Fluorescence- and luminescence-based methods for the determination of affinity and activity of neuropeptide Y2 receptor ligands

https://doi.org/10.1016/j.ejphar.2006.08.075Get rights and content

Abstract

With respect to the discovery and characterization of neuropeptide Y2 receptor ligands as pharmacological tools or potential drugs, fluorescence- and luminescence-based assays were developed to determine both the affinity and the activity of receptor agonists and antagonists. A flow cytometric binding assay is described for the hY2 receptor stably expressed in CHO cells using cy5-labeled porcine neuropeptide Y and compared with a radioligand binding assay. Binding of the fluorescent ligand was visualized by confocal microscopy. Stable co-transfection with the chimeric G protein Gqi5 enabled the establishment of a spectrofluorimetric fura-2 and a flow cytometric fluo-4 calcium assay. Further stable expression of apoaequorin targeted to the mitochondria allowed the establishment of an aequorin assay which could be performed in the 96-well format. The shape of the concentration–response curves of porcine neuropeptide Y in the presence of the Y2-selective receptor antagonist BIIE0246, characteristic of either competitive or insurmountable antagonism, depended on the period of incubation with the cells. Functional data of Y2 receptor agonists and antagonists determined in the fluorescence- and luminescence-based assays were in good agreement.

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.

References (38)

  • J.W. Lynch et al.

    A pertussis toxin-insensitive calcium influx mediated by neuropeptide Y2 receptors in a human neuroblastoma cell line

    J. Biol. Chem.

    (1994)
  • P.M. Rose et al.

    Cloning and functional expression of a cDNA encoding a human type 2 neuropeptide Y receptor

    J. Biol. Chem.

    (1995)
  • J. Stables et al.

    A bioluminescent assay for agonist activity at potentially any G-protein-coupled receptor

    Anal. Biochem.

    (1997)
  • J. Stables et al.

    Recombinant aequorin as reporter of changes in intracellular calcium in mammalian cells

    Methods Enzymol.

    (2000)
  • M.D. Ungrin et al.

    An automated aequorin luminescence-based functional calcium assay for G-protein-coupled receptors

    Anal. Biochem.

    (1999)
  • R.L. Batterham et al.

    Gut hormone PYY(3–36) physiologically inhibits food intake

    Nature

    (2002)
  • A.G. Beck-Sickinger et al.

    A novel cyclic analog of neuropeptide Y specific for the Y2 receptor

    Eur. J. Biochem.

    (1992)
  • J.R. Blinks

    Applications of calcium-sensitive photoproteins in experimental biology

    Photochem. Photobiol.

    (1978)
  • Y. Cheng et al.

    Relationship between the inhibition constant (KI) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction

    Biochem. Pharmacol.

    (1973)
  • Cited by (35)

    • Probing the pharmacology of G protein-coupled receptors with fluorescent ligands

      2015, Neuropharmacology
      Citation Excerpt :

      Furthermore, the presence of an adenosine receptor agonist increased the number of these projections demonstrating a previously unknown role of the A3 receptor in neutrophil function (Corriden et al., 2013). Many GPCRs are known to internalise in response to agonist exposure, and fluorescent agonists for GPCRs can also internalise receptors or result in clustering of the fluorescent ligand occupied receptors within the cell and on the cell membrane, as demonstrated for the μ and δ opioid receptor (Arttamangkul et al., 2000) adenosine-A2A receptor (Brand et al., 2008), AT1 angiotensin receptor (Hunyady et al., 2002) and neuropeptide Y1 receptor (Ziemek et al., 2006) heterologously expressed in non-natively expressing systems. More excitingly, this opens up the possibility of visualising reorganisation and regulation of endogenously expressed GPCRs.

    • Dimeric argininamide-type neuropeptide y receptor antagonists: Chiral discrimination between Y<inf>1</inf> and Y<inf>4</inf> receptors

      2013, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      IC50 values were converted to Ki values according to the Cheng–Prusoff equation35 using a Kd value of 1.2 nM20 (SK-N-MC cells) or 2.9 nM20 (MCF-7-Y1 cells). Flow cytometric binding assays on CHO-hY2 and HEC-1B-hY5 cells using Cy5-pNPY (Kd = 5.2 nM (Y2R), Ki = 4.4 nM (Y5R)) as labeled ligands were performed as described previously.23,24 Two or three independent experiments were performed in duplicate or triplicate on a FACSCalibur™ flow cytometer (Becton Dickinson, Heidelberg, Germany), equipped with an argon laser (488 nm) and a red diode laser (635 nm).

    • Red-fluorescent argininamide-type NPY Y<inf>1</inf> receptor antagonists as pharmacological tools

      2011, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      This procedure turned out to be reliable at concentrations up to 1 μM. The binding assays on CHO-hY2, HEC-1B-hY5 and CHO-hY4 cells using Cy5-pNPY (Kd 5.2 nM (Y2R), Ki 4.4 nM (Y5R)), Dy-635-pNPY (Ki 5.5 nM, Y2R) and Cy5-[K4]-hPP (Kd 5.6 nM, Y4R) as labelled ligands were essentially performed as previously described2,5,38,39 with minor modifications. For Y4R binding studies, instead of S0586-[K4]-hPP,5 Cy5-[K4]-hPP was used at concentrations of 5 nM and 3 nM in case of 54 and 66, respectively.

    View all citing articles on Scopus

    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.

    View full text