High-throughput screening of G protein-coupled receptor antagonists using a bioluminescence resonance energy transfer 1-based beta-arrestin2 recruitment assay

J Biomol Screen. 2005 Aug;10(5):463-75. doi: 10.1177/1087057105275344.


In this study, the authors developed HEK293 cell lines that stably coexpressed optimal amounts of beta-arrestin2-Rluc and VENUS fusions of G protein-coupled receptors (GPCRs) belonging to both class A and class B receptors, which include receptors that interact transiently or stably with beta-arrestins. This allowed the use of a bioluminescence resonance energy transfer (BRET) 1- beta-arrestin2 translocation assay to quantify receptor activation or inhibition. One of the developed cell lines coexpressing CCR5-VENUS and beta-arrestin2- Renilla luciferase was then used for high-throughput screening (HTS) for antagonists of the chemokine receptor CCR5, the primary co-receptor for HIV. A total of 26,000 compounds were screened for inhibition of the agonist-promoted beta-arrestin2 recruitment to CCR5, and 12 compounds were found to specifically inhibit the agonist-induced beta-arrestin2 recruitment to CCR5. Three of the potential hits were further tested using other functional assays, and their abilities to inhibit CCR5 agonist-promoted signaling were confirmed. This is the 1st study describing a BRET1-beta-arrestin recruitment assay in stable mammalian cells and its successful application in HTS for GPCRs antagonists.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Arrestins / chemistry
  • Arrestins / metabolism*
  • Automation
  • Calcium / metabolism
  • Cell Line
  • Dose-Response Relationship, Drug
  • Drug Evaluation, Preclinical / methods*
  • Energy Transfer
  • Genes, Reporter
  • Green Fluorescent Proteins / metabolism
  • HIV / metabolism
  • Humans
  • Luciferases, Renilla / metabolism
  • Luminescent Measurements
  • Macromolecular Substances / metabolism
  • Microscopy, Fluorescence
  • Plasmids / metabolism
  • Protein Transport
  • Receptors, CCR5 / metabolism
  • Receptors, G-Protein-Coupled / antagonists & inhibitors*
  • Renilla
  • Time Factors
  • beta-Arrestins


  • Arrestins
  • Macromolecular Substances
  • Receptors, CCR5
  • Receptors, G-Protein-Coupled
  • beta-Arrestins
  • Green Fluorescent Proteins
  • Luciferases, Renilla
  • Calcium