Fluorescence- and Radiolabeling of [Lys⁴,Nle^17,30]hPP Yields Molecular Tools for the NPY Y₄ Receptor

The neuropeptide Y (NPY) Y₄ 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, [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 Y₅ 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: [³H]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 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.