Characterization of [³H]Propionylated Human Peptide YY-A New Probe for Neuropeptide Y Y₂ Receptor Binding Studies

The neuropeptide Y (NPY) Y₂ receptor (Y₂R) is a G-protein-coupled receptor that is involved in the regulation of various physiological processes such as neurotransmitter release, bone metabolism, and memory. Consequently, the Y₂R represents a potential drug target, e.g., for the treatment of epilepsy and mood disorders. Until now, the determination of the Y₂R binding affinities of Y₂R ligands has primarily been performed using ¹²⁵I-labeled derivatives of the endogenous Y₂R agonists NPY and peptide YY (PYY). A tritium-labeled NPY derivative has also been used; however, its suitability for binding assays in sodium-containing buffer is doubtful. We synthesized a tritium-labeled PYY derivative by [³H]propionylation at Lys⁴ ([³H]2). The radioligand was characterized by saturation binding, association, and dissociation kinetics and was applied in competition binding assays. Specific binding of [³H]2 at intact Chinese hamster ovary cells expressing the hY₂R was saturable in both sodium-free buffer (apparent K _d = 0.016-0.067 nM) and sodium-containing buffer (175 mM Na⁺, apparent K _d = 0.16-0.18 nM). Competition binding experiments with Y₂R reference ligands yielded K _i values, which are in good agreement with the reported Y₂R binding affinities, showing that [³H]2 represents a useful tritiated tool compound for the determination of Y₂R binding affinities also in buffers containing sodium at physiological concentrations.