title: Quantitative analysis of G-protein-coupled receptor internalization using DnaE intein-based assay.
abstract: G-protein-coupled receptors (GPCRs), the largest family of cell surface receptors, are involved in many physiological processes. They represent highly important therapeutic targets for drug discovery. Currently, there are numerous cell-based assays developed for the pharmacological profiling of GPCRs and the identification of novel agonists and antagonists. However, the development of new, faster, easier, and more cost-effective approaches to detect GPCR activity remains highly desirable. β-arrestin-dependent internalization has been demonstrated to be a common mechanism for most GPCRs. Here we describe a novel assay for quantitative analysis of GPCR internalization based on DnaE intein-mediated reconstitution of fragmented Renilla luciferase or Firefly luciferase when activated GPCRs interact with β-arrestin2 or Rab5. Further validation, using functionally divergent GPCRs, showed that EC50 values obtained for the known agonists and antagonists were in close agreement with the results of previous reports. This suggests that this assay is sensitive enough to permit quantification of GPCR internalization. Compared with conventional assays, this novel assay system is cost-effective, rapid, and easy to manipulate. These advantages may allow this assay to be used universally as a functional cell-based system for GPCR characterization and in the screening process of drug discovery.
authors.affiliation: Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Bin
authors.initials: B
authors.lastname: Lu
authors.affiliation: Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Linjie
authors.initials: L
authors.lastname: Chen
authors.affiliation: Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Shi
authors.affiliation: Institute of Biochemistry, College of Life Sciences, Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China.
authors.forename: Naiming
authors.initials: N
authors.lastname: Zhou
mesh_terms: D004258:DNA Polymerase III / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007301:Insect Hormones / Q000494:pharmacology; D047668:Inteins; D049409:Luciferases, Firefly / Q000378:metabolism; D049410:Luciferases, Renilla / Q000378:metabolism; D009525:Niacin / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D007301:Insect Hormones; D009842:Oligopeptides; D043562:Receptors, G-Protein-Coupled; C044833:adipokinetic hormone; D009525:Niacin; D049410:Luciferases, Renilla; D049409:Luciferases, Firefly; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III; D011761:Pyrrolidonecarboxylic Acid
keywords: Agonist; Cell-based assay; DnaE intein; G-protein-coupled receptor; Internalization; Luciferase; Protein splicing; β-Arrestins