title: The BRET2/arrestin assay in stable recombinant cells: a platform to screen for compounds that interact with G protein-coupled receptors (GPCRS).
abstract: In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395nm) using appropriate dual channel luminometry instruments (e.g., Fusion Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein-Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of beta-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2:beta-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2:beta-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R:RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand-GPCR interactions which can be applied to ligand identification for orphan receptors.
authors.affiliation: BioSignal Packard Inc, 1744 William, Montreal, Quebec, H3J IR4, Canada.
authors.forename: Lucie
authors.initials: L
authors.lastname: Bertrand
authors.forename: Stéphane
authors.initials: S
authors.lastname: Parent
authors.forename: Mireille
authors.initials: M
authors.lastname: Caron
authors.forename: Mireille
authors.initials: M
authors.lastname: Legault
authors.forename: Erik
authors.initials: E
authors.lastname: Joly
authors.forename: Stéphane
authors.initials: S
authors.lastname: Angers
authors.forename: Michel
authors.initials: M
authors.lastname: Bouvier
authors.forename: Mike
authors.initials: M
authors.lastname: Brown
authors.forename: Benoit
authors.initials: B
authors.lastname: Houle
authors.forename: Luc
authors.initials: L
authors.lastname: Ménard
mesh_terms: D019390:Arrestins / Q000378:metabolism*; D015374:Biosensing Techniques; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D008156:Luciferases / Q000032:analysis; D008163:Luminescent Measurements; D008164:Luminescent Proteins / Q000032:analysis*; D017483:Receptors, Vasopressin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012680:Sensitivity and Specificity; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins
chemical_list: C000605254:ARRB2 protein, human; D019390:Arrestins; D008164:Luminescent Proteins; D017483:Receptors, Vasopressin; D011993:Recombinant Fusion Proteins; D000071560:beta-Arrestin 2; D000071557:beta-Arrestins; D049452:Green Fluorescent Proteins; D008156:Luciferases; D019204:GTP-Binding Proteins