Improved donor/acceptor BRET couples for monitoring beta-arrestin recruitment to G protein-coupled receptors.
We report highly sensitive bioluminescence resonance energy transfer (BRET) assays with optimized donor/acceptor couples. We combined the energy donors Renilla luciferase (Rluc) and the Rluc8 variant with the energy acceptors yellow fluorescent protein, the YPet variant and the Renilla green fluorescent protein (RGFP). Different donor/acceptor couples were tested in well-established assays measuring ligand-induced beta-arrestin (betaARR) intramolecular rearrangements and recruitment to G protein-coupled receptors. We show increased sensitivity with Rluc8/YPet and Rluc8/RGFP couples and measured previously undetectable BRET signals. These tools improve existing betaARR assays and offer new options for the development of future BRET assays.
Kamal
Maud
M
Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France.
Marquez
Marcel
M
Vauthier
Virginie
V
Leloire
Audrey
A
Froguel
Philippe
P
Jockers
Ralf
R
Couturier
Cyril
C
D019390:Arrestins / Q000032:analysis* / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008163:Luminescent Measurements / Q000379:methods*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000737:chemistry*; D000071557:beta-Arrestins
D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins