The hormone arginine vasopressin (AVP) regulates body water balance by mediating plasma membrane expression of the water channel aquaporin-2 (AQP2) in kidney epithelial principal cells. Membrane accumulation is regulated by AVP induced phosphorylation at S256 and S269. Both events favour membrane retention of the protein, a process that can involve altered protein:protein interactions (PPIs). In contrast, ubiquitination at K270 mediates AQP2 internalization. We are examining the role of acute posttranslational modifications (PTM) on AQP2 trafficking. In double mutated AQP2 expressing cells, AQP2-256D/269A and AQP2- 256D/269D, the protein predominantly localized in the plasma membrane, whereas 256A/269A-AQP2 and 256A/269D-AQP2 had an intracellular localization. This suggests that pS256 is dominant in determining AQP2 plasma membrane localization. AQP2-256D/269A or -256D/269D expressing cells had decreased AQP2 internalization compared to AQP2-WT; with 256D/269D-AQP2 being internalized slowest. This suggests that altered PPIs may modulate each site individually. Treatment of AQP2-WT cells with phosphatase inhibitors resulted in a decreased rate of AQP2 endocytosis. In parallel, pS269-AQP2 levels were increased and prolonged, whereas only minor changes in pS256-AQP2 were observed. Various AQP2 mutants with reduced endocytic rates were ubiquitinated following either forskolin or combined forskolin and TPA treatment. Phosphatase inhibitor treatment generally increased levels of ubiquitination of AQP2-WT. The ubiquitin deficient mutant AQP2- 270R had similar S256 and S269 phosphorylation levels as AQP2-WT. Our data indicate that: 1) Phosphorylation at S269 is likely a major PTM involved in slowing down AQP2 endocytosis, 2) S269-phosphorylation and K270-ubiquitination can occur in parallel, and 3) under certain conditions, S269-phosphorylation mediated membrane retention can override the ubiquitin signal for endocytosis.