Characterization of the putative phosphorylation sites of the AQP2 c-terminus and their role in AQP2 trafficking in LLC-PK1 cells.

Characterization of the putative phosphorylation sites of the AQP2 c-terminus and their role in AQP2 trafficking in LLC-PK1 cells. Am J Physiol Renal Physiol. 2015 Aug 19;:ajprenal.00152.2015 Authors: Arthur J, Huang J, Nomura N, Jin WW, Li W, Cheng X, Brown D, Lu HJ Abstract Vasopressin (VP) stimulates a signaling cascade resulting in phosphorylation and apical membrane accumulation of AQP2, leading to water reabsorption by kidney collecting ducts. However, the roles of most C-terminal phosphorylation events in stimulated and constitutive AQP2 recycling are incompletely understood. Here, we generated LLC-PK1 cells containing point mutations of all potential phosphorylation sites in the AQP2 C-terminus: S226, S229, T244, S256, S261, S264, and S269, to determine their impact on AQP2 trafficking. We produced an All Null AQP2 construct in which these serine (S) or threonine (T) residues were mutated to alanine (A) or glycine (G), and we then re-introduced the phosphorylation mimic, aspartic acid (D) individually to each site in the All Null mutant. As expected, the All Null mutant does not accumulate at the plasma membrane in response to VP, but still undergoes constitutive recycling as shown by its membrane accumulation when endocytosis is blocked by methyl-β-cyclodextrin (MβCD), and accumulation in a perinuclear patch at low temperature (20oC). Single phosphorylation mimics S226D, S229D, T244D, S261D, S264D, and S269D were insuffici...
Source: Am J Physiol Renal P... - Category: Urology & Nephrology Authors: Tags: Am J Physiol Renal Physiol Source Type: research