Regulation of V2 vasopressin receptor degradation by agonist-promoted ubiquitination.
The seven-transmembrane-spanning vasopressin V2 receptor (V2R) is a Gs-coupled receptor that is rapidly phosphorylated and internalized following stimulation with the agonist, arginine-vasopressin. Herein, we show that the V2R is ubiquitinated following agonist stimulation. V2R-ubiquitination is not observed in a beta-arrestin1,2 deleted mouse fibroblast cell line and is restored following introduction of beta-arrestin2, thus indicating that beta-arrestin2 is required for the ubiquitination of V2R. A mutant V2R (K268R) that is not ubiquitinated still activates Gs and internalizes with similar kinetics as the wild type receptor. Unstimulated wild type and K268R mutant receptors degrade at similar rates and have comparable half-lives of 217 +/- 17 and 245 +/- 29 min as determined by pulse-chase experiments. However, following agonist stimulation, the rate of receptor degradation for the wild type is enhanced (half-life of 69 +/- 19 min), whereas that of the mutant is only minimally affected (half-life of 188 +/- 11 min). These data suggest that V2R levels are regulated through at least two processes. In the absence of agonist stimulation, a slow degradative pathway operates that is independent of receptor ubiquitination. However, receptor stimulation leads to rapid beta-arrestin2-dependent ubiquitination of the receptor and increased degradation.
Martin
Negin P
NP
Howard Hughes Medical Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Durham, North Carolina 27710, USA.
Lefkowitz
Robert J
RJ
Shenoy
Sudha K
SK
D000595:Amino Acid Sequence; D000818:Animals; D019390:Arrestins / Q000235:genetics; D015153:Blotting, Western; D019556:COS Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008239:Lysine / Q000737:chemistry; D051379:Mice; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D017483:Receptors, Vasopressin / Q000096:biosynthesis* / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D025801:Ubiquitin / Q000378:metabolism*; D000071557:beta-Arrestins
D019390:Arrestins; D017483:Receptors, Vasopressin; D025801:Ubiquitin; D000071557:beta-Arrestins; D000242:Cyclic AMP; D008239:Lysine