Different binding epitopes on the NK sub(1) receptor for substance P and a non-peptide antagonist

Non-peptide ligands for peptide receptors have been discovered in several systems through file screening programs, but the mechanism of action for these candidate drugs is obscure. The compound CP 96345 is a high-affinity, non-peptide antagonist of the substance P (NK sub(1)) receptor. Here we identify epitopes on the NK sub(1) receptor responsible for the specific binding of CP 96345 by systematic exchange of corresponding segments between the NK sub(1) receptor and the homologous NK sub(3) (neurokinin B) receptor, which does not bind the non-peptide ligand. Non-conserved residues, in two epitopes around the top of transmembrane segment V and in one epitope at the top of transmembrane segment VI, are essential for the specific action of CP 96345 on the NK sub(1) receptor, but are surprisingly not important for the binding of the natural peptide ligand, substance P. Susceptibility to the non-peptide antagonists can be conveyed to the previously unresponsive NK sub(3) receptor by mutational transfer of this discontinuous epitope from the NK sub(1) receptor.