Aminoglycoside Pretreatment Partially Restores the Function of Truncated V2 Vasopressin Receptors Found in Patients with Nephrogenic Diabetes Insipidus

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Title: Aminoglycoside Pretreatment Partially Restores the Function of Truncated V2 Vasopressin Receptors Found in Patients with Nephrogenic Diabetes Insipidus
Authors: Schulz, Angela; Sangkuhl, Katrin; Lennert, Thomas; Wigger, Marianne; Price, David Anthony; Nuuja, Anja; Gruters, Annette; Schultz, Prof. Dr. Med. Gunter; Schoneberg, Torsten
Publisher: Journal of Clinical Endocrinology and Metabolism
Date Published: November 01, 2002
Reference Number: 570
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By screening patients with X-linked nephrogenic diabetes insipidus (NDI) for mutations within the V(2) vasopressin receptor (AVPR2) gene, we have identified six novel and two recurrent mutations. Additionally, one patient revealed a genomic deletion of 3.2 kb encompassing most of the AVPR2 gene and the last exon/3'-region of C1 gene, which is in close proximity to the AVPR2 locus. In-depth characterization of the mutant AVPR2s by a combination of functional and immunological techniques allowed to gain further insight into molecular mechanisms leading to the receptor dysfunction. Aiming at the functional reconstitution of mutant G protein-coupled receptors, several strategies of potential therapeutic usefulness have been tested. Because the functional rescue of truncated receptors is most challenging, we addressed this issue by applying an aminoglycoside approach. Here, we demonstrate that the misreading capacity of the aminoglycoside antibiotic geneticin was sufficient to restore function of mutant AVPR2s harboring premature stop codons in an in vitro expression system.
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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 arginine vasopressin 2 receptor (AVPR2) is a protein. It is comprised of a string of amino acid residues and functions as a receptor that the hormone, vasopressin (AVP), binds to. It can happen that the AVPR2 gene, which helps produce the AVPR2 protein, is mutated. When this happens, the AVPR2 is often structurally, and thus functionally, impaired. This is one of the molecular bases for Nephrogenic Diabetes Insipidus (NDI).

Schulz, et al., screened the AVPR2 gene of 12 NDI patients, discovering 8 mutations, two of which had never been previously reported in NDI literature. Two of the mutations were of the type called nonsense mutations. This type of mutation causes a premature termination of protein synthesis, and this results in a truncated protein, one that does not achieve its full length or its proper form, and thus is missing some of the amino acid residues that are present in the normal AVPR2 protein. A truncated protein generally cannot perform the function for which it was intended because its structure is incapable of doing so.

Schulz, et al., attempted to restore the function of the two truncated AVPR2 mutations they found in their screening. They did this by adding geneticin, a member of the aminoglycoside family of antibiotics, to laboratory cell cultures containing AVPR2 genes. The reason a nonsense mutation results in a truncated protein is that one of the three terminator codons that is used to signal the end of the protein appears in the middle of the synthesizing process. When it is read, the synthesizing stops prematurely. Geneticin can increase the frequency of misreading of these codons, thereby permitting the protein synthesizing process to continue to the normal end of the protein.

Both of the nonsense mutants gained some functional ability in the presence of geneticin. They were able to initiate a molecular sequence that normal AVPR2s do, and there was a larger number of the mutants reaching the normal length of an AVPR2. Interestingly, the AVPR2 proteins generated from one of the geneticin treated mutant AVPR2 genes were less potent than normal AVPR2s, whereas the other achieved normal potency. The researchers speculated that the efficiency of the geneticin treatment strategy is dependent on the precise position of the mutation within the AVPR2 gene. A further cause for notice was that although there was a significant restoration of function in one of the mutant AVPR2 genes, the AVPR2 proteins did not sufficiently appear in the cell membranes. And this is where they must be if they are to perform their function as a receptor for AVP.

Finally, the research team noted that aminoglycosides are toxic and thus not suitable for long term use at their present stage of development.