SEPARABLE BINDING-SITES FOR THE NATURAL AGONIST ENDOTHELIN-1 AND THE NONPEPTIDE ANTAGONIST BOSENTAN ON HUMAN ENDOTHELIN-A RECEPTORS

A three-dimensional model for the transmembrane domains of human endot helin-A receptor was built using,structural information from bacterior hodopsin and sequence alignment to other guanine-nucleotide-binding re gulatory(G) protein-coupled receptors. Based on this model, 18 amino a cids located at the inside of the receptor were mutated and analyzed f or binding of the natural ligand endothelin-1 and bosentan, a recently described potent orally active endothelin antagonist [Clozel, M., Bre u, V., Gray, G., Kalina, B., Loffler, B.-M., Burri, K., Cassal, J.-M., Hirth, G., Muller, M., Neidhart, W. & Ramuz, H. (1994) Pharmacologica l 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 tra nsmembrane region 1, 2, 3, 3, and 6, respectively, caused reduced spec ific binding of I-125-labelled endothelin-1, despite an expression lev el similar to wild-type endothelin-A receptor Mutation of Tyr263, Arg3 26 and Asp351 preserved endothelin-1 binding but caused reduced bindin g 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 obser vations demonstrate a dissociation of the binding site for the peptidi c natural agonist endothelin-1 and the synthetic non-peptide antagonis t bosentan. They provide the molecular basis for bosentan being a spec ific antagonist for both, endothelin-A as well as endothelin-B recepto rs and may in combination with studies on structure/activity relations hip support the design of novel and more potent endothelin receptor an tagonists.