Regulation of the vasopressin V2 receptor by vasopressin in polarized renal collecting duct cells

• Published in: Molecular biology of the cell Vol. 15; no. 12; pp. 5693 - 5699
• Main Authors: Robben, J H, Knoers, N V A M, Deen, P M T
• Format: Journal Article
• Language: English
• Published: United States The American Society for Cell Biology 01.12.2004
• Subjects: Animals; Cell Line; Cell Polarity; Dogs; Glycosylation; Kidney Tubules, Collecting - cytology; Kidney Tubules, Collecting - drug effects; Kidney Tubules, Collecting - metabolism; Lysosomes - drug effects; Lysosomes - metabolism; Organelles - metabolism; Protein Processing, Post-Translational - drug effects; Receptors, Vasopressin - genetics; Receptors, Vasopressin - metabolism; Time Factors; Vasopressins - pharmacology
• ISSN: 1059-1524; 1939-4586
• DOI: 10.1091/mbc.E04-04-0337

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.