Bioluminescence resonance energy transfer (bret) to detect the interactions between kappa opioid receptor and nonvisual arrestins

Bioluminescence resonance energy transfer (BRET) is a very sensitive technique employed to study protein–protein interactions, including G-protein-coupled receptor (GPCR) hetero- and homo-dimerization. Recently, BRET has also been used to investigate the interaction between GPCRs (e.g.: α2 adrenergic receptor, muscarinic M2 receptor, dopaminergic D2 receptor) and nonvisual arrestins. Within the last decade an increasing interest arose toward opioid agonists with limited activation of arrestin-dependent signaling pathways, as they are believed to be effective analgesics with reduced adverse effects. Here a BRET protocol is described to investigate interactions between the kappa opioid receptor (KOR) and nonvisual arrestins (arrestin-2 and arrestin-3) in HEK-293 cells, both under basal conditions and after exposure to KOR ligands.