The BRET2/arrestin assay in stable recombinant cells:: A platform to screen for compounds that interact with G protein-coupled receptors (GPCRS)

被引:88
作者
Bertrand, L
Parent, S
Caron, M
Legault, M
Joly, E
Angers, S
Bouvier, M
Brown, M
Houle, B
Ménard, L
机构
[1] BioSignal Packard Inc, Montreal, PQ H3J 1R4, Canada
[2] Univ Montreal, Dept Biochem, Montreal, PQ H3C 3J7, Canada
[3] Univ Montreal, Grp Rech Syst Nerveux Autonome, Montreal, PQ H3C 3J7, Canada
来源
JOURNAL OF RECEPTOR AND SIGNAL TRANSDUCTION RESEARCH | 2002年 / 22卷 / 1-4期
关键词
D O I
10.1081/RRS-120014619
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP(2)) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC(TM), RLuc emits blue light at 395 nm. If the GFp(2) is brought into close proximity to RLuc via a specific biomolecular interaction, the GFp(2) 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(TM) 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 GFp(2): 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/GFp(2): 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.
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页码:533 / 541
页数:9
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