title: Identification of residues involved in ligand binding to the neurokinin-2 receptor.
abstract: Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and location of these receptor residues suggest that the distance between bound peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thus receptor subtype selectivity. While many single-residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists.
authors.affiliation: Department of Molecular Pharmacology and Biochemistry, Merck Research Laboratories, Rahway, New Jersey 07065, USA.
authors.forename: R R
authors.initials: RR
authors.lastname: Huang
authors.forename: P P
authors.initials: PP
authors.lastname: Vicario
authors.forename: C D
authors.initials: CD
authors.lastname: Strader
authors.forename: T M
authors.initials: TM
authors.lastname: Fong
mesh_terms: D000595:Amino Acid Sequence; D001549:Benzamides / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D008969:Molecular Sequence Data; D015288:Neurokinin A / Q000037:antagonists & inhibitors / Q000378:metabolism; D015287:Neurokinin B / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010880:Piperidines / Q000378:metabolism*; D018041:Receptors, Neurokinin-2 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011994:Recombinant Proteins; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D013373:Substance P / Q000378:metabolism; D015320:Tachykinins / Q000378:metabolism*
chemical_list: D001549:Benzamides; D008024:Ligands; D010446:Peptide Fragments; D010716:Phosphatidylinositols; D010880:Piperidines; D018041:Receptors, Neurokinin-2; D011994:Recombinant Proteins; D015320:Tachykinins; D013373:Substance P; C073839:SR 48968; D015288:Neurokinin A; D015287:Neurokinin B