Palmitoylation of human endothelin(B) - Its critical role in G protein coupling and a differential requirement for the cytoplasmic tail by G protein subtypes

By site-directed mutagenesis, three cysteine residues (amino acids 402, 403, and 405) in the carboxyl terminus of human endothelin(B) (ETB) were identified as potential palmitoylation sites. Substitutions of all of the three cysteine residues with serine gave an unpalmitoylated mutant, C2S/C3S/C5S, When expressed in Chinese hamster ovary cells, C2S/C3S/C5S was localized on the cell surface, retained high affinities to ET-1 and ET-3, and was rapidly internalized when bound to the ligand, However, unlike the wild-type ETB, C2S/C3S/C5S 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 subtypes. Truncation of the carboxyl terminus including Cys(403)/Cys(405) gave a deletion mutant Delta 403 that was palmitoylated ton Cys(402) and lacked the carboxyl terminus downstream to the palmitoylation site. Delta 403 did transmit a stimulatory effect on phospholipase C via a pertussis toxin-insensitive G protein but it failed to transmit an inhibitory effect on adenylate cyclase. These results indicated a differential requirement for the carboxyl terminus downstream to the palmitoylation site in the coupling with G protein subtypes, i.e. it is required for the coupling with G(i) but not for that with G(q).