An impaired routing of wild-type aquaporin-2 after tetramerization with an aquaporin-2 mutant explains dominant nephrogenic diabetes insipidus

Abstract

Autosomal recessive and dominant nephrogenic diabetes insipidus (NDI), a disease in which the kidney is unable to concentrate urine in response to vasopressin, are caused by mutations in the aquaporin-2 (AQP2) gene. Missense AQP2 proteins in recessive NDI have been shown to be retarded in the endoplasmic reticulum, whereas AQP2-E258K, an AQP2 mutant in dominant NDI, was retained in the Golgi complex. In this study, we identified the molecular mechanisms underlying recessive and dominant NDI. Sucrose gradient centrifugation of rat and human kidney proteins and subsequent immunoblotting revealed that AQP2 forms homotetramers. When expressed in oocytes, wild-type AQP2 and AQP2-E258K also formed homotetramers, whereas AQP2-R187C, a mutant in recessive NDI, was expressed as a monomer. Upon co-injection, AQP2-E258K, but not AQP2-R187C, was able to heterotetramerize with wild-type AQP2. Since an AQP monomer is the functional unit and AQP2-E258K is a functional but misrouted water channel, heterotetramerization of AQP2-E258K with wild-type AQP2 and inhibition of further routing of this complex to the plasma membrane is the cause of dominant NDI. This case of NDI is the first example of a dominant disease in which the 'loss-of-function' phenotype is caused by an impaired routing rather than impaired function of the wild-type protein.

The Proteasome Is Involved in the Degradation of Different Aquaporin-2 Mutants Causing Nephrogenic Diabetes Insipidus

“…29 In the present study we confirm the Golgi localization of E258K AQP2 by using double confocal immunofluorescence to detect the Golgi marker mannosidase II. However, by using additional double confocal immunofluorescence microscopy to detect the late endosome/ lysosome marker LAMP 1, co-distribution of E258K AQP2 with LAMP 1 could be unequivocally demonstrated in the clone 9 hepatocytes.…”

“…The reason was that mixed tetramer formation between wt and E258K AQP2 has been reported that resulted in the retention of the wt protein and causing dominant NDI. 24,29 As shown in Figure 2B, AQP9 RNA was undetectable in clone 9 hepatocytes in contrast to rat liver. Thus, the possible formation of mixed tetramers composed of wt AQP9 and E258K AQP2 can be excluded.…”

Glycosylation Is Important for Cell Surface Expression of the Water Channel Aquaporin-2 but Is Not Essential for Tetramerization in the Endoplasmic Reticulum

“…Miscellaneous Methods-Methods for cRNA injection in Xenopus oocytes, water permeability measurements, immunofluorescence microscopy, cell surface biotinylation, and velocity gradient centrifugation have been described previously (17,19,28). Confocal laser scanning immunofluorescence microscopy of MDCK cells was done as described previously (29).…”

“…AQP2 was detected by immunoprecipitation of the gradient fractions and quantitated by phosphorimaging. Previous studies validated this method to separate AQP2 monomers and tetramers (19). Already after 30 min of labeling, most of AQP2 distributed in the high density fractions of the gradient, corresponding to a position where tetrameric AQP2 sediments.…”

“…18) through two putative mechanisms. Recessive nonfunctional mutants are thought to be retained in the ER and fail to tetramerize, whereas dominant AQP2 mutants oligomerize with the product of the wild-type allele, forming tetramers that are improperly targeted and do not reach the apical cell surface (19,20). Given the consequences of mutant AQP2 alleles for the health of affected individuals, surprisingly little is known about biosynthetic maturation and sorting of AQP2.…”