MUTATIONAL ANALYSIS OF THE ENDOTHELIN TYPE-A RECEPTOR (ET(A)) - INTERACTIONS AND MODEL OF THE SELECTIVE ET(A) ANTAGONIST EMS-182874 WITH THE PUTATIVE ET(A) RECEPTOR-BINDING CAVITY

Endothelin (ET) receptor antagonism is a potential therapeutic interve ntion in the treatment of vascular diseases. To elucidate the mechanis m of antagonist-ET receptor complex formation, the interactions of fou r chemically distinct antagonists were investigated using a combinatio n of genetic and biochemical approaches. By site-specific mutagenesis we previously demonstrated that Tyr129 in the second transmembrane dom ain 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 indancarboxyli c acid SE 209670, and the naphthalenesulfonamide BMS-182874 were decre ased 20-1000-fold in Tyr129Ala, Tyr129Ser, and Tyr129His ET(A) recepto r mutants. Substitution of Tyr129 with Phe or Trp did not alter the hi gh-affinity binding of BQ-123, bosentan, or SE 209670. EMS-182874 bind ing affinity was decreased 10-fold in Tyr129Phe and Tyr129Trp ET recep tors. These data indicate a role of aromatic interactions in the bindi ng of these antagonists to ETA receptors and, in the case of EMS-18287 4, also suggested a hydrogen bond with the tyrosine hydroxyl. This hyp othesis was supported bq structure-activity data with analogs of EMS-1 82874 that varied the C-5 dimethylamino substituent on the naphthalene ring. Mutation of Asp126 and Asp133 also altered binding of EMS-18287 4 and C-5 analogs. In all cases, naphthalenesulfonamide binding was mo re severely affected by mutation of Asp 133 than by mutation of Asp126 . Phosphoinositide hydrolysis and extracellular acidification rate stu dies demonstrated the importance of Tyr129 to ET(A)-mediated signal tr ansduction. On the basis of these data, two plausible models of the do cked 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.