title: Separable binding sites for the natural agonist endothelin-1 and the non-peptide antagonist bosentan on human endothelin-A receptors.
abstract: A three-dimensional model for the transmembrane domains of human endothelin-A receptor was built using structural information from bacteriorhodopsin and sequence alignment to other guanine-nucleotide-binding regulatory(G) protein-coupled receptors. Based on this model, 18 amino acids located at the inside of the receptor were mutated and analyzed for binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Breu, V., Gray, G., Kalina, B., Löffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Müller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacological characterization of bosentan, a new potent orally active nonpeptide endothelin receptor antagonist, J. Pharmacol. Exp. Ther. 270, 228-235]. Mutation of Gly97, Lys140, Lys159, Gln165 and Phe315, located in transmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced specific binding of 125I-labelled endothelin-1, despite an expression level similar to wild-type endothelin-A receptor. Mutation of Tyr263, Arg326 and Asp351 preserved endothelin-1 binding but caused reduced binding of bosentan. These amino acids, located on transmembrane regions 5, 6 and 7, respectively, are conserved among endothelin-A and endothelin-B receptors but not in other G-protein-coupled receptors. These observations demonstrate a dissociation of the binding site for the peptidic natural agonist endothelin-1 and the synthetic non-peptide antagonist bosentan. They provide the molecular basis for bosentan being a specific antagonist for both, endothelin-A as well as endothelin-B receptors and may in combination with studies on structure/activity relationship support the design of novel and more potent endothelin receptor antagonists.
authors.affiliation: Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
authors.forename: V
authors.initials: V
authors.lastname: Breu
authors.forename: K
authors.initials: K
authors.lastname: Hashido
authors.forename: C
authors.initials: C
authors.lastname: Broger
authors.forename: C
authors.initials: C
authors.lastname: Miyamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Furuichi
authors.forename: A
authors.initials: A
authors.lastname: Hayes
authors.forename: B
authors.initials: B
authors.lastname: Kalina
authors.forename: B M
authors.initials: BM
authors.lastname: Löffler
authors.forename: H
authors.initials: H
authors.lastname: Ramuz
authors.forename: M
authors.initials: M
authors.lastname: Clozel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000077300:Bosentan; D002460:Cell Line; D065128:Endothelin Receptor Antagonists; D016232:Endothelins / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D017433:Protein Structure, Secondary; D017466:Receptors, Endothelin / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D013449:Sulfonamides / Q000378:metabolism*
chemical_list: D065128:Endothelin Receptor Antagonists; D016232:Endothelins; D017466:Receptors, Endothelin; D013449:Sulfonamides; D019204:GTP-Binding Proteins; D000077300:Bosentan