Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands☆
Introduction
Neuropeptide Y is a member of the so-called pancreatic polypeptide or neuropeptide Y family that also includes peptide YY and pancreatic polypeptide (Michel et al., 1998). Neuropeptide Y is widely distributed in the brain and the peripheral nervous system, and is implicated in various physiological processes including regulation of food intake, anxiety, mood and memory, blood pressure and circadian rhythm. Neuropeptide Y and related peptides exert their biological actions by interacting with at least five different G protein-coupled receptors, designated Y1, Y2, Y4, Y5 and y6 (Hazelwood, 1993, Michel, 2004). Their main signal transduction pathway is a coupling to pertussis toxin sensitive G proteins of the Gi/o family, leading to an inhibition of adenylyl cyclase.
The neuropeptide Y2 receptor is considered the most abundant neuropeptide Y receptor in the human brain and to be involved, for instance, in memory and learning. As recent studies reported on an anorectic effect of the Y2 preferring agonist peptide YY(3–36) after peripheral application in rodents and humans (Abbott et al., 2005, Batterham et al., 2002), the Y2 receptor has also become an attractive drug target for the treatment of eating disorders.
Binding data of Y2 receptor ligands are usually determined in radioligand binding assays, requiring a filtration step in order to separate bound from unbound ligand. Although homogenous binding assays using the scintillation proximity assay technique have been described (Dautzenberg, 2005, Dautzenberg et al., 2005), the use of radio-labeled ligands is still indispensable, causing high costs and radioactive waste. Recently, BODIPY-labeled neuropeptide Y analogues with high affinity and selectivity for the Y1, Y2, Y4 and Y5 receptors have been described (Dumont et al., 2005). The use of fluorescent ligands in a flow cytometric binding assay has been previously described for the chemokine receptor CXCR4 (Hatse et al., 2004), the epidermal growth factor (Stein et al., 2001) and the formylpeptide receptor (Edwards et al., 2005).
As a functional assay, the establishment of calcium mobilization by co-transfection of neuropeptide Y receptors and chimeric G proteins Gqo5, Gqi5 and Gqi9 into HEK293 cells has been reported (fluorometric imaging plate reader (FLIPR) assay) (Dautzenberg et al., 2005). However, calcium mobilization assays using non-ratiometric fluorescent indicator dyes like fluo-4 have the drawback of dye leakage and the use of ratiometric indicator dyes such as fura-2 is often not amenable to the application in the multiplate reader format.
The photoprotein aequorin has been widely used for many years to visualize changes in intracellular calcium (Blinks, 1978), but the purified protein had to be microinjected, limiting its use as a calcium indicator. The cloning of the apoaequorin cDNA (Inouye et al., 1985) and the recombinant expression of the protein by various cell types has greatly improved the use of the bioluminescent protein. Reconstitution of aequorin can be accomplished by simple addition of the co-factor coelenterazine to the cell culture medium (Torfs et al., 2002). In contrast to fluorescence indicator dyes used at high concentrations (usually 20–200 μM) aequorin (usually recombinantly expressed < 1 μM) does not significantly affect endogenous Ca2+ buffer capacity (Brini et al., 1995), and no ester hydrolysis products, which may alter the physiological response, are released in the cell. Therefore, recombinantly expressed aequorin has been often used for the functional screening of various G protein-coupled receptors (Button and Brownstein, 1993, Dupriez et al., 2002, Le Poul et al., 2002, Schaeffer et al., 1999, Stables et al., 1997, Torfs et al., 2002, Ungrin et al., 1999). The most robust bioluminescence signals after receptor activation were obtained with mitochondrially targeted aequorin (Stables et al., 1997, Stables et al., 2000). The use of cells stably co-expressing mitochondrial apoaequorin, the promiscuous Galpha16 protein and various G protein-coupled receptors have been previously described (Dupriez et al., 2002, Stables et al., 1997).
Here we report on the establishment of a flow cytometric binding assay and the stepwise stable transfection of cells with the Gqi5 and mtAEQ constructs for the development of functional fluorescence- and luminescence-based assays for the neuropeptide Y2 receptor.
Section snippets
Materials, peptides, reagents and radiochemicals
The peptides porcine neuropeptide Y, porcine [L31, P34]-neuropeptide Y and porcine neuropeptide Y(13–36) were synthesized as described previously (Cabrele et al., 2001). The peptides were used with a purity higher than 90% as determined by analytical HPLC. Porcine peptide YY was purchased from Novabiochem, Switzerland. Porcine [3H]propionyl-neuropeptide Y (specific activity 2.07, 3.96 TBq/mmol respectively) was from Amersham Biosciences (Little Chalfont, UK). The vectors pcDNA3.1/hygro
Flow cytometric binding assay
CHO cells stably expressing the hY2 receptor bound the fluorescent ligand cy5-labeled porcine neuropeptide Y with high affinity (Kd = 5.2 ± 2.1 nM; Fig. 2). Thus, 5 nM of the fluorescent ligand were used for competition binding experiments. The peptides porcine peptide YY (Ki = 0.4 ± 0.1 nM), porcine neuropeptide Y (Ki = 0.8 ± 0.2 nM), porcine neuropeptide Y(13–36) (Ki = 1.7 ± 0.4 nM) bound to the CHO-hY2 cells with typical Y2 receptor pharmacology, whereas porcine [L31, P34]-neuropeptide Y, a ligand with
Discussion
The use of flow cytometry has been shown to be a valuable tool for the determination of receptor binding data (Edwards et al., 2004). In this study, we established a flow cytometric binding assay for the hY2 receptor performed in equilibrium without the necessity of separating bound from unbound ligand. As the natural ligand neuropeptide Y is a bulky peptide, the coupling to the fluorescent dye cy5 is well tolerated, and the labeled peptide retains a reasonable affinity for the hY2 receptor
Acknowledgements
We thank Prof. Dr. B. Conklin for the kind gift of the Gqi5 construct, Prof. Dr. S. Thayer for the pMTAEQ vector, PD Dr. T. Dobner for the pcDNA3.1/hygro and pcDNA3.1/zeo vectors, Prof. Dr. P. Rose for the hY2 receptor cDNA and the Deutsche Forschungsgemeinschaft for financial support of the Research Training Group (Graduiertenkolleg) GRK 760.
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Presented on the occasion of the 2nd Summer School Medicinal Chemistry, Regensburg, Germany, October 5–7, 2004, and the Annual Meeting of the divisions of Medicinal Chemistry of the GDCh and DPhG, Frontiers in Medicinal Chemistry, Leipzig, Germany, March 13–16, 2005.