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	A high-throughput screening system for g-protein-coupled receptors using beta-lactamase enzyme complementation technology
	Zhao, Chuan-ke1,2; Yin, Qi1,2; Li, Shi-you1
刊名	Acta pharmacologica sinica
	2010-12-01
卷号	31期号:12页码:1618-1624
关键词	G-protein-coupled receptor Beta-lactamase Enzyme fragment complementation High-throughput screening
ISSN号	1671-4083
DOI	10.1038/aps.2010.154
通讯作者	Li, shi-you(lishiyou@big.ac.cn)
英文摘要	Aim: to establish a system for monitoring the activation of g-protein-coupled receptors (gpcrs) using beta-lactamase enzyme fragment complementation (efc) technology. methods: two inactive beta-lactamase deletion fragments, bla(a) and bla(b), were fused to beta-arrestin and gpcr, respectively. a stable cell line named hek/293-beta 2a2, which expressed two fusion proteins, gpcr/bla(b) and beta-arrestin2/bla(a), was generated under antibiotic selection. a natural compound library of high performance liquid chromatography (hplc)-fractionated samples from the ethanol extracts of chinese medicinal herbs was used for high-throughput screening (hts) of beta 2-adrenoceptor (beta 2ar) agonists against the cell line hek/293-beta 2a2. the interested hits were validated by the measurement of second-messenger cyclic adenosine monophosphate (camp) production. results: the stable cell line hek/293-beta 2a2 responded to beta 2ar agonist/antagonist in a dose-dependent manner. the ec(50) value obtained for isoproterenol was 15.5 nmol/l, and the ic(50) value obtained for propranolol was 51.9 nmol/l. furthermore, hts was performed to identify beta 2ar agonists from the natural compound library we established. the z' factor value was determined to be 0.68. three hits were identified from primary screening and found to be as potent as isoproterenol in a camp assay. conclusion: a cell-based high-throughput functional assay was established to directly monitor the activation of gpcrs based on the interaction between agonist-activated gpcr and beta-arrestin using beta-lactamase efc technology, which can be used to search for leads in the natural compound library.
WOS关键词	7-TRANSMEMBRANE RECEPTORS ; OPIOID RECEPTOR ; LIVING CELLS ; ASSAY ; IDENTIFICATION ; ACTIVATION ; AGONISTS ; PHOSPHORYLATION ; INTERNALIZATION ; VALIDATION
WOS研究方向	Chemistry ; Pharmacology & Pharmacy
WOS类目	Chemistry, Multidisciplinary ; Pharmacology & Pharmacy
语种	英语
出版者	SHANGHAI INST MATERIA MEDICA
WOS记录号	WOS:000284936500013
内容类型	期刊论文
URI标识	http://www.corc.org.cn/handle/1471x/2409661
专题	中国科学院大学
通讯作者	Li, Shi-you
作者单位	1.Chinese Acad Sci, Beijing Inst Genom, Key Lab Genome Sci & Informat, Beijing 100029, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100039, Peoples R China
推荐引用方式 GB/T 7714	Zhao, Chuan-ke,Yin, Qi,Li, Shi-you. A high-throughput screening system for g-protein-coupled receptors using beta-lactamase enzyme complementation technology[J]. Acta pharmacologica sinica,2010,31(12):1618-1624.
APA	Zhao, Chuan-ke,Yin, Qi,&Li, Shi-you.(2010).A high-throughput screening system for g-protein-coupled receptors using beta-lactamase enzyme complementation technology.Acta pharmacologica sinica,31(12),1618-1624.
MLA	Zhao, Chuan-ke,et al."A high-throughput screening system for g-protein-coupled receptors using beta-lactamase enzyme complementation technology".Acta pharmacologica sinica 31.12(2010):1618-1624.

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