Irreversible Binding Kinetics of Neuropeptide Y Ligands to Y<sub>2</sub> but Not to Y<sub>1</sub> and Y<sub>5</sub> Receptors

Abstract

Neuropeptide Y (NPY) receptors type 1 (Y<sub>1</sub>), type 2 Y<sub>2</sub>) and type 5 (Y<sub>5</sub>) were tested for their kinetic properties to bind radiolabeled NPY or PYY. Rapid association and dissociation was observed with recombinant (HEK293 cells) and endogenous (SK-N-MC cells) human Y<sub>1</sub> and recombinant mouse Y<sub>5</sub> receptors. Recombinant (HEK293) and endogenous (SMS-KAN) human Y<sub>2</sub> receptors bound both radiolabels comparable to the Y<sub>1</sub> receptors, but only minimal (∼20%) dissociation of both radiolabels was observed after long incubation time (>8 h). Furthermore, neither peptide nor small molecule Y<sub>2</sub> ligands efficiently competed for binding to Y<sub>2</sub> receptors once association binding had been initiated. The Y<sub>2</sub>-selective antagonist BIIE0246 behaved as an insurmountable antagonist in functional assays when pre-incubated for 30 min before agonist addition, but was a competitive antagonist when co-applied with the agonist. These data show that Y<sub>2</sub> receptors in contrast to Y<sub>1</sub> and Y<sub>5</sub> receptors bind their ligands in an irreversible manner.