学上海医学院药理研究中心，上海 200032；²复旦大学脑科学研究院，上海 200032

摘要

大量β-肾上腺素受体(β-adrenergic receptor, β-AR)药物被应用于心血管疾病等临床疾病的治疗，但许多常用的β-AR药物对下游β-arrestin偏向性信号通路的激活特性和作用并不清楚。本文旨在检测临床常用的β-AR药物对β-arrestin的招募作用。我们采用TANGO (transcriptional activation following arrestin translocation)方法在稳定转染四环素反式激活因子(tetracycline transactivator protein, tTA)依赖荧光素酶报告子和β-arrestin2-TEV融合基因的HEK293细胞系(HTLA细胞)中检测β-AR配体对β-arrestin2的招募情况。β-AR被其配体激活后，β-arrestin2被募集到受体的C端，随后G蛋白耦联受体(G protein-coupled receptors, GPCRs)融合蛋白在TEV蛋白酶裂解位点发生裂解。裂解导致tTA的释放，tTA转移到细胞核后可激活荧光素酶报告基因的转录。结果显示，非选择性β-AR激动剂肾上腺素、去甲肾上腺素和异丙肾上腺素能通过β1-AR和β2-AR招募β-arrestin2。选择性β1-AR激动剂多巴酚丁胺和得诺巴明能通过β1-AR招募β-arrestin2。选择性β2-AR激动剂沙丁胺醇和丙卡特罗能通过β2-AR招募β-arrestin2。非选择性β-AR拮抗剂阿普洛尔和吲哚洛尔能通过β1-AR招募β-arrestin2。选择性β1-AR拮抗剂塞利洛尔和贝凡洛尔能够通过β1-AR招募β-arrestin2。选择性β2-AR拮抗剂布托沙明能够通过β1-AR产生β-arrestin2招募作用。上述结果为β-AR药物的潜在作用研究提供线索，也为后续β-arrestin偏向性β-AR药物的筛选奠定研究基础。

关键词： β-肾上腺素受体; β-arrestin2; G蛋白耦联受体; 荧光素酶报告基因

β-arrestin2 recruitment by β-adrenergic receptor agonists and antagonists 

WANG Yi-Ran¹, CHENG De-Qin², MA Lan¹, LIU Xing^1,*

¹ Pharmacology Research Center, Shanghai Medical College, Fudan University, Shanghai 200032, China；²Institutes of Brain Science, Fudan University, Shanghai 200032, China

Abstract

A large number of β-adrenergic receptor (β-AR) agonists and antagonists are widely used in the treatment of cardiovascular diseases and other diseases. Nonetheless, it remains unclear whether these commonly used β-AR drugs can activate downstream β- arrestin-biased signaling pathways. The objective of this study was to investigate β-arrestin2 recruitment effects of β-AR agonists and antagonists that were commonly used in clinical practice. We used TANGO (transcriptional activation following arrestin translocation) assay to detect the β-arrestin2 recruitment by β-AR ligands in HEK293 cell line (HTLA cells) stably transfected with tetracycline transactivator protein (tTA) dependent luciferase reporter and β-arrestin2-TEV fusion gene. Upon activation of β-AR by a β-AR ligand, β-arrestin2 was recruited to the C terminus of the receptor, followed by cleavage of the G protein-coupled receptors (GPCRs) fusion protein at the TEV protease-cleavage site. The cleavage resulted in the release of tTA, which, after being transported to the nucleus, activated transcription of the luciferase reporter gene. The results showed that β-AR non-selective agonists epinephrine, noradrenaline and isoprenaline all promoted β-arrestin2 recruitment at β1-AR and β2-AR. β1-AR selective agonists dobutamine and denopamine both promoted β-arrestin2 recruitment at β1-AR. β2-AR selective agonists procaterol and salbutamol promoted β-arrestin2 recruitment at β2-AR. β-AR non-selective antagonists alprenolol and pindolol promoted β-arrestin2 recruitment at β1-AR. β1-AR selective antagonists celiprolol and bevantolol showed β-arrestin2 recruitment at β1-AR. β2-AR selective antagonists butoxamine showed β-arrestin2 recruitment at β1-AR. These results provide some clues for the potential action of β-AR drugs, and lay a foundation for the screening of β-arrestin-biased β-AR ligands.

Key words: β-adrenergic receptors; β-arrestin2; G protein-coupled receptors; luciferase reporter gene

收稿日期：　　录用日期：

通讯作者：刘星　　E-mail: xingliu@fudan.edu.cn

引用本文：

王怡然, 程德琴, 马兰, 刘星. 临床常用β-肾上腺素受体激动剂和拮抗剂对β-arrestin2招募作用的研究[J]. 生理学报 2022; 74 (6): 993-1004.

WANG Yi-Ran, CHENG De-Qin, MA Lan, LIU Xing. β-arrestin2 recruitment by β-adrenergic receptor agonists and antagonists . Acta Physiol Sin 2022; 74 (6): 993-1004 (in Chinese with English abstract).