Biochemical Basis of Partial Nephrogenic Diabetes Insipidus Phenotypes

Title: Biochemical Basis of Partial Nephrogenic Diabetes Insipidus Phenotypes
Authors: Bichet, Daniel G.; Robertson, Gary; Sadeghi, Hamid; Birnbaumer, Mariel; Innamorati, Giulio
Publisher: Molecular Endocrinology
Date Published: November 01, 1997
Reference Number: 151
Biochemical properties of mutant type 2 vasopressin receptors (V2Rs) causing a partial phenotype of nephrogenic diabetes insipidus were investigated in transiently transfected HEK 293 cells. Cell surface expression of the V2R was not altered by substituting Asp85 in the second transmembrane region by Asn as determined by saturation binding assays. Although the affinity of the mutant V2R for arginine vasopressin (AVP) was reduced only 6-fold, the response of adenylyl cyclase activity to AVP revealed a 50-fold right shift in EC50 and a decreased maximum response for the mutant V2R. These data indicated that replacement of Asp85 by Asn affected coupling of the receptor to Gs, a conclusion substantiated by a 20-fold decrease in the calculated coupling efficiency of this receptor. The Gly201Asp mutation in the second extracellular loop, also found associated with an NDI partial phenotype, decreased cell surface expression of the V2R with minor reduction in ligand-binding affinity and coupling efficiency to Gs. A pronounced difference was observed for this mutant V2R between the stimulation