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 [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, delta 402 and delta 403. Despite the absence of the cytoplasmic tail, both delta 402 and delta 403 showed essentially the same features as C2/3/5S, except that delta 403 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.
Okamoto
Y
Y
Department of Pharmacology, Faculty of Medicine, Kyoto University, Japan.
Ninomiya
H
H
Tanioka
M
M
Sakamoto
A
A
Miwa
S
S
Masaki
T
T
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011487:Protein Conformation; D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes
D044023:Receptor, Endothelin B; D017466:Receptors, Endothelin; D013462:Sulfur Radioisotopes; D004247:DNA; D019204:GTP-Binding Proteins; D003545:Cysteine