Abstract
The neuropeptide Y (NPY) Y-4 receptor (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, [Lys(4)]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the ...
Abstract
The neuropeptide Y (NPY) Y-4 receptor (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, [Lys(4)]hPP proved to be an appropriate full-length PP analog to prepare a fluorescent ligand by derivatization at the epsilon-amino group. To preclude oxidation upon long-term storage, we replaced the two methionine residues in [Lys(4)]hPP by norleucine and prepared the corresponding [H-3]propionylated ([H-3]12) and cyanine labeled (13) peptides, which were characterized and compared with a set of reference compounds in binding (Y-1, Y-2, Y-4, and Y-5 receptors) 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: [H-3]12 affinity (K-d in Na+-free buffer: 1.1 nM) clearly decreased with increasing sodium ion concentration, whereas dissociation and Y4R-mediated internalization of 13 (K-d in Nat-free buffer: 10.8 nM) were strongly affected by the osmolarity of the buffer as demonstrated by confocal microscopy. Displacement of [H-3]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 [H-3]propionyl-pNPY were investigated in saturation binding and competition binding.
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