title: Fluorescence- and Radiolabeling of [Lys4,Nle17,30]hPP Yields Molecular Tools for the NPY Y4Receptor.
abstract: The neuropeptide Y (NPY) Y4receptor (Y4R) is involved in energy homeostasis and considered a potential drug target for the treatment of obesity. Only a few molecular tools, i.e., radiolabeled and fluorescent ligands, for the investigation of the Y4R were reported. Previously, [Lys4]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ε-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys4]hPP by norleucine and prepared the corresponding [3H]propionylated ([3H]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y1, Y2, Y4, and Y5receptors) and functional (luciferase gene reporter, beta-arrestin-1,2) Y4R assays. Both molecular probes proved to be useful in radiochemical and flow cytometric saturation and competition Y4R binding experiments. Most strikingly, there was a different influence of the composition of buffer on equilibrium binding and kinetics: [3H]12 affinity (Kdin Na+-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and Y4R-mediated internalization of 13 (Kdin Na+-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [3H]12 and 13 revealed a tendency to higher apparent affinities for a set of reference peptides in hypotonic (Na+-free) compared to isotonic buffers. The differences were negligible in the case of hPP but up to 270-fold in the case of GW1229 (GR231118). By contrast, no relevant influence of Na+on Y5R affinity became obvious, when the radioligands [H]12 and [3H]propionyl-pNPY were investigated in saturation binding and competition binding.
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Dukorn
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Timo
authors.initials: T
authors.lastname: Littmann
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Max
authors.identifier: 0000-0002-8095-8627
authors.initials: M
authors.lastname: Keller
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Kilian
authors.initials: K
authors.lastname: Kuhn
authors.affiliation: Division of Chemistry and Bioanalytics, Department of Molecular Biology, University of Salzburg , Billrothstraße 11, 5020 Salzburg, Austria.
authors.forename: Chiara
authors.initials: C
authors.lastname: Cabrele
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Baumeister
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Günther
authors.initials: G
authors.lastname: Bernhardt
authors.affiliation: Institute of Pharmacy, University of Regensburg , Universitätsstraße 31, 93053 Regensburg, Germany.
authors.forename: Armin
authors.identifier: 0000-0002-9709-1433
authors.initials: A
authors.lastname: Buschauer
mesh_terms: D001667:Binding, Competitive; D016503:Drug Delivery Systems / Q000379:methods*; D004355:Drug Stability; D005453:Fluorescence; D006801:Humans; D008024:Ligands; D009765:Obesity / Q000188:drug therapy; D011485:Protein Binding; D019275:Radiopharmaceuticals / Q000138:chemical synthesis*; D017476:Receptors, Neuropeptide Y / Q000378:metabolism*
chemical_list: D008024:Ligands; D019275:Radiopharmaceuticals; D017476:Receptors, Neuropeptide Y