Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin InteractionsReport as inadecuate




Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions - Download this document for free, or read online. Document in PDF available to download.

1

Department of Chemistry, School of Science, The University of Tokyo, Bunkyo-ku, Hongo, Tokyo 113, Japan

2

PRESTO, Japan Science and Technology Agency, Tokyo, Japan





*

Author to whom correspondence should be addressed.



Abstract G protein-coupled receptors GPCRs are crucial elements in mammalian signal transduction, and are considered to represent potent drug targets. We have previously developed a GPCR assay system in cultured cells based on complementation of split fragments of click beetle Pyrearinus termitilluminans luciferase. The interaction of GPCRs with its target, β-arrestin, resulted in strong emission of bioluminescence upon stimulation with its specific ligand. In this study, we improved precision of the GPCR assay system by using railroad worm Phrixothrix hirtus luciferase as an internal control. We generated stable cell lines harboring the railroad worm luciferase and quantitatively evaluate the extent of GPCR-β-arrestin interactions. We showed concentration-dependent bioluminescence responses for four GPCRs: β2-adrenoceptor, endothelin receptor type A, α2-adrenoceptor and human μ-opioid receptor. We also demonstrated that the variation of responses was reduced significantly by normalizing the data with bioluminescence from railroad worm luciferase. This assay system represents a simple and reliable approach for screening drug candidates in a high throughput manner. View Full-Text

Keywords: GPCR; luciferase; protein interaction GPCR; luciferase; protein interaction





Author: A.K.M. Kafi 1, Mitsuru Hattori 1, Naomi Misawa 1 and Takeaki Ozawa 1,2,*

Source: http://mdpi.com/



DOWNLOAD PDF




Related documents