Missorting of the Aquaporin-2 mutant E258K to multivesicular bodies/lysosomes in dominant NDI is associated with its monoubiquitination and increased phosphorylation by PKC but is due to the loss of E258

Abstract

To stimulate renal water reabsorption, vasopressin induces phosphorylation of Aquaporin-2 (AQP2) water channels at S256 and their redistribution from vesicles to the apical membrane, whereas vasopressin removal results in AQP2 ubiquitination at K270 and its internalization to multivesicular bodies (MVB). AQP2-E258K causes dominant nephrogenic diabetes insipidus (NDI), but its subcellular location is unclear, and the molecular reason for its involvement in dominant NDI is unknown. To unravel these, AQP2-E258K was studied in transfected polarized Madin-Darby canine kidney (MDCK) cells. In MDCK cells, AQP2-E258K mainly localized to MVB/lysosomes (Lys). Upon coexpression, wild-type (wt) AQP2 and AQP2-E258K formed multimers, which also localized to MVB/Lys, independent of forskolin stimulation. Orthophosphate labeling revealed that forskolin increased phosphorylation of wt-AQP2 and AQP2-E258K but not AQP2-S256A, indicating that the E258K mutation does not interfere with the AQP2 phosphorylation at S256. In contrast to wt-AQP2 but consistent with the introduced protein kinase C (PKC) consensus site, AQP2-E258K was phosphorylated by phorbol esters. Besides the 29-kDa band, however, an additional band of about 35 kDa was observed for AQP2-E258K only, which represented AQP2-E258K uniquely monoubiquitinated at K228 only. Analysis of several mutants interfering with AQP2-E258K phosphorylation, and/or ubiquitination, however, revealed that the MVB/lysosomal sorting of AQP2-E258K occurred independent of its monoubiquitination or phosphorylation by PKC. Instead, our data reveal that the loss of the E258 in AQP2-E258K is fundamental to its missorting to MVB/Lys and indicate that this amino acid has an important role in the proper structure formation of the C-terminal tail of AQP2.

Repulsion between Lys258 and upstream arginines explains the missorting of the AQP2 mutant p.Glu258Lys in nephrogenic diabetes insipidus

“…Altogether, these data (and those reported [Kamsteeg et al, 2008]) reveal that E258 itself is essential in AQP2 sorting and that any change of E258 targets AQP2 to MVB/lysosomes. Moreover, our data also show that the conserved glutamic acid may have a role in sorting of AQP1 and AQP5, but not of AQP4, to the plasma membrane.…”

“…1A). Because the replacement of E258 in AQP2 by Ala, Gln, Lys, or Arg results in MVB sorting as shown by almost complete colocalization with Lamp2 [Kamsteeg et al, 2008], we explored the effect of substitutions for the glutamic acids in AQP1, 4, and 5. For readability, the mutants created by in vitro mutagenesis described in the remainder of the document are given at the amino acid level (one-letter code) only.…”

“…In our search to determine the cause for missorting of AQP2-E258K to MVBs, we showed that missorting of AQP2-E258K is not due to the lack of S256 phosphorylation or to its different form of ubiquitination as compared to wt-AQP2 [Kamsteeg et al, 2008;Mulders et al, 1998]. Here, however, we discovered that missorting of AQP2-E258K is partially caused by the charge-based repulsion between the patient's mutation (Lys258) and a triplet of arginines in a novel 8-amino acid motif around Lys258.…”

“…Although the mutant AQP2 proteins all oligomerize with wt-AQP2, their missorting is diverse. AQP2-E258K is missorted to late multivesicular bodies (MVBs) [Hirano et al, 2003;Kamsteeg et al, 2008;Procino et al, 2003;Robben et al, 2006], p.His260fs to the basolateral plasma membrane [Kamsteeg et al, 2003], p.Arg254Leu to recycling vesicles [De Mattia et al, 2005] and the 11 frame-shift mutants reveal a combination of basolateral and lysosomal sorting combined with rapid degradation [Asai et al, 2003;Marr et al, 2002]. The p.His260fs mutant is missorted due to the introduction of basolateral sorting signals and the p.Arg254Leu mutant because of its inability to become phosphorylated by PKA, thereby remaining vesicular.…”

Endocytic regulation of alkali metal transport proteins in mammals, yeast and plants

“…Interestingly, defects in MVB have been associated with several kidney diseases, indicating the pivotal role of this system in regulating ion and water fluxes. Mutant alleles of the aquaporin, AQP2, which accumulate in the MVB instead of the plasma membrane, cause nephrogenic diabetes insipidus (Kamsteeg, et al 2008). Moreover, defects in MVB formation are used as biomarkers of focal segmental glomerulosclerosis (Kim, et al 2003).…”