Split luciferase-based assay for simultaneous analyses of the ligand concentration- and time-dependent recruitment of...

Functional selectivity of agonists has gained increasing interest in G protein-coupled receptor (GPCR) research, e.g. due to expectations of drugs with reduced adverse effects. Different agonist-dependent GPCR conformations are conceived to selectively activate a balanced or imbalanced intracellular signalling response, involving e.g. different Gα subtypes, Gβγ-subunits and β-arrestins. To discriminate between the different signalling pathways (bias), sensitive techniques are needed that do not interfere with signalling. We applied split luciferase complementation to the GPCR/β-arrestin2 interaction and thoroughly analysed the influence of its implementation on intracellular signalling. This led to an assay enabling the functional characterization of ligands at the hH₁R, the hM_1,5R and the hNTS₁R in live HEK293T cells. As demonstrated at the hM_1,5R, the assay was sensitive enough to identify iperoxo as a superagonist. Time-dependent analyses of the recruitment of β-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with β-arrestin2 and their recycling to the cell membrane. The developed β-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and β-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.