Agonist-promoted ubiquitination differentially regulates receptor trafficking of endothelin type A and type B receptors.
Two types of G protein-coupled receptors for endothelin-1 (ET-1), ET type A receptor (ETAR) and ETBR, closely resemble each other, but upon ET-1 stimulation, they follow totally different intracellular trafficking pathways; ETAR is recycled back to plasma membrane, whereas ETBR is targeted to lysosome for degradation. However, the mechanisms for such different fates are unknown. Here we demonstrated that ETBR but not ETAR was ubiquitinated on the cell surface following ET-1 stimulation and that ETBR was internalized and degraded in lysosome more rapidly than ETAR. The mutant ETBR (designated "5KR mutant") in which 5 lysine residues in the C-tail were substituted to arginine was not ubiquitinated, and its rates of internalization and degradation after ET-1 stimulation became slower, being comparable with those of ETAR. Confocal microscopic study showed that following ET-1 stimulation, ETAR and 5KR mutant of ETBR were co-localized mainly with Rab11, a marker of recycling endosome, whereas ETBR was co-localized with Rab7, a marker of late endosome/lysosome. In the 5KR mutant, ET-1-induced ERK phosphorylation and an increase in the intracellular Ca(2+) concentration upon repetitive ET-1 stimulation were larger. A series of ETBR mutants (designated "4KR mutant"), in which either one of 5 arginine residues of the 5KR mutant was reverted to lysine, were normally ubiquitinated, internalized, and degraded, with ERK phosphorylation being normalized. These results demonstrate that agonist-induced ubiquitination at either lysine residue in the C-tail of ETBR but not ETAR switches intracellular trafficking from recycling to plasma membrane to targeting to lysosome, causing decreases in the cell surface level of ETBR and intracellular signaling.
From the Departments of Cellular Pharmacology and.
Koji
K
Terada
From the Departments of Cellular Pharmacology and.
Takahiro
T
Horinouchi
Cell Physiology, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan.
Yoichiro
Y
Fujioka
From the Departments of Cellular Pharmacology and.
Tsunehito
T
Higashi
From the Departments of Cellular Pharmacology and.
Prabha
P
Nepal
From the Departments of Cellular Pharmacology and.
Mika
M
Horiguchi
From the Departments of Cellular Pharmacology and.
Sarita
S
Karki
From the Departments of Cellular Pharmacology and.
Chizuru
C
Hatate
From the Departments of Cellular Pharmacology and.
Akimasa
A
Hoshi
From the Departments of Cellular Pharmacology and.
Takuya
T
Harada
From the Departments of Cellular Pharmacology and.
Yosuke
Y
Mai
Cell Physiology, Hokkaido University Graduate School of Medicine, North 15, West 7, Kita-ku, Sapporo 060-8638, Japan.
Yusuke
Y
Ohba
From the Departments of Cellular Pharmacology and smiwa@med.hokudai.ac.jp.
Soichi
S
Miwa
D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D019332:Endothelin-1 / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D018613:Microscopy, Confocal; D009154:Mutation; D010766:Phosphorylation; D021381:Protein Transport / Q000187:drug effects; D044022:Receptor, Endothelin A / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D044023:Receptor, Endothelin B / Q000819:agonists / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination / Q000187:drug effects; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins
D019332:Endothelin-1; D048049:Extracellular Signal-Regulated MAP Kinases; D044022:Receptor, Endothelin A; D044023:Receptor, Endothelin B; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human
Endocytosis; Endothelin; Lysosome; Trafficking; Ubiquitination