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.