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Nat Commun. 2016 Jul 11;7:12178. doi: 10.1038/ncomms12178.

Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET.

Author information

1
Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, Québec, Canada H4A 3J1.
2
Department of Biochemistry and Institute for Research in Immunology and Cancer (IRIC), Université de Montréal, Montréal, Québec, Canada H3C 1J4.
3
Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada H3G 1Y6.
4
Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada H3A 0C7.

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.

PMID:
27397672
PMCID:
PMC4942582
DOI:
10.1038/ncomms12178
[Indexed for MEDLINE]
Free PMC Article

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