Distinct subdomains of human endothelin receptors determine their selectivity to endothelin(A)-selective antagonist and endothelin(B)-selective agonists

The endothelin (ET) family of peptides acts via two subtypes of G-protein- coupled heptahelical receptors termed ET(A) and ET(B), which have distinct rank orders of affinity to endothelin receptor agonists and antagonists. To delineate which portions of the receptor molecules determine ligand selectivity, we have constructed a series of chimeras between human ET(A) and ET(B) receptors and characterized the chimeric receptors expressed in heterologous cell lines by competitive radioligand binding analysis and by measuring agonist-induced transients of intracellular Ca²⁺. We demonstrate that the binding determinant for the ET(B)-selective agonists ET-3, BQ3020, and IRL1620 resides within the region spanning the putative transmembrane helices IV-VI and the adjacent loop regions. In contrast, the transmembrane helices I, II, III, and VII plus the intervening loop regions specify the selectivity for BQ123, an ET(A)-selective antagonist. BQ123 exhibited no detectable agonistic activity in all wild-type and chimeric receptors tested. A chimeric receptor that has the transmembrane helices IV-VI (and adjacent loops) from the ET(B) receptor inserted into the remaining regions from the ET(A) receptor binds both the ET(A)- and ET(B)-selective ligands with high affinities. Moreover, BQ123 competitively inhibits the binding of the amino- terminally truncated ET(B) agonists, ¹²⁵I-BQ3020 and ¹²⁵I-IRL1620, to this chimeric receptor, suggesting that BQ123 is a mimic of the carboxyl- terminal linear portion of endothelins. These findings indicate that there are at least two separable ligand interaction subdomains within the endothelin receptors.