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 C

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• 作者：Maria L. Webb ; Pramathesh S. Patel ; Patricia M. Rose ; Eddie C. K. Liu ; Philip D. Stein ; Joel Barrish ; David A. Lach ; Terry Stouch ; Susan M. Fisher ; Ophelia Hadjilambris ; Helen Lee ; Stephen Skwish ; Ken
• 刊名：Biochemistry
• 出版年：1996
• 出版时间：February 27, 1996
• 年：1996
• 卷：35
• 期：8
• 页码：2548 - 2556
• 全文大小：472K
• 年卷期：v.35,no.8(February 27, 1996)
• ISSN：1520-4995

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 (ET_A) 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 ET_A 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 ET_Areceptors 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 ET_A-mediated signal transduction.On the basis of these data, two plausiblemodels of the docked conformation of BMS-182874 in the ET_Areceptor are proposed as a starting pointfor further delineation of interactions that underlieantagonist-ET_A receptor complex formation.