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

The potent vasoactive peptide hormone endothelin (ET) binds to receptors which belong to the G-protein coupled receptor family. The availability of non-peptide antagonists for ET receptors allows investigation of the relationship among the binding sites for peptide and non-peptide ligands. In this study, a lysine residue, conserved within transmembrane domain 3 (TM3) of the ET(A) and ET(B) receptor subtypes, is implicated in agonist and antagonist binding by its analogous position within 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) variants with unaltered affinities for agonist peptide ET-1 but which have affinities for peptide agonists ET-2, ET-3, sarafotoxin 6C, and IRL 1736 which are between 1-3 orders of magnitude lower than their corresponding wild-type hET(B) values. Significantly, the affinities of non-peptide antagonists, (+/-)-SB 209670 and its analogs as well as Ro 46-2005, are abrogated. The results suggest that an interaction of K182 of hET(B) with the indan 2-carboxyl of (+/-)-SB 209670 may contribute to the high-affinity binding of the diarylindan antagonists. The results indicate that TM3 of hET(B) is a region of overlap among the binding sites of non-peptide antagonists and the affected peptide agonists.