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Author Notes:

Address for reprint requests and other correspondence: Guangping Chen, Renal Division, Emory University School of Medicine, WMB Rm. 338, 1639 Pierce Dr., Atlanta, GA 30322; Email: gchen3@emory.edu

Author contributions: H.S. and G.C. conception and design of research; H.S., C.B.C., and O.L. performed experiments; H.S., J.M.S., and G.C. analyzed data; H.S. and G.C. prepared figures; J.M.S. and G.C. interpreted results of experiments; J.M.S. and G.C. edited and revised manuscript; G.C. drafted manuscript; G.C. approved final version of manuscript.

No conflicts of interest, financial or otherwise, are declared by the author(s).


Research Funding:

This work was supported by National Institutes of Health Grants R01-DK087838 (to G. Chen) and R01-DK041707 (to J. M. Sands).


  • urine-concentrating mechanism
  • protein kinase A
  • phosphorylation
  • clathrin
  • vasopressin

Forskolin stimulation promotes urea transporter UT-A1 ubiquitination, endocytosis, and degradation in MDCK cells


Journal Title:

AJP - Renal Physiology


Volume 303, Number 9


, Pages F1325-F1332

Type of Work:

Article | Post-print: After Peer Review


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.

Copyright information:

© 2012 the American Physiological Society

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