Cysteine residues in the carboxyl terminal domain of the endothelin-B receptor are required for coupling with G-proteins
Okamoto, Y.; Ninomiya, H.; Tanioka, M.; Sakamoto, A.; Miwa, S.; Masaki, T.
Journal of Cardiovascular Pharmacology 31(Suppl): S230-S232
1998
ISSN/ISBN: 0160-2446 PMID: 9595445 Document Number: 493337
We demonstrate that the human endothelin-B (ETB) receptor incorporates (3H) palmitic acid. Mutation of three putative palmitoylated cysteine residues (amino 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 toxin-insensitive G-protein, most likely a member(s) of the Gq family. 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.