title: Development of a BRET2 screening assay using beta-arrestin 2 mutants.
abstract: This study has focused on enhancing the signal generated from the interaction between a G-protein-coupled receptor (GPCR) and beta-arrestin 2 (beta-arr2), measured by the bioluminescence resonance energy transfer (BRET(2)) technology. Both class A (beta(2)-adrenergic receptor [beta(2)-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 BRET(2) signal can be enhanced by using (1) beta-arr2 phosphorylation-independent mutant (beta-arr2 R169E) and (2) beta-arr2 mutants deficient in their ability to interact with the components of the clathrin-coated vesicles (beta-arr2 R393E, R395E and beta-arr2 373 stop). For the class B receptor, there was no major difference in the agonist-promoted BRET(2) signal when comparing results obtained with wild-type (wt) and mutant beta-arr2. However, with the class A receptor, a more than 2-fold increase in the BRET(2) signal was observed with beta-arr2 mutants lacking the AP-2 or both AP-2 and clathrin binding sites. This set of data suggests that the inability of these beta-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 BRET(2) signal. The beta-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 BRET(2)-based agonist and antagonist screening assays.
authors.affiliation: Institute of Anatomy, Histology & Embryology, Veterinary Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors.forename: Milka
authors.initials: M
authors.lastname: Vrecl
authors.forename: Rasmus
authors.initials: R
authors.lastname: Jorgensen
authors.forename: Azra
authors.initials: A
authors.lastname: Pogacnik
authors.forename: Anders
authors.initials: A
authors.lastname: Heding
mesh_terms: D000818:Animals; D019390:Arrestins / Q000235:genetics* / Q000378:metabolism; D019556:COS Cells; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D006801:Humans; D066298:In Vitro Techniques; D008163:Luminescent Measurements / Q000379:methods; D008954:Models, Biological; D009154:Mutation; D043562:Receptors, G-Protein-Coupled / Q000145:classification / Q000378:metabolism; D018040:Receptors, Neurokinin-1 / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D043562:Receptors, G-Protein-Coupled; D018040:Receptors, Neurokinin-1; D011993:Recombinant Fusion Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins