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

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

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。

A large number ofβ-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 usedβ-AR drugs can activate downstreamβ-arrestin-biased signaling pathways.The objective of this study was to investigateβ-arrestin2 recruitment effects ofβ-AR agonists and antagonists that were commonly used in clinical practice.We used TANGO(transcriptional activation following arrestin translocation)assay to detect theβ-arrestin2 recruitment byβ-AR ligands in HEK293 cell line(HTLA cells)stably transfected with tetracycline transactivator protein(tTA)dependent luciferase reporter andβ-arrestin2-TEV fusion gene.Upon activation ofβ-AR by aβ-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 thatβ-AR non-selective agonists epinephrine,noradrenaline and isoprenaline all promotedβ-arrestin2 recruitment atβ1-AR andβ2-AR.β1-AR selective agonists dobutamine and denopamine both promotedβ-arrestin2 recruitment atβ1-AR.β2-AR selective agonists procaterol and salbutamol promotedβ-arrestin2recruitment atβ2-AR.β-AR non-selective antagonists alprenolol and pindolol promotedβ-arrestin2 recruitment atβ1-AR.β1-AR selective antagonists celiprolol and bevantolol showedβ-arrestin2 recruitment atβ1-AR.β2-AR selective antagonists butoxamine showedβ-arrestin2 recruitment atβ1-AR.These results provide some clues for the potential action ofβ-AR drugs,and lay a foundation for the screening ofβ-arrestin-biasedβ-AR ligands.