Cysteine residues in the carboxyl terminal domain of the endothelin-B receptor are required for coupling with G-proteins

We demonstrate that the human endothelin-B (ETB) receptor incorporates [H-3]palmitic acid. Mutation of three putative palmitoylated cysteine residues (an-Lino acids 402, 403 and 405) in the carboxyl terminus into serine residues (C2/3/5S) completely prevented palmitoylation of ETB. When expressed in CHO cells, C2/3/5S was localized on the cell surface. retained high affinity for ET-1 and ET-3, and was rapidly internalized when bound to the ligand. However, unlike the wild-type ETB C2/3/5S transmitted neither an inhibitory effect on adenylate cyclase nor a stimulatory effect on phospholipase C, indicating a critical role of palmitoylation in the coupling with G-proteins. regardless of the G-protein subtype. Truncation of the carboxyl terminus, including all or a part of the three cysteine residues, gave palmitoylation-negative and -positive deletion mutants, Delta402 and Delta403. Despite the absence of the cytoplasmic tail, both Delta402 and Delta403 showed essentially the same features as C2/3/5S, except that Delta403 did transmit a stimulatory effect on phospholipase C via a pertussis to.,,in-insensitive G-protein, most likely a member(s) of the Gq fan-Lily. These results indicated a differential requirement for the carboxyl terminus downstream from the palmitoylation site in the coupling with G-protein subtypes, i.e., it is required for the coupling with Gi but not for that with Gq.