Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells.
Binding of arginine-vasopressin (AVP) to its V2 receptor (V2R) in the basolateral membrane of principal cells induces Aquaporin-2-mediated water reabsorption in the kidney. To study the regulation of the V2R by dDAVP in a proper model, a polarized renal cell line stably-expressing V2R-GFP was generated. Labeled AVP-binding studies revealed an equal basolateral vs. apical membrane distribution for V2R-GFP and endogenous V2R. In these cells, GFP-V2R was expressed in its mature form and localized for 75% in the basolateral membrane and for 25% to late endosomes/lysosomes. dDAVP caused a dose- and time-dependent internalization of V2R-GFP, which was completed within 1 h with 100 nM dDAVP, was prevented by coincubation with a V2R antagonist, and which reduced its half-life from 11.5 to 2.8 h. Semiquantification of the V2R-GFP colocalization with E-cadherin (basolateral membrane), early endosomal antigen-1 (EEA-1) and lysosome-associated membrane protein-2 (LAMP-2) in time revealed that most dDAVP-bound V2R was internalized via early endosomes to late endosomes/lysosomes, where it was degraded. The dDAVP-internalized V2R did not recycle to the basolateral membrane. In conclusion, we established the itinerary of the V2R in a polarized cell model that likely resembles the in vivo V2R localization and regulation by AVP to a great extent.
Department of Physiology, Nijmegen Center for Molecular Life Sciences, Radboud University Nijmegen Medical Center, 6500 HB Nijmegen, The Netherlands.
J H
JH
Robben
N V A M
NV
Knoers
P M T
PM
Deen
D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D004285:Dogs; D006031:Glycosylation; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D015388:Organelles / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017483:Receptors, Vasopressin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014667:Vasopressins / Q000494:pharmacology*
D017483:Receptors, Vasopressin; D014667:Vasopressins