An endothelin type A receptor-expressing cell to characterize endothelin-1 binding and screen antagonist

Endothelin-1 (ET-1) induces contraction of vascular smooth muscle through binding to endothelin type A receptor (ETAR). COS-7 cells stably expressing high levels of the ETAR were established (designated COS-7(ETAR)). The COS-7(ETAR) cell bound [125I]ET-1 with a Kd of 932 ± 161 pM and a Bmax of 74 ± 13 fmol/2 × 105 cells. [125I]ET-1 binding was inhibited by ET-1 and the ETAR antagonist BQ-610, but not by the endothelin type B receptor (ETBR) antagonist BQ-788. In clones expressing two ETAR mutants containing D46N or R53Q substitutions in the first extracellular domain of the receptor, [125I]ET-1 binding activity was dramatically reduced. This suggests that these single amino acid substitutions alter the three-dimensional structure of the ligand-binding domain of the ETAR. Using COS-7(ETAR) cell, we showed that Ca2+ or Mg2+ was essential for ET-1 binding to the ETAR and that ET-1 treatment induced postreceptor signaling, that is, intracellular accumulation of cyclic AMP (cAMP) and Ca2+ mobilization. The COS-7(ETAR) established in this study will be a useful tool for screening ET-1 antagonists for treating hypertension.