title: Inhibition of endocytosis causes phosphorylation (S256)-independent plasma membrane accumulation of AQP2.
abstract: Inhibition of clathrin-mediated endocytosis by expression of a GTPase-deficient dynamin mutant (dynamin-2/K44A) for 16 h results in an accumulation of plasma membrane aquaporin-2 (AQP2) in epithelial cells stably transfected with wild-type AQP2. We now show a similar effect of K44A dynamin in LLC-PK1 cells transfected with an S256 phosphorylation-deficient AQP2 mutant, AQP2(S256A), and in AQP2-transfected inner medullary collecting duct (IMCD) cells. More acute blockade of endocytosis in these cells with the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD; 10 mM) resulted in a rapid and extensive cell-surface accumulation of both wild-type AQP2 and AQP2 (S256A) within 15 min after treatment. This effect was similar to that induced by treatment of the cells with vasopressin. Blockade of endocytosis by mbetaCD was confirmed using quantitative analysis of FITC-dextran uptake and AQP2 membrane insertion was verified by cell-surface biotinylation. These data indicate that AQP2 recycles constitutively and rapidly between intracellular stores and the cell surface in LLC-PK1 and IMCD cells. The constitutive trafficking process is not dependent on phosphorylation of the serine-256 residue of AQP2, which is, however, an essential step for regulated vasopressin/cAMP-mediated translocation of AQP2. Our data show that rapid and extensive plasma membrane accumulation of AQP2 can occur in a vasopressin receptor (V2R)- and phosphorylation-independent manner, pointing to a potential means of bypassing the mutated V2R in X-linked nephrogenic diabetes insipidus to achieve cell surface expression of AQP2.
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins / Q000235:genetics / Q000378:metabolism*; D019921:Biotinylation; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000378:metabolism; D003505:Cyclodextrins / Q000494:pharmacology; D034285:Dynamin II / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D006825:Hybridomas; D007685:Kidney Tubules, Collecting / Q000378:metabolism; D018374:LLC-PK1 Cells; D010766:Phosphorylation; D012076:Renal Agents / Q000494:pharmacology; D012694:Serine / Q000378:metabolism; D013552:Swine; D014667:Vasopressins / Q000494:pharmacology; D047392:beta-Cyclodextrins
chemical_list: D051399:Aquaporin 2; D051403:Aquaporin 6; D020346:Aquaporins; D003505:Cyclodextrins; D012076:Renal Agents; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D014667:Vasopressins; D012694:Serine; D002784:Cholesterol; D034285:Dynamin II