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

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 (ET(A)) 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 SE 209670, and the naphthalenesulfonamide BMS-182874 were decreased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ET(A) receptor mutants. Substitution of Tyr129 with Phe or Trp did not alter the high-affinity binding of BQ-123, bosentan, or SE 209670. EMS-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 EMS-182874, also suggested a hydrogen bond with the tyrosine hydroxyl. This hypothesis was supported bq structure-activity data with analogs of EMS-182874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of EMS-182874 and C-5 analogs. In all cases, naphthalenesulfonamide binding was more severely affected by mutation of Asp 133 than by mutation of Asp126. Phosphoinositide hydrolysis and extracellular acidification rate studies demonstrated the importance of Tyr129 to ET(A)-mediated signal transduction. On the basis of these data, two plausible models of the docked conformation of EMS-182874 in the ET(A) receptor are proposed as a starting point for further delineation of interactions that underlie antagonist-ET(A) receptor complex formation.