Palmitoylation of human endothelinB. 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 endothelinB (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 Cys403/Cys405 gave a deletion mutant Delta403 that was palmitoylated on Cys402 and lacked the carboxyl terminus downstream to the palmitoylation site. Delta403 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 Gi but not for that with Gq.
Okamoto
Y
Y
Department of Pharmacology, Faculty of Medicine, Kyoto University, Kyoto 606, Japan.
Ninomiya
H
H
Tanioka
M
M
Sakamoto
A
A
Miwa
S
S
Masaki
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002451:Cell Compartmentation; D006224:Cricetinae; D003545:Cysteine / Q000737:chemistry; D003593:Cytoplasm / Q000737:chemistry; D019332:Endothelin-1 / Q000378:metabolism; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D008024:Ligands; D008969:Molecular Sequence Data; D010168:Palmitates / Q000378:metabolism*; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D017384:Sequence Deletion; D015398:Signal Transduction
D019332:Endothelin-1; D008024:Ligands; D010168:Palmitates; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D003545:Cysteine