LYSINE-182 OF ENDOTHELIN-B RECEPTOR MODULATES AGONIST SELECTIVITY ANDANTAGONIST AFFINITY - EVIDENCE FOR THE OVER-LAP OF PEPTIDE AND NONPEPTIDE LIGAND-BINDING SITES

The potent vasoactive peptide hormone endothelin (ET) binds to recepto rs which belong to the G-protein coupled receptor family. The availabi lity of non-peptide antagonists for ET receptors allows investigation of the relationship among the binding sites for peptide and non-peptid e ligands. In this study, a lysine residue, conserved within transmemb rane domain 3 (TM3) of the ET(A) and ET(B) receptor subtypes, is impli cated in agonist and antagonist binding by its analogous position with in TM3 to a binding site aspartate residue conserved within bioactive amine receptors. Replacement of this lysine within hET(B) by arginine, alanine, methionine, aspartate, or glutamate results in hET(B) varian ts with unaltered affinities for agonist peptide ET-1 but which have a ffinities for peptide agonists ET-2, ET-3, sarafotoxin 6C, and IRL 173 6 which are between 1-3 orders of magnitude lower than their correspon ding wild-type hET(B) values. Significantly, the affinities of non-pep tide antagonists, (+/-)-SB 209670 and its analogs as well as Ro 46-200 5, are abrogated. The results suggest that an interaction of K182 of h ET(B) with the indan 2-carboxyl of (+/-)-SB 209670 may contribute to t he high-affinity binding of the diarylindan antagonists. The results i ndicate that TM3 of hET(B) is a region of overlap among the binding si tes of non-peptide antagonists and the affected peptide agonists.