Two Vasopressin Type 2 Receptor Gene Mutations R143P and Delta V278 in Patients with Nephrogenic Diabetes Insipidus Impair Ligand Binding of the Receptor
| Title: | Two Vasopressin Type 2 Receptor Gene Mutations R143P and Delta V278 in Patients with Nephrogenic Diabetes Insipidus Impair Ligand Binding of the Receptor |
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| Authors: | Tsukaguchi, Hiroyasu; Matsubara, Hiroaki; Mori, Yasukiyo; Yoshimasa, Yasunao; Yoshimasa, Takaaki; Nakao, Kazuwa; Inada, Mitsuo |
| Publisher: | Biochemical and Biophysical Research Communications |
| Date Published: | June 26, 1995 |
| Reference Number: | 62 |
We recently identified vasopressin type 2 receptor gene mutations in two unrelated Japanese families with X-linked nephrogenic diabetes insipidus, which were a missense mutation from Arg143 to Pro (R143P) and a single amino acid deletion of Val278 or 279 (delta V278). To investigate the mechanism by which the mutations cause arginine vasopressin (AVP) resistance in this disorder, we expressed them in mammalian cells and analyzed their functional properties. Stable expression study with Chinese hamster ovary (CHO) cells demonstrated that the R143P mutation reduced receptor binding capacity to 10% of normal. However, the R143P mutant itself had a normal affinity for AVP and stimulated adenylyl cyclase production at up to 50% of the wild-type level, suggesting that the mutant receptors could function normally despite their reduced surface expression. In contrast, the delta V278 mutation totally abolished receptor-ligand binding and subsequent adenylyl cyclase stimulation, indicating that delta V278 mutant is virtually nonfunctional receptor. Northern blotting revealed that mutant CHO cell lines produced levels of receptor mRNA similar to the wild-type cell line. Our results suggest that the two mutations impair binding activity of the receptor without affecting mRNA accumulation, thereby causing AVP resistance through a posttranscriptional mechanism.
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)
Normally, AVP binds with V2R which is coupled to a stimulatory G-protein. This stimulates adenylyl cyclase which, in turn, increases the production of cAMP, an important metabolic regulator. This stimulates a process (that probably includes protein kinase A) that causes the water-transporting protein, aquaporin-2 (AQP2) to insert itself in the apex of the membranes of the principal cells of the kidney collecting ducts. The increased ability of the principal cells to transport water which results from the insertion is what allows the kidney to reabsorb water and concentrate urine.
To date, over 60 different mutations of the V2R gene that prevent this sequence from being completed have been discovered. A mutated gene can produce a defective protein, one that has a different structure than normal and therefore cannot perform its intended function effectively, if at all. Geneticists pay close attention to the structure-function relationship caused by mutations, and in the present article, Tsukaguchi, et al., report on their research into how two specific V2R gene mutations -- one labeled R143P and one labeled ^V278 --affect their respective V2Rs' structure and ability to function. They wanted to discover the mechanism by which these two mutations prevent the molecular sequence that results in the kidney concentrating urine.
To do so they expressed the mutant V2R strains in mammalian cell cultures and studied how they functioned. The authors found that the R143P mutant created V2Rs that still exhibited a normal attraction for AVP, and they were able to stimulate adenylyl cyclase production at up to 50% of normal V2Rs. This resulted in a significant increase in cAMP. The problem was the actual number of these V2Rs was drastically lower than the normal number of V2Rs that normally exist. These V2Rs had normal binding affinity with AVP despite a reduction of V2R number to about ten percent of normal. Their significantly decreased cell surface expression prevented them from functioning effectively.
In contrast, study of the V2Rs produced by the ^V278 mutant V2R gene revealed that these V2Rs were completely incapable of binding with AVP and therefore could not stimulate adenylyl cyclase at all. These V2Rs, then, were completely nonfunctional.
The authors state that their research results cannot fully explain how these mutations impair binding activity. They are currently studying to see if the impaired binding is due to a pure biding problem or an abnormality of the V2R synthesis or an inability of the defective V2R to travel to the cell surface.