Binding-, Intracellular Transport-, and Biosynthesis-Defective Mutants of Vasopressin Type 2 Receptor in Patients with X-Linked Nephrogenic Diabetes Insipidus

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Title: Binding-, Intracellular Transport-, and Biosynthesis-Defective Mutants of Vasopressin Type 2 Receptor in Patients with X-Linked Nephrogenic Diabetes Insipidus
Authors: Tsukaguchi, Hiroyasu; Matsubara, Hiroaki; Taketani, Shigeru; Mori, Yasukiyo; Seido, Tsutomu; Inada, Mitsuo
Publisher: Journal of Clinical Investigation
Date Published: October 01, 1995
Reference Number: 53
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Nephrogenic diabetes insipidus (NDI) is most often an X-linked disorder in which urine is not concentrated due to renal resistance to arginine vasopressin. We recently identified four vasopressin type 2 receptor gene mutations in unrelated X-linked NDI families, including R143P, delta V278, R202C, and 804insG. All these mutations reduced ligand binding activity to < 10% of the normal without affecting mRNA accumulation. To elucidate whether the receptors are expressed on the cell surface, we analyzed biosynthesis and localization of tagged or untagged receptors stably expressed in Chinese hamster ovary (CHO) cells, using two antibodies directed against distinct termini. Whole-cell and surface labeling studies revealed that the R202C clone had both surface-localized (50-55 kD) and intracellular proteins (40 and 75 kD), similar to the wild-type AVPR2 clone, whereas the R143P and delta V278 clones lacked the surface receptors, despite relatively increased intracellular components. The 804insG mutant cell produced no proteins despite an adequate mRNA level. Immunoflourescence staining confirmed that the R202C mutant reaches the cell surface, whereas the R143P and delta V278 mutants are retained within the cytoplasmic compartment. Thus, R202C, R143P/delta V278, and 804insG result in three distinct phenotypes, that is, a simple binding impairment at the cell surface, blocked intracellular transport, and ineffective biosynthesis or/and accelerated degradation of the receptor, respectively, and therefore are responsible for NDI. This phenotypic classification will help understanding of molecular pathophysiology of this disorder.
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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)

Tsukaguchi, et al., identified four V2R gene mutations associated with nephrogenic diabetes insipidus (NDI) in four unrelated Japanese families: a R143P, a ^V278, a R202C and an 804insG mutation. Tsukaguchi, et al., analyzed how the mutational defects in the individual V2R genes affected the biological function of the V2Rs they produced.

The authors found that all four mutations reduced the ability of their respective V2Rs to bind with the antidiuretic hormone, arginine vasopressin (AVP)by 90%. If V2R cannot bind with AVP, AVP cannot initiate the molecular sequence which allows the kidney to reabsorb water, concentrate urine and thus balance body water. The result of this failure to bind is NDI.

There could be different reasons the four V2R gene mutations either greatly reduced or completely eliminated their respective V2Rs from binding with AVP. One possibility was that the mutations affected the V2R genes' ability to synthesize their V2Rs. If they could not do this, then there would be no V2Rs to bind with AVP. To find out if this was so, the researchers analyzed the mutant genes' biosynthesizing abilities in laboratory cell cultures. Another reason for the low binding rates could be that the V2Rs could not transport to the cell membranes where the AVP/V2R binding takes place. The authors also tested for this.

They found that the R202C mutated V2R was located both on the cell surface and within the cell, much like normal V2R. The R143P and ^V278 V2Rs, on the other hand, were not found on the cell surface, though they were located inside the cell. And the 804insG mutant V2R could not be located at all, which meant the 804insG mutant V2R gene, despite having an adequate mRNA level, could not synthesize any V2Rs.

Another level of analysis confirmed that the R202C mutant reaches the cell surface, whereas the R143P and ^V278 are kept within the cell, away from the cell surface. So the researchers found that although all their NDI patients expressed the same symptom: polyuria (chronic passage of large amounts of urine) and polydipsia (chronic, excessive thirst), and though these symptoms all were due to mutated V2R genes, the different mutations of the V2R genes produced V2Rs that dysfunctioned in different ways on the molecular level.

The researchers' analysis showed that V2R mutations can be classified into at least three distinct phenotypes, or modes of expression. The first phenotype, exemplified by the R202C mutant, is where the V2R can reach the cell surface, but it loses its binding capacity. The authors speculate that this phenotype fails to bind because it fails to achieve the proper shape required for binding with AVP. The second phenotype, as exemplified by R143P and ^V278, is when the receptors are retained within the cell and cannot get to the cell surface to bind with AVP. The third phenotype, exemplified by 804insG, is when either the mutant gene cannot synthesize a receptor or it can synthesize a receptor, but that receptor rapidly degrades before it can perform its function.

The authors state that use of their phenotypic classification will serve as a powerful tool to promote a clearer understanding of the structure-function relationship of V2R, an understanding which will lead to improved therapy for NDI.