This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and β-arrestin 2 (β-arr2), measured by the bioluminescence resonance energy transfer (BRET2) technology. Both class A (β2-adrenergic receptor [β2-AR]) and class B (neurokinin-type 1 receptor [NK1-R]) GPCRs, classified based on their internalization characteristics, have been analyzed. It was evaluated whether the BRET2 signal can be enhanced by using (1) β-arr2 phosphorylation-independent mutant (β-arr2 R169E) and (2) β-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (β-arr2 R393E, R395E and β-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET2 signal when comparing results obtained with wild-type (wt) and mutant β-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET2 signal was observed with β-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these β-arr2 mutants to interact with the components of the clathrin-coated vesicle probably prevents their rapid dissociation from the receptor, thus yielding an increased and more stable BRET2 signal. The β-arr2 R393E, R395E mutant also enhanced the signal window with other members of the GPCR family (neuropeptide Y type 2 receptor [NPY2-R] and TG1019 receptor) and was successfully applied in full-plate BRET2-based agonist and antagonist screening assays.
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