文摘
Endothelin (ET) receptor antagonism is a potential therapeuticintervention in the treatmentof vascular diseases. To elucidate the mechanism ofantagonist-ET receptor complex formation, theinteractions of four chemically distinct antagonists were investigatedusing a combination of genetic andbiochemical approaches. By site-specific mutagenesis we previouslydemonstrated that Tyr129 in thesecond transmembrane domain was critical for high-affinity,subtype-selective binding to the A subtypeof ET (ETA) receptors [Krystek et al. (1994) J.Biol. Chem. 269, 12383-12386]. Affinitiesof theconstrained cyclic pentapeptide BQ-123, thepyrimidinylbenzenesulfonamide bosentan, theindancarboxylicacid SB 209670, and the naphthalenesulfonamide BMS-182874 weredecreased 20-1000-fold inTyr129Ala, Tyr129Ser, and Tyr129His ETA receptor mutants.Substitution of Tyr129 with Phe or Trpdid not alter the high-affinity binding of BQ-123, bosentan, or SB209670. BMS-182874 binding affinitywas decreased 10-fold in Tyr129Phe and Tyr129Trp ET receptors.These data indicate a role of aromaticinteractions in the binding of these antagonists to ETAreceptors and, in the case of BMS-182874, alsosuggested a hydrogen bond with the tyrosine hydroxyl. Thishypothesis was supported by structure-activity data with analogs of BMS-182874 that varied the C-5dimethylamino substituent on the naphthalenering. Mutation of Asp126 and Asp133 also altered binding ofBMS-182874 and C-5 analogs. In allcases, naphthalenesulfonamide binding was more severely affected bymutation of Asp133 than by mutationof Asp126. Phosphoinositide hydrolysis and extracellularacidification rate studies demonstrated theimportance of Tyr129 to ETA-mediated signal transduction.On the basis of these data, two plausiblemodels of the docked conformation of BMS-182874 in the ETAreceptor are proposed as a starting pointfor further delineation of interactions that underlieantagonist-ETA receptor complex formation.
