title: Mutation of peptide binding site in transmembrane region of a G protein-coupled receptor accounts for endothelin receptor subtype selectivity.
abstract: The molecular basis for endothelin (ET) isopeptide selectivity between ETA and ETB receptors was studied by examining ligand binding to several site-specific mutants of the human ETA receptor. Based on a computer-built three-dimensional model of the ETA receptor, five non-conserved amino acids, clustered around the putative ligand binding site, were targeted for mutation to alanine. Expression of the wild-type and mutant ETA receptors in COS-7 cells revealed that the binding profile of one of the ETA mutants, Tyr129-->Ala, was characteristic of the ETB receptor. In the Tyr129-->Ala ETA receptor mutant the affinity of two ETB-selective agonists, endothelin-3 and sarafotoxin S6c, was increased 10-200-fold, whereas that for two ETA-selective antagonists, BQ-123 and BMS-182874, was reduced 350-2,000-fold. Thus, mutation of a single amino acid in the second transmembrane region of the wild-type ETA receptor results in subtype conversion. In addition, these data represent the first example of peptide interactions with a transmembrane region of a G protein-coupled receptor and indicate that Tyr129, located in the second transmembrane region of the ETA receptor, is a critical component for determination of endothelin receptor subtype-selective ligand binding.
authors.affiliation: Department of Macromolecular Modeling, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 08543.
authors.forename: S R
authors.initials: SR
authors.lastname: Krystek
authors.forename: P S
authors.initials: PS
authors.lastname: Patel
authors.forename: P M
authors.initials: PM
authors.lastname: Rose
authors.forename: S M
authors.initials: SM
authors.lastname: Fisher
authors.forename: B K
authors.initials: BK
authors.lastname: Kienzle
authors.forename: D A
authors.initials: DA
authors.lastname: Lach
authors.forename: E C
authors.initials: EC
authors.lastname: Liu
authors.forename: J S
authors.initials: JS
authors.lastname: Lynch
authors.forename: J
authors.initials: J
authors.lastname: Novotny
authors.forename: M L
authors.initials: ML
authors.lastname: Webb
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000378:metabolism; D017466:Receptors, Endothelin / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D009842:Oligopeptides; D017466:Receptors, Endothelin; D011994:Recombinant Proteins; D019204:GTP-Binding Proteins