Reversed Polarized Delivery of an Aquaporin-2 Mutant Causes Dominant Nephrogenic Diabetes Insipidus

Title: Reversed Polarized Delivery of an Aquaporin-2 Mutant Causes Dominant Nephrogenic Diabetes Insipidus
Authors: Kamsteeg, Erik-Jan; Bichet, Daniel G.; Konings, Irene B. M.; Nivet, Hubert; Lonergan, Michele; Arthus, Marie-Francoise; van Os, Carel; Deen, Peter M.T.
Publisher: Journal of Cell Biology
Date Published: December 08, 2003
Reference Number: 639
Vasopressin regulates body water conservation by redistributing aquaporin-2 (AQP2) water channels from intracellular vesicles to the apical surface of renal collecting ducts, resulting in water reabsorption from urine. Mutations in AQP2 cause autosomal nephrogenic diabetes insipidus (NDI), a disease characterized by the inability to concentrate urine. Here, we report a frame-shift mutation in AQP2 causing dominant NDI. This AQP2 mutant is a functional water channel when expressed in Xenopus oocytes. However, expressed in polarized renal cells, it is misrouted to the basolateral instead of apical plasma membrane. Additionally, this mutant forms heterotetramers with wild-type AQP2 and redirects this complex to the basolateral surface. The frame shift induces a change in the COOH terminus of AQP2, creating both a leucine- and a tyrosine-based motif, which cause the reversed sorting of AQP2. Our data reveal a novel cellular phenotype in dominant NDI and show that dominance of basolateral sorting motifs in a mutant subunit can be the molecular basis for disease.

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This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

The majority of cases of congenital NDI that are caused by mutations in the aquaporin-2 (AQP2) gene are inherited in an autosomal recessive manner. That is, both parents must contribute a mutated AQP2 allele. (The gene of any particular location on a chromosome can exist in any of various forms, called alleles.) However, a minority of cases caused by AQP2 gene mutations are inherited in a dominant manner. That is, only one parent need contribute a mutated AQP2 gene for NDI to occur in their offspring.

Upon stimulation from a molecular sequence initiated by the hormone, arginine vasopressin (AVP), a normal AQP2 protein is able to travel from the cell interior to the cell’s apical membrane. Once inserted there, it allows water to pass through the membrane. The apical membrane is that section of the cell membrane that is at the top pole of the cell. The basolateral section of the membrane is the base and sides of the cell.

Kamsteeg, et al., were presented with an extended family in which 7 members had inherited NDI caused by an AQP2 mutation inherited in a dominant fashion. Analysis of the AQP2 genes of 5 of the patients revealed 3 mutations in the AQP2 allele. The mutated AQP2 gene’s effect was to extend the tail end of the AQP2 protein (the C-terminus) synthesized from it. The research team referred to the mutant protein as AQP2-insA.

The researchers conducted a series of experiments with AQP2–insA using different laboratory cell cultures in order to determine how AQP2–insA exerted its dominant effect. They discovered AQP2–insA could function as a water channel. That is, it could let water flow through cell membranes. But when tested in epithelial cell cultures (the kidney collecting ducts cells where AQP2 occurs are epithelial cells), the AQP2–insA did not move to the apical membrane upon stimulation. Instead, they moved to the basolateral membrane.

The researchers then combined AQP2–insA with normal AQP2s in the cell culture and observed that the mutant AQP2s would combine with the normal AQP2s. When this occurred, the combined form of AQP2–insA and normal AQP2 would be directed toward the basolateral membrane. This explains how the patients only had to receive the mutated AQP2 gene from one parent, as the AQP2 protein that would result from it would combine with normal AQP2s produced from the normal AQP2 gene inherited from the other parent and the combined AQP2/ AQP2–insA would be directed not to the apical membrane where it should go, but to the basolateral membrane.

Further study indicated that AQP2–insA is directed to the basolateral membrane because its C-terminus, which was much longer than normal due to the addition of amino acids, contained amino acids that acted as two signals that signaled the cell to direct it to the basolateral membrane. Further, AQP2–insA has a sequence of amino acids in its C-terminus referred to as the YXXO motif that induces a decrease in its expression on the cell surface. Refining their observations yet further, the researchers discovered there was a leucine based and a tyrosine amino acid based motif in the AQP2–insA which cause the basolateral sorting of AQP2–insA.