title: Mutations along transmembrane segment II of the NK-1 receptor affect substance P competition with non-peptide antagonists but not substance P binding.
abstract: Mutational analysis of the NK-1 receptor indicates that residues involved in non-peptide antagonist binding cluster around the outer portion of transmembrane segments (TM) V and VI. In contrast mutations affecting the binding of the natural peptide agonist, substance P, are scattered in the exterior part of the receptor. Recently it was reported that a number of mutations in TM-II also seriously impair substance P binding. Here we confirm that Ala substitutions for these residues located on a hydrophilic helical face of TM-II basically eliminate substance P binding to the NK-1 receptor, provided that a radiolabeled non-peptide antagonist is used as radioligand. Surprisingly, radiolabeled substance P bound well to all these mutant receptors and was displaced with only slightly reduced affinity by the unlabeled peptide and by the non-peptide antagonists. The wild-type homologous NK-2 receptor displayed properties similar to those observed in the mutated NK-1 receptors, i.e. concomitant high affinity binding of radiolabeled agonist peptide (in this case neurokinin A), yet low affinity, G-protein independent competition of unlabeled peptide with radiolabeled non-peptide antagonist. It is concluded that substitutions in TM-II of the NK-1 receptor do not affect the high affinity binding of substance P but instead block the ability of the peptides to compete for non-peptide antagonist binding. It is suggested that certain mutations can impair interchange between receptor conformations that each bind different ligands with high affinity.
authors.affiliation: University Department of Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark.
authors.forename: M M
authors.initials: MM
authors.lastname: Rosenkilde
authors.forename: M
authors.initials: M
authors.lastname: Cahir
authors.forename: U
authors.initials: U
authors.lastname: Gether
authors.forename: S A
authors.initials: SA
authors.lastname: Hjorth
authors.forename: T W
authors.initials: TW
authors.lastname: Schwartz
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008961:Models, Structural; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000378:metabolism; D064729:Neurokinin-1 Receptor Antagonists; D017433:Protein Structure, Secondary; D018040:Receptors, Neurokinin-1 / Q000737:chemistry* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D013373:Substance P / Q000031:analogs & derivatives / Q000378:metabolism* / Q000494:pharmacology*; D014162:Transfection
chemical_list: D064729:Neurokinin-1 Receptor Antagonists; D018040:Receptors, Neurokinin-1; D011994:Recombinant Proteins; D013373:Substance P; D015288:Neurokinin A