Phosphorylation of aquaporin-2 regulates its endocytosis and protein-protein interactions.
The water channel aquaporin-2 (AQP2) is essential for urine concentration. Vasopressin regulates phosphorylation of AQP2 at four conserved serine residues at the COOH-terminal tail (S256, S261, S264, and S269). We used numerous stably transfected Madin-Darby canine kidney cell models, replacing serine residues with either alanine (A), which prevents phosphorylation, or aspartic acid (D), which mimics the charged state of phosphorylated AQP2, to address whether phosphorylation is involved in regulation of (i) apical plasma membrane abundance of AQP2, (ii) internalization of AQP2, (iii) AQP2 protein-protein interactions, and (iv) degradation of AQP2. Under control conditions, S256D- and 269D-AQP2 mutants had significantly greater apical plasma membrane abundance compared to wild type (WT)-AQP2. Activation of adenylate cyclase significantly increased the apical plasma membrane abundance of all S-A or S-D AQP2 mutants with the exception of 256D-AQP2, although 256A-, 261A-, and 269A-AQP2 mutants increased to a lesser extent than WT-AQP2. Biotin internalization assays and confocal microscopy demonstrated that the internalization of 256D- and 269D-AQP2 from the plasma membrane was slower than WT-AQP2. The slower internalization corresponded with reduced interaction of S256D- and 269D-AQP2 with several proteins involved in endocytosis, including Hsp70, Hsc70, dynamin, and clathrin heavy chain. The mutants with the slowest rate of internalization, 256D- and 269D-AQP2, had a greater protein half-life (t(1/2) = 5.1 h and t(1/2) = 4.4 h, respectively) compared to WT-AQP2 (t(1/2) = 2.9 h). Our results suggest that vasopressin-mediated membrane accumulation of AQP2 can be controlled via regulated exocytosis and endocytosis in a process that is dependent on COOH terminal phosphorylation and subsequent protein-protein interactions.
Moeller
Hanne B
HB
Water and Salt Research Center, Department of Anatomy, Aarhus University, DK-8000 Aarhus C, Denmark.
Praetorius
Jeppe
J
Rützler
Michael R
MR
Fenton
Robert A
RA
D000818:Animals; D051399:Aquaporin 2 / Q000235:genetics / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D004705:Endocytosis / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding; D014162:Transfection; D014667:Vasopressins / Q000378:metabolism
D051399:Aquaporin 2; D014667:Vasopressins; D001710:Biotin