Breu V, Hashido K, Broger C, Miyamoto C, Furuichi Y, Hayes A, Kalina B, Löffler B M, Ramuz H, Clozel M
Pharma Division, F. Hoffmann-La Roche Ltd., Basel, Switzerland.
Eur J Biochem. 1995 Jul 1;231(1):266-70.
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.
利用来自细菌视紫红质的结构信息以及与其他鸟嘌呤核苷酸结合调节（G）蛋白偶联受体的序列比对，构建了人内皮素A受体跨膜结构域的三维模型。基于该模型，对位于受体内侧的18个氨基酸进行了突变，并分析了它们对天然配体内皮素-1和波生坦（一种最近描述的强效口服活性内皮素拮抗剂）的结合情况[克洛泽尔，M.，布勒，V.，格雷，G.，卡利纳，B.，勒夫勒，B.-M.，伯里，K.，卡萨勒，J.-M.，希尔特，G.，米勒，M.，奈德哈特，W.和拉穆兹，H.（1994年）波生坦的药理学特性，一种新型强效口服活性非肽内皮素受体拮抗剂，《药理学与实验治疗学杂志》270，228 - 235]。分别位于跨膜区1、2、3、3和6的甘氨酸97、赖氨酸140、赖氨酸159、谷氨酰胺165和苯丙氨酸315的突变，尽管表达水平与野生型内皮素A受体相似，但导致125I标记的内皮素-1特异性结合减少。酪氨酸263、精氨酸326和天冬氨酸351的突变保留了内皮素-1的结合，但导致波生坦的结合减少。这些氨基酸分别位于跨膜区5、6和7，在内皮素A和内皮素B受体中保守，但在其他G蛋白偶联受体中不保守。这些观察结果表明肽类天然激动剂内皮素-1和合成非肽拮抗剂波生坦的结合位点存在解离。它们为波生坦作为内皮素A和内皮素B受体的特异性拮抗剂提供了分子基础，并可能与构效关系研究相结合，支持新型更强效内皮素受体拮抗剂的设计。
