Novel mutation in the AVPR2 gene in a Danish male with nephrogenic diabetes insipidus caused by ER retention and subsequent lysosomal degradation of the mutant receptor

Abstract

Mutations in the arginine vasopressin receptor 2 (AVPR2) gene can cause X-linked nephrogenic diabetes insipidus (NDI) characterized by the production of large amounts of urine and an inability to concentrate urine in response to the antidiuretic hormone vasopressin. We have identified a novel mutation in the AVPR2 gene (L170P) located in the fourth transmembrane domain in a Danish NDI male. Analysis of the mutant receptor in Madin-Darby Canine Kidney cell culture revealed that AVPR2-L170P was retained in the endoplasmic reticulum, and the expression was dramatically downregulated compared to wild-type AVPR2. Inhibition of the lysosome resulted in increased intracellular accumulation of AVPR2-L170P, indicating that AVPR2-L170P is downregulated via the lysosome. Inhibition of the proteasome resulted in plasma membrane localization of AVPR2-L170P, although the overall levels of AVPR2-L170P were unchanged.

Fig. 1.

Pedigree of NDI relatives. Open circles, women; open squares, men. Roman numerals, the generation; circles with a central dot, obligatory carriers; closed squares, affected subjects. A slash (/) through a symbol indicates that the subject is deceased. The proband is shown by an arrow. The year of birth is beneath the subject.

Fig. 2.

(A) Wild-type AVPR2-GFP transiently expressed in MDCK cells and stained with the Golgi marker G58K. Wild-type AVPR2-GFP localizes to cell–cell contacts of MDCK cells and to the Golgi region. (B). AVPR2-L170P-GFP transiently expressed in MDCK cells and stained with the Golgi marker G58K. AVPR2-L170P-GFP does not seem to localize to the Golgi compartment. (C) AVPR2-L170P-GFP transiently expressed in MDCK cells and stained with the ER marker calreticulin. AVPR2-L170P-GFP localizes to intracellular structures with some overlap with the ER marker calreticulin. (D) AVPR2-L170P-GFP transiently expressed in MDCK cells. Cells were loaded with Lysotracker Red before fixation, which shows that cells expressing AVPR2-L170P-GFP had larger lysosomes than surrounding cells, although AVPR2-L170P-GFP did not colocalize, which could be due to degradation of the GFP-tag in lysosomes. Please note that samples B, C and D have been stained with antibody against the GFP tag since they were otherwise not bright enough for imaging. Scale bar is 10 μm.

Fig. 3.

(A) Representative images of AVPR2-L170P-GFP stably expressed in MDCK cells 16 h after induction with tetracycline followed by 4 h of incubation with DMSO or chloroquine (100 μM). There is a marked accumulation of AVPR2-L170P-GFP in cells treated with chloroquine compared to controls (DMSO). Moreover, following chloroquine treatment, AVPR2-L170P-GFP is localized in larger structures, which colocalize with the lysosomal marker Cathepsin D. (B) Western blot analysis of equal amount of protein from cells stably expressing AVPR2-L170P-GFP, either not induced or induced by tetracycline, and transiently expressing wild-type AVPR2-GFP probed with a GFP antibody. A 68-kDa band was observed in all samples as well as a 46-kDa band corresponding to the lysosomal degradation product. The 68-kDa band was decreased in intensity and the 46-kDa degradation product was increased in intensity in AVPR2-L170P-GFP compared to wild-type AVPR2-GFP; however, this was reversed by inhibition of lysosome degradation by chloroquine. Moreover, a broad 57-kDa band in wild-type AVPR2-GFP was completely absent from AVPR2-L170P-GFP. Cells not induced by tetracycline still show significant levels of AVPR2-L170P-GFP, indicating a leaky promoter. Scale bar is 10 μm.

Fig. 4.

(A) Representative images of AVPR2-L170P-GFP stably expressed in MDCK cells 16 h after induction with tetracycline followed by 4 h of incubation with DMSO or MG-132. There is a marked relocalization of AVPR2-L170P-GFP from the ER to the plasma membrane following inhibition of the proteasome with MG-132 compared to DMSO. (B) Representative western blot analysis using GFP antibodies. MDCK cells stably expressing AVPR2-L170P-GFP induced by tetracycline followed by a 4-h treatment with either DMSO or the proteasome inhibitor MG-132. A 68-kDa band corresponding to the non-glycosylated form of AVPR2-L170P-GFP was present in all lanes as well as a 46-kDa band presumed to be a degradation product. There was no significant difference in the intensity of the bands between DMSO and MG-132-treated cells. Scale bar is 10 μm.