Mutational Analysis of the Endothelin Type A Receptor (ETA):  Interactions and Model of the Selective ETA Antagonist BMS-182874 with the Putative ETA Receptor Binding Cavity

• Published in: Biochemistry (Easton) Vol. 35; no. 8; pp. 2548 - 2556
• Main Authors: Webb, Maria L, Patel, Pramathesh S, Rose, Patricia M, Liu, Eddie C. K, Stein, Philip D, Barrish, Joel, Lach, David A, Stouch, Terry, Fisher, Susan M, Hadjilambris, Ophelia, Lee, Helen, Skwish, Stephen, Dickinson, Kenneth E. J, Krystek, Stanley R
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 27.02.1996
• Subjects: Amino Acid Sequence; Animals; Binding Sites; Cell Line; Dansyl Compounds - chemistry; Dansyl Compounds - metabolism; Dansyl Compounds - pharmacology; Endothelin Receptor Antagonists; Endothelins - chemistry; Endothelins - genetics; Endothelins - metabolism; Humans; Hydrogen Bonding; In Vitro Techniques; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Receptor, Endothelin A; Receptors, Endothelin - chemistry; Receptors, Endothelin - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi951836v

Endothelin (ET) receptor antagonism is a potential therapeutic intervention in the treatment of vascular diseases. To elucidate the mechanism of antagonist−ET receptor complex formation, the interactions of four chemically distinct antagonists were investigated using a combination of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane domain was critical for high-affinity, subtype-selective binding to the A subtype of ET (ETA) receptors [Krystek et al. (1994) J. Biol. Chem. 269, 12383−12386]. Affinities of the constrained cyclic pentapeptide BQ-123, the pyrimidinylbenzenesulfonamide bosentan, the indancarboxylic acid SB 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20−1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SB 209670. BMS-182874 binding affinity was decreased 10-fold in Tyr129Phe and Tyr129Trp ET receptors. These data indicate a role of aromatic interactions in the binding of these antagonists to ETA receptors and, in the case of BMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported by structure−activity data with analogs of BMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of BMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ETA-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of BMS-182874 in the ETA receptor are proposed as a starting point for further delineation of interactions that underlie antagonist−ETA receptor complex formation.