Characterization of [(3)H]Propionylated Human Peptide YY-A New Probe for Neuropeptide Y Y(2) Receptor Binding Studies.

The neuropeptide Y (NPY) Y(2) receptor (Y(2)R) 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 Y(2)R represents a potential drug target, e.g., for the treatment of epilepsy and mood disorders. Until now, the determination of the Y(2)R binding affinities of Y(2)R ligands has primarily been performed using (125)I-labeled derivatives of the endogenous Y(2)R 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 [(3)H]propionylation at Lys(4) ([(3)H]2). The radioligand was characterized by saturation binding, association, and dissociation kinetics and was applied in competition binding assays. Specific binding of [(3)H]2 at intact Chinese hamster ovary cells expressing the hY(2)R 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 Y(2)R reference ligands yielded K (i) values, which are in good agreement with the reported Y(2)R binding affinities, showing that [(3)H]2 represents a useful tritiated tool compound for the determination of Y(2)R binding affinities also in buffers containing sodium at physiological concentrations.