MUTATIONS ALONG TRANSMEMBRANE SEGMENT-II OF THE NK-1 RECEPTOR AFFECT SUBSTANCE-P COMPETITION WITH NONPEPTIDE ANTAGONISTS BUT NOT SUBSTANCE-P BINDING

Mutational analysis of the NK-1 receptor indicates that residues invol ved in non-peptide antagonist binding cluster around the outer portion of transmembrane segments (TM) V and VI. In contrast mutations affect ing the binding of the natural peptide agonist, substance P, are scatt ered in the exterior part of the receptor. Recently it was reported th at a number of mutations in TM-II also seriously impair substance P bi nding. Here we confirm that Ala substitutions for these residues locat ed on a hydrophilic helical face of TM-II basically eliminate substanc e P binding to the NK-1 receptor, provided that a radiolabeled non-pep tide antagonist is used as radioligand. Surprisingly, radiolabeled sub stance P bound well to all these mutant receptors and was displaced wi th only slightly reduced affinity by the unlabeled peptide and by the non-peptide antagonists. The wild-type homologous NK-2 receptor displa yed properties similar to those observed in the mutated NK-1 receptors , i.e. concomitant high affinity binding of radiolabeled agonist pepti de (in this case neurokinin A), yet low affinity, G-protein independen t competition of unlabeled peptide with radiolabeled non-peptide antag onist. It is concluded that substitutions in TM-II of the NK-1 recepto r do not affect the high affinity binding of substance P but instead b lock the ability of the peptides to compete for non-peptide antagonist binding. It is suggested that certain mutations can impair interchang e between receptor conformations that each bind different ligands with high affinity.