Phosphorylation of aquaporin-2 regulates its endocytosis and protein-protein interactions

被引:147
作者
Moeller, Hanne B. [1 ]
Praetorius, Jeppe [1 ]
Rutzler, Michael R. [1 ]
Fenton, Robert A. [1 ]
机构
[1] Aarhus Univ, Water & Salt Res Ctr, Dept Anat, DK-8000 Aarhus C, Denmark
基金
新加坡国家研究基金会; 英国医学研究理事会;
关键词
Madin-Darby canine kidney cells; trafficking; vasopressin; water homeostasis; water channel; AQP2; TRAFFICKING; WATER; VASOPRESSIN; MEMBRANE; IDENTIFICATION; SERINE-261; INHIBITION; EXOCYTOSIS; EXPRESSION; DOMINANT;
D O I
10.1073/pnas.0910683107
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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 toWT-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.
引用
收藏
页码:424 / 429
页数:6
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