Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells.
The adenylyl cyclase stimulator forskolin (FSK) stimulates UT-A1 phosphorylation, membrane trafficking, and urea transport activity. Here, we found that FSK stimulation induces UT-A1 ubiquitination in UT-A1 Madin-Darby canine kidney (MDCK) cells. This suggests that phosphorylation by FSK also triggers the protein degradation machinery for UT-A1. UT-A1-MDCK cells were treated with 100 μg/ml cycloheximide to inhibit protein synthesis, with or without 10 μM FSK. Total UT-A1 protein abundance was significantly reduced after FSK treatment, concomitantly ubiquitinated UT-A1 was increased. We then specifically investigated the effect of FSK on UT-A1 expressed on the cell plasma membrane. FSK treatment accelerated UT-A1 removal from the cell plasma membrane by increasing UT-A1 endocytosis as judged by biotinylation/MesNa treatment and confocal microscopy. We further found that inhibition of the clathrin-mediated endocytic pathway, but not the caveolin-mediated endocytic pathway, significantly blocks FSK-stimulated UT-A1 endocytosis. The PKA inhibitor H89 and the proteasome inhibitors MG132 and lactacystin reduced FSK-induced membrane UT-A1 reduction. Our study shows that FSK activates the UT-A1 urea transporter and the activation/phosphorylation subsequently triggers the downregulation of UT-A1, which represents an important mechanism for the cell to return to the basal conditions after vasopressin stimulation.
Department of Medicine, Renal Division, Emory University School of Medicine, Atlanta, GA 30322, USA.
Hua
H
Su
Conner B
CB
Carter
Oskar
O
Laur
Jeff M
JM
Sands
Guangping
G
Chen
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects; D002966:Clathrin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology*; D003513:Cycloheximide / Q000494:pharmacology; D004285:Dogs; D004705:Endocytosis / Q000187:drug effects*; D007668:Kidney / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism*; D023421:Models, Animal; D059748:Proteolysis / Q000187:drug effects*; D015398:Signal Transduction / Q000187:drug effects; D054875:Ubiquitination / Q000187:drug effects*; D000096982:Urea Transporters
D002966:Clathrin; D005576:Colforsin; D003513:Cycloheximide; D026901:Membrane Transport Proteins; D000096982:Urea Transporters