Abstract
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 G alpha subtypes, G beta gamma-subunits and ...
Abstract
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 G alpha subtypes, G beta gamma-subunits and beta-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/beta-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 hH(1)R, and the hM(1,5)R and the hNTS(1)R in live HEK293T cells. As demonstrated at the hM(1,5)R, the assay was sensitive enough to identify iperoxo as a superagonist. Time-dependent analyses of the recruitment beta-arrestin2 became possible, allowing the identification of class A and class B GPCRs, due to the differential duration of their interaction with beta-arrestin2 and their recycling to the cell membrane. The developed beta-arrestin2 recruitment assay, which provides concentration- and time-dependent information on the interaction between GPCRs and beta-arrestin2 upon stimulation of the receptor, should be broadly applicable and of high value for the analysis of agonist bias.
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