1. Innovative Bioluminescence Resonance Energy Transfer Assay Reveals Differential Agonist-Induced D2 Receptor Intracellular Trafficking and Arrestin-3 Recruitment
- Author
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Brice Ronsin, Luc De Vries, Frédéric Finana, Didier Cussac, and Claudie Cathala
- Subjects
0301 basic medicine ,Bioluminescence Resonance Energy Transfer Techniques ,Endosome ,Arrestins ,media_common.quotation_subject ,Endocytic cycle ,Green Fluorescent Proteins ,CHO Cells ,Proto-Oncogene Proteins p21(ras) ,03 medical and health sciences ,0302 clinical medicine ,Cricetulus ,Dopamine receptor D2 ,Lysosome ,medicine ,Arrestin ,Animals ,Internalization ,Late endosome ,media_common ,Pharmacology ,Chemistry ,Receptors, Dopamine D2 ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,rab GTP-Binding Proteins ,Molecular Medicine ,Dopamine Antagonists ,Haloperidol ,Signal transduction ,Lysosomes ,030217 neurology & neurosurgery ,Signal Transduction - Abstract
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 bioluminescence resonance energy transfer (BRET) assays that measure relative distances between Renilla luciferase8-tagged D2R and green fluorescent protein 2 (GFP2)-tagged K-Ras (plasma membrane marker), and between luciferase8-tagged D2R 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 luciferase8-tagged D2R 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 in internalizing D2R than most of the antiparkinsonian agents. However, antipsychotics were nearly as efficacious as antiparkinsonians in directing the D2R toward early and recycling endosomes. The Rab7 marker for the late endosome/lysosome route was also capable of discriminating 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 decipher D2R ligand-induced arrestin-3 recruitment and trafficking, with potentially more selective therapeutic profiles and fewer undesired side effects.
- Published
- 2019