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Am J Physiol Cell Physiol (January 26, 2005). doi:10.1152/ajpcell.00353.2004
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Submitted on July 20, 2004
Accepted on January 12, 2005

Downregulation of the vasopressin type 2 receptor (V2R) after vasopressin-induced internalization: involvement of a lysosomal degradation pathway

Richard Bouley1*, Herbert Y. Lin1, Malay K. Raychowdhury1, Vladimir Marshansky1, Dennis Brown1, and Dennis A. Ausiello1

1 Program in Membrane Biology and Renal Unit, Massachusetts General Hospital, Boston, MA, USA

* To whom correspondence should be addressed. E-mail: bouley{at}receptor.mgh.harvard.edu.

Vasopressin (VP) increases urinary concentration by signaling through the vasopressin receptor (V2R) in collecting duct principal cells. After downregulation, V2R reappears at the cell surface via an unusually slow (several hours) "recycling"" pathway. To examine this pathway, we expressed V2R-GFP (green fluorescent protein) in LLC-PK1a cells. V2R-GFP showed similar characteristics to wild type V2R, including high affinity for VP and adenylyl cyclase stimulation. V2R-GFP was located mainly in the plasma membrane in unstimulated cells, but after VP-induced internalization it colocalized with the lysosomal marker, Lysotracker. Western blotting of V2R-GFP showed a broad 57-68 kDa band and a doublet at 46 kDa and 52 kDa prior to VP treatment. After 4h VP exposure, the 57-68 kDa band lost 50% of its intensity whereas the lower 46 kDa band increased by 200%. The lysosomal inhibitor chloroquine abolished this VP effect whereas lactacystin, a proteosome inhibitor, had no effect. Incubating cells at 20°C to block trafficking from the trans-Golgi network (TGN) reduced V2R membrane fluorescence and a perinuclear patch developed. Cycloheximide reduced the intensity of this patch, showing that newly synthesized V2R-GFP contributed significantly to its appearance. Cycloheximide also inhibited the reappearance of cell surface V2R after downregulation. We conclude that after downregulation, V2R-GFP is delivered to lysosomes and degraded. Reappearance of V2R at the cell surface depends on new protein synthesis, partially explaining the long time lag needed to fully reestablish V2R at the cell surface after downregulation. This degradative pathway may be an adaptive response to allow receptor-ligand association in the hypertonic and acidic environment of the renal medulla.




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