Dual-Color Bioluminescence Analysis for Quantitatively Monitoring G-Protein-Coupled Receptor and β-Arrestin Interactions

Pharmaceuticals

A.K.M. Kafi¹  Mitsuru Hattori¹  Naomi Misawa¹ 

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

关键词: GPCR;    luciferase;    protein interaction;   

DOI  :  10.3390/ph4030457

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2011 by the authors; licensee MDPI, Basel, Switzerland.