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An internalization assay for Dopamine D2(short) Receptors using Bioluminescence Resonance Energy Transfer (BRET) technology
Author(s) -
De Vries Luc,
Finana Frederic,
Cathala Claudie,
Ronsin Brice,
Cussac Didier,
GattiMcArthur Silvia
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb567
Subject(s) - internalization , dopamine receptor d2 , endosome , receptor , agonist , microbiology and biotechnology , biology , dopamine receptor , g protein coupled receptor , biochemistry
Internalization and subsequent degradation of GPCRs located at the plasmamembrane represent a desired therapeutic approach to eliminate those receptors whose activation leads to unwanted cellular behaviour. In order to follow the trafficking of endocytosed receptors and thus the efficacy of agents directing these receptors into the internalization/degradation pathway, markers for their localisation at the plasmamembrane (K‐Ras), early (Rab5), recycling (Rab4) and late (Rab7) endosomes, and lysosomes (Rab7) have been described previously. We reasoned that such markers could be used in BRET biosensor assays (live cells) to time‐dependently follow the destination of internalized receptors and quantify the efficacy of agents that force the receptors into lysosomes. We therefore started by coexpressing the Renilla luciferase8‐tagged D2 dopamine receptor (D2‐R), with GFP2‐tagged K‐Ras, Rab5, Rab4 or Rab7 in CHO cells. We observed a basal BRET signal between D2‐R and K‐Ras that was robustly diminished by a 1 h agonist treatment (1 μM dopamine) and completely antagonized by 10 μM haloperidol treatment. At the same time point the BRET signals between D2‐R/Rab5, D2‐R/Rab4 and D2‐R/Rab7 were enhanced by the agonist and again neutralized by the antagonist. Using this technology we were thus able to follow the trafficking of the D2‐R from the plasmamembrane to the endosomal/lysosomal compartments. It also allowed us to perform a comparative pharmacological/functional study for different antipsychotic and anti‐Parkinson agents with affinities for the D2‐R receptor.