| Autor: |
Bertrand, Lucie, Parent, Stéphane, Caron, Mireille, Legault, Mireille, Joly, Erik, Angers, Stéphane, Bouvier, Michel, Brown, Mike, Houle, Benoit, Ménard, Luc |
| Předmět: |
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| Zdroj: |
Journal of Receptors & Signal Transduction; Nov2002, Vol. 22 Issue 1-4, p533-541, 9p, 3 Charts |
| Abstrakt: |
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 510 nm. 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 β-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP² β-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:β-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V[sub 2]R) fused to RLuc (V[sub 2]R: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. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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