title: Development of a novel DnaE intein-based assay for quantitative analysis of G-protein-coupled receptor internalization.
abstract: G-protein-coupled receptor (GPCR) internalization provides a G-protein-subtype-independent method for assaying agonist-stimulated activation of receptors. We have developed a novel assay that allows quantitative analysis of GPCR internalization based on the interaction between activated GPCRs and β-arrestin2 and on Nostoc punctiforme DnaE intein-mediated reconstitution of Renilla luciferase fragments. This assay system was validated using four functionally divergent GPCRs treated with agonists and antagonists. The EC(50) values obtained for the known agonists and antagonists are in close agreement with the results of previous reports, indicating that this assay system is sensitive enough to permit quantification of GPCR internalization. This rapid and quantitative assay, therefore, could be used universally as a functional cell-based assay for GPCR high-throughput screening during drug discovery.
authors: Institute of Biochemistry, College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, China.
authors: Yaping
authors: Y
authors: Zhang
authors: Wen
authors: W
authors: Yang
authors: Linjie
authors: L
authors: Chen
authors: Ying
authors: Y
authors: Shi
authors: Guo
authors: G
authors: Li
authors: Naiming
authors: N
authors: Zhou
mesh_terms: D001483:Base Sequence; D001681:Biological Assay / Q000379:methods*; D000242:Cyclic AMP / Q000378:metabolism; D004258:DNA Polymerase III / Q000378:metabolism*; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D047668:Inteins / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D046937:Nostoc / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018027:Receptors, Glucagon / Q000378:metabolism; D014162:Transfection
chemical_list: D050505:Mutant Proteins; D043562:Receptors, G-Protein-Coupled; D018027:Receptors, Glucagon; D000242:Cyclic AMP; D008156:Luciferases; C074904:DNA polymerase III, alpha subunit; D004258:DNA Polymerase III