TY - JOUR T1 - Innovative BRET assay reveals differential agonist-induced D2 receptor intracellular trafficking and arrestin-3 recruitment JF - Molecular Pharmacology JO - Mol Pharmacol DO - 10.1124/mol.119.115998 SP - mol.119.115998 AU - Luc De Vries AU - Frederic Finana AU - Claudie Cathala AU - Brice Ronsin AU - Didier Cussac Y1 - 2019/01/01 UR - http://molpharm.aspetjournals.org/content/early/2019/07/02/mol.119.115998.abstract N2 - The dopamine D2 receptor (D2R) mediates ligand-biased signaling with potential therapeutic implications. However, internalization, choice of endocytic routes and degradation of the D2R in lysosomes may also participate in agonist-directed trafficking. We developed BRET assays that measure relative distances between Renilla Luciferase8-tagged D2R (D2R-Luc) and Green Fluorescent Protein 2 (GFP2)-tagged K-Ras (plasmamembrane marker), and between D2R-Luc and GFP2-Rab5 (early), GFP2-Rab4 (recycling) or GFP2-Rab7 (late) endosomal markers. The BRET signal between D2R-Luc and GFP2-K-Ras was robustly diminished after receptor internalization induced by dopamine, with subsequent BRET signals increasing when D2R-Luc approached GFP2-Rab proteins in endosomal compartments. All BRET signals were blocked by the selective D2R antagonist haloperidol and were decreased by low temperature and high sucrose blocks, two parameters interfering with internalization. Some antipsychotic drugs such as aripiprazole are less efficacious to internalize D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians to direct the D2R towards early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable to discriminate between D2R compounds. We could show that some drugs engaged the D2R either to interact preferentially with arrestin-3 or to internalize. Our study revealed that D2R trafficking in cells was differentially regulated by antipsychotic- and antiparkinsonian drugs. Taken together, the BRET assays reported here could further help deciphering D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and less undesired side effects.SIGNIFICANCE STATEMENT The effects of antipsychotic and antiparkinson compounds on the D2 dopamine receptor were revealed using innovative BRET assays based on K-Ras and Rab probes. The pharmacological characteristics of the compounds could be well defined with these assays. ER -