Vasopressin increases S261 phosphorylation in AQP2-P262L, a mutant in recessive nephrogenic diabetes insipidus

Abstract

Background. Mutations in the aquaporin-2 (AQP2) gene cause nephrogenic diabetes insipidus (NDI), a renal disorder characterized by polyuria due to a lacking antidiuretic response to vasopressin. While most AQP2 mutants in recessive NDI are misfolded and retained in the endoplasmic reticulum, AQP2-P262L in NDI was impaired in its vasopressin-dependent translocation from vesicles to the plasma membrane. Methods. Vasopressin-induced translocation of AQP2 coincides with AQP2 phosphorylation at S256, S264 and T269 and dephosphorylation at S261. Since P262 lies adjacent to S261, we tested whether a changed phosphorylation could underlie AQP-P262L missorting in NDI. Results. In polarized cells, AQP2-P262L expressed as a double 29/30 kDa band, whereas wt-AQP2 expressed only as a 29 kDa band. Phosphatase treatment revealed that the 30 kDa AQP2-P262L band was due to changed phosphorylation. The use of newly developed phospho-specific antibodies showed that forskolin not only increased pS256 and pT269, but, in contrast to wt-AQP2, also pS261 in AQP2-P262L. The expression of AQP2-P262L proteins in which S261 phosphorylation was prevented (S261A), however, was still missorted to vesicles/basolateral membrane, despite the absence of the 30 kDa band. Conclusions. Together, our data reveal that vasopressin induces instead of reduces the phosphorylation of S261 in AQP2-P262L, but it remains to be established whether the changed phosphorylation causes its missorting in NDI.

Small-Molecule Screening Identifies Modulators of Aquaporin-2 Trafficking

“…Because 4AD inhibits only the S256 phosphorylation, our data corroborate the many earlier studies in providing further evidence that the S256 phosphorylation is the crucial trigger for the membrane insertion of AQP2. [7][8][9]15,25 Our data also show that the cAMP-dependent dephosphorylation at S261 and the phosphorylation at S269 are independent from the S256 phosphorylation by PKA. Tamma et al found that the cAMP/PKA-dependent S261 phosphorylation also occurs in the AQP2-S256A mutant, which cannot be phosphorylated at S256, and that forskolin induces dephosphorylation of S261 in this mutant.…”

“…In line with these observations, 4AD prevented the forskolin-induced increase of AQP2 phosphorylation at S256 (AQP2-pS256) and S269 (AQP2-pS269) at the plasma membrane ( Figure 3). The reason for the nuclear staining with the anti-pS269 antibody in the absence of forskolin is unclear; it also occurred with a custom-made antibody 25 (data not shown), suggesting that the antibodies cross-react with a nuclear protein containing a similar epitope. The effective concentrations of 4AD did not induce any visible signs of toxicity in MCD4 or IMCD cells (24-hour toxicity assay) (Supplemental Figure 3B).…”

“…AQP2-pS256 was detected with phospho-specific custom-made polyclonal rabbit antibody (Eurogentec Deutschland GmbH, Germany; 1:200 and 1:25, respectively) and antirabbit Cy3-coupled secondary antibodies. 19,25 AQP2-pS269 was detected with phospho-specific primary antibody (p112-269, Phosphosolutions, Aurora, CO) and antirabbit Cy3-coupled secondary antibodies. All secondary antibodies were purchased from Dianova (Hamburg, Germany) and used in a dilution of 1:600.…”

“…AQP2-pS261 was detected with phospho-specific polyclonal phospho-S261 rabbit antibody (Abcam; Cambridge, UK). AQP2-pS256 was detected with phospho-specific custom-made polyclonal rabbit antibody (see above), 19,25 and AQP2-pS269 with a commercially available antibody (Phosphosolutions, see above). We detected a-tubulin with a CalBiochem antibody (CP06).…”

Negative feedback from CaSR signaling to aquaporin-2 sensitizes vasopressin to extracellular Ca2+

“…AQP2-pS256 antibodies were a kind gift from Peter Deen (Department of Physiology, Radboud University Medical Center, Nijmegen, The Netherlands) and has been described previously (Trimpert et al, 2012). AQP2-pS261 antibodies were purchased from Novus Biological (DBA, Milan, Italy).…”