THE BRET 2 =ARRESTIN ASSAY IN STABLE RECOMBINANT CELLS: A PLATFORM TO SCREEN FOR COMPOUNDS THAT INTERACT WITH G PROTEIN-COUPLED RECEPTORS (GPCRS)*

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1 JOURNAL OF RECEPTORS AND SIGNAL TRANSDUCTION Vol. 22, Nos. 1 4, pp , 2002 NEW METHODOLOGY VENDOR SECTION THE BRET 2 =ARRESTIN ASSAY IN STABLE RECOMBINANT CELLS: A PLATFORM TO SCREEN FOR COMPOUNDS THAT INTERACT WITH G PROTEIN-COUPLED RECEPTORS (GPCRS)* Lucie Bertrand, 1 Stéphane Parent, 1 Mireille Caron, 1 Mireille Legault, 1 Erik Joly, 1 Stéphane Angers, 2 Michel Bouvier, 2 Mike Brown, 1 Benoit Houle, 1 and Luc Ménard 1 1 BioSignal Packard Inc, 1744 William, Montréal, Québec, H3J 1R4, Canada 2 Department of Biochemistry and Groupe de recherche sur le système nerveux autonome, Université de Montréal, Montréal, QC, Canada ABSTRACT In BRET 2 (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 510 nm. BRET 2 signals are therefore easily determined by measuring the ratio of green over blue light (510=395 nm) using appropriate dual channel luminometry instruments (e.g., Fusion TM Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET 2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET 2, 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by agonists leads to the interaction of b-arrestin (a protein that is *This article was submitted by a vendor and has not been peer-reviewed. DOI: =RRS Copyright # 2002 by Marcel Dekker, Inc (Print); (Online)

2 534 BERTRAND ET AL. involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP 2 : b-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET 2 =arrestin assays. In addition, using the HEK 293=GFP 2 : b-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V 2 R) fused to RLuc (V 2 R : RLuc) and used it for the pharmacological characterization of compounds in BRET 2 =arrestin assays. This approach yields genuine pharmacology and supports the BRET 2 =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. PRINCIPLE OF BIOLUMINESCENE RESONANCE ENERGY TRANSFER Expression of Renilla luciferase (RLuc) alone, in the presence of the cellpermeant substrate DeepBlueC TM, gives an emission spectrum that peaks at 395 nm (solid line; Fig. 1). In the absence of Green Fluorescent Protein (GFP 2 ), the signal at 510 nm is approximately 5% of the signal at 395 nm Bioluminescence Resonance Energy Transfer (BRET 2 signal of 0.05). When GFP 2 is brought into close proximity to RLuc (less than 100 Å) via a specific biomolecular interaction, it will absorb part of the energy from RLuc and reemit light at 510 nm. The GFP 2 : RLuc fusion construct allows efficient energy transfer between RLuc and GFP 2 resulting in two major peaks at 395 nm and 510 nm (dashed line) to give a BRET 2 (510=395 nm) signal of Rapid GPCR desensitization is a tightly regulated process involving specific proteins. One of the main pathways takes place in two steps. First, the agonistactivated receptor is specifically recognized and phosphorylated by a family of kinases known as G protein-coupled receptor kinases (GRKs). The GRK-mediated phosphorylation leads to the binding of cytosolic proteins called arrestins. The GPCR arrestin interaction prevents further activation of G proteins and targets the Figure 1. Principle of BRET 2 (details see text).

