Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET
Nat Commun. 2016 Jul 11;7:12178. doi: 10.1038/ncomms12178.
Yoon Namkung 1, Christian Le Gouill 2, Viktoria Lukashova 2, Hiroyuki Kobayashi 2, Mireille Hogue 2, Etienne Khoury 1, Mideum Song 1, Michel Bouvier 2, Stéphane A Laporte 1 3 4
PMID: 27397672 PMCID: PMC4942582 DOI: 10.1038/ncomms12178
Abstract
Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however, robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligand-promoted recruitment or sequestration of RLuc-tagged proteins to, or from, specific cell compartments, as well as sensitive subcellular BRET imaging for protein translocation visualization. These sensors are scalable to high-throughput formats and allow quantitative pharmacological studies of GPCR trafficking in real time, in live cells, revealing ligand-dependent biased trafficking of receptor/β-arrestin complexes.