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

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 quantitat...

Ausführliche Beschreibung

Gespeichert in:
Bibliographische Detailangaben
Veröffentlicht in:Nature communications 2016-07, Vol.7 (1), p.12178-12178, Article 12178
Hauptverfasser: Namkung, Yoon, Le Gouill, Christian, Lukashova, Viktoria, Kobayashi, Hiroyuki, Hogue, Mireille, Khoury, Etienne, Song, Mideum, Bouvier, Michel, Laporte, Stéphane A.
Format: Artikel
Sprache:eng
Schlagworte:
Online-Zugang:Volltext
Tags: Tag hinzufügen
Keine Tags, Fügen Sie den ersten Tag hinzu!
Beschreibung
Zusammenfassung: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. Cellular signaling processes often involve trafficking of receptors and other proteins between subcellular compartments. Here the authors demonstrate a method based on the concept of Enhanced bystander Bioluminescence Resonance Energy Transfer (EbBRET) that allows efficient real time monitoring of endocytosis and trafficking.
ISSN:2041-1723
2041-1723
DOI:10.1038/ncomms12178