3 BRET 2 =ARRESTIN ASSAY 535 receptor to clathrin-coated pits, and subsequent internalization. Arrestins represent a family of six cytosolic proteins of about 48 kda, with b-arrestin 1 and b-arrestin 2 (also known as arrestin 2 and arrestin 3, respectively) showing the widest tissue distribution. Although not all GPCRs internalize through clathrin-coated pits, it is estimated that the majority do so via an arrestin-dependent mechanism. THE BIOLUMINESCENE RESONANCE ENERGY TRANSFER=ARRESTIN ASSAY We have developed a BRET 2 =arrestin assay to monitor the interaction between agonist-stimulated GPCRs and arrestin proteins. For this assay, the receptor is fused to Renilla luciferase to generate a GPCR : RLuc fusion protein, whereas the human b-arrestin 2 is fused to GFP 2, yielding GFP 2 : b-arrestin 2 (Fig. 2). Upon interaction of the b-arrestin moiety with the phosphorylated chimeric receptor, RLuc is brought into close proximity with GFP 2. This interaction can be easily quantified by measuring the energy transfer between RLuc and GFP 2 upon addition of DeepBlueC TM. BRET 2 signals can then be determined by measuring the ratio of green over blue light (515=410 nm) using appropriate dual channel luminometry instruments (such as the Fusion TM Universal Microplate Analyzer, Packard BioScience). Using the ecdysone-inducible system (Invitrogen=Life Technologies) we generated a stable HEK 293 cell line expressing the GFP 2 : b-arrestin 2 fusion Figure 2. Principle of the BRET 2 =arrestin assay (details see text).

4 536 BERTRAND ET AL. Figure 3. Generation of a clonal cell line expressing GFP 2 : b-arrestin 2 under the control of an inducible promoter. protein (HEK 293=GFP 2 : b-arrestin 2). Forty different clones were isolated and tested for GFP 2 : b-arrestin expression upon stimulation with saturating concentration of ponasterone (an analog of ecdysone; Fig. 3). Several positive clones were identified and one was further characterized. A 24-h treatment with increasing concentrations of ponasterone induces a dose-dependent increase in GFP 2 : b-arrestin 2 expression. APPLICATION TO THE ANALYSIS OF THREE G PROTEIN-COUPLED RECEPTORS HEK 293 cells stably expressing GFP 2 : b-arrestin 2 were transiently transfected with three GPCRs fused to RLuc (G q -coupled orexin 1 receptor (OX1R), Figure 4. Measurement of agonist-induced GPCR activation in HEK 293=GFP 2 : b-arrestin 2 cells.

5 BRET 2 =ARRESTIN ASSAY 537 Figure 5. Generation of a double-stable cell line co-expressing GFP 2 : b-arrestin 2 and V2R : RLuc.

6 538 BERTRAND ET AL. Figure 6. Pharmacological characterization of V 2 R agonists using the V 2 R : Rluc=GFP 2 : b-arrestin 2 double cell line. G s -coupled vasopressin 2 receptor (V 2 R) and G i -coupled N-formyl peptide receptor-like 1 (FPRL-1)). The agonist-dependent increase in the BRET 2 signal observed with these receptors demonstrates the applicability of the HEK 293=GFP 2 : b-arrestin 2 cell line to study the activation of various GPCRs (Fig. 4). The HEK 293=GFP 2 : b-arrestin 2 clonal cell line was used as the recipient cell line for the stable expression of the V 2 R : RLuc receptor. Forty different clones were tested for an agonist-stimulated BRET 2 signal. Several positive clones were isolated and one was further characterized. As observed with the recipient GFP 2 : b-arrestin 2 cell line (Fig. 3), expression of GFP 2 : b-arrestin 2 required induction with ponasterone (Fig. 5, panel A). As expected, the agonist-dependent increase in the BRET 2 signal was maximal after induction of GFP 2 : b-arrestin 2 expression with 10 mm ponasterone (Fig. 5, panel B). Dose-response curves were performed for all compounds (n ¼ 2 6) and the results demonstrate that each agonist induced a dose-dependent BRET 2 signal (Fig. 6). Table 1. Comparison of pk i and BRET 2 pec 50 Values for Agonists Agonists pk i (binding) a BRET 2 pec 50 SD dvdavp AVP Desmopressin Oxytocin a Data from Thibonnier et al. (Vasopressin and Oxytocin, ed. Zingg et al., Plenum Press, NY, p. 251, 1998).

7 BRET 2 =ARRESTIN ASSAY 539 Figure 7. Pharmacological characterization of V 2 R antagonists using the V 2 R : Rluc=GFP 2 : b-arrestin 2 double stable cell line. Table 1 compares the calculated pec 50 values from the BRET 2 dose-response curves and the published pk i values obtained in binding experiments. Dose-response curves with 8-AVP were generated in the presence of various concentrations of antagonists. Figure 7 shows the results obtained with the V2 antagonist. As expected, the EC 50 value was increased in the presence of the antagonist. The EC 50 values for 8-AVP, as measured in the presence of each concentration of antagonist, were used to generate Schild plots and calculate pk B values. Table 2 shows the calculated pk B s along with reported pk i (or pa 2 ) values. Following induction with ponasterone, cells stably expressing V 2 R : RLuc and GFP 2 : b-arrestin 2 were plated in 90 wells of a 96-well plate, stimulated with 5 nm 8-AVP ( EC 50 ), 1 mm 8-AVP (saturating concentration) or with buffer alone (30 wells per condition), and the BRET 2 signal was measured. Six wells contained untransfected cells and were used as negative controls. A Z 0 value of 0.56 was calculated for cells stimulated with 1 mm 8-AVP (Fig. 8). Table 2. Comparison of pk i and BRET 2 pk B Values for Antagonists Antagonists pk i (or pa 2 ) a pk B SD Ada-VP 8.11 ( pa 2 ) V2 antagonist Phaa-VP a The binding pk i values for V2 antagonist and Phaa-VP are from Thibonnier et al. (Vasopressin and Oxytocin, ed. Zingg et al., Plenum Press, NY, p. 251, 1998). The pa 2 value for Ada-VP is from Manning et al. (Nature 329: 839, 1987).

8 540 BERTRAND ET AL. Figure 8. Robustness of the BRET 2 =arrestin assay. METHODS BRET 2 =arrestin assay on the double-stable cell line: Induce expression of GFP 2 : b-arrestin 2 by treating the cells with 10 mm ponasterone A for 24 h. Plate cells in a 96-well Optiplate microplate (Packard BioScience) in triplicate. Add agonist and incubate at room temperature for 20 min (see Table 3). Add DeepBlueC TM to a final concentration of 5 mm. Read immediately using a Fusion TM Universal Microplate Analyzer (Packard BioScience). Table 3. Tested Compounds for the V2 Receptor Name Agonist=Antagonist Abbreviation Arg 8 -Vasopressin Agonist 8-AVP Oxytocin Agonist Oxytocin (d(ch 2 ) 1 5, D-Ile 2, Ile 4, Antagonist V 2 antagonist Arg 8, Ala-NH 9 2 )-Vasopressin Deamino-Cys 1,Val 4, Agonist dvdavp D-Arg 8 -Vasopressin Deamino-Cys 1, Agonist Desmopressin D-Arg 8 -Vasopressin 1-Adamantaneacetyl 1, D-Tyr(Et) 2,Val 4, Abu 6, Arg 8,9 -Vasopressin Antagonist Ada-VP

9 BRET 2 =ARRESTIN ASSAY 541 Additional information: For Fig. 4, the agonists used for OX1R, V 2 R and FPRL1 were orexin A (1 mm), 8-AVP (50 nm) and the synthetic peptide WKYMVm (1 mm), respectively. For antagonist dose-response curves (Fig. 7), cells were incubated first with the antagonist for 15 min and then with various concentrations of 8-AVP for 20 min. All experiments shown are representative of at least two experiments. CONCLUSION We generated a stable cell line expressing GFP 2 : b-arrestin 2 under the control of the inducible ecdysone promoter. Using this cell line, the agonistpromoted interaction of the GFP 2 : b-arrestin 2 fusion protein with various GPCR : RLuc fusion proteins was easily detected using transient assays as demonstrated with OX1R, V 2 R and FPRL-1 receptors. In addition, this stable line was used as a recipient cell line to generate a V 2 R : Rluc=GFP 2 : b-arrestin 2 double-stable line. Pharmacological characterization of this double cell line using BRET 2 showed genuine pharmacological properties for the V2 receptor. Overall, our results showed that this approach offers the following advantages: Ease of set up and use. Intact cell assay Applicability to a variety of GPCRs. Coupling pathway independent (suitable for G s,g q and G i receptors). Robust : very good Z 0 values (>0.5). Genuine pharmacology (agonists, antagonists). Suitable for compound characterization and screening. Well-suited for orphan GPCR screening. Detection of receptor expression in the absence of ligand (from RLuc activity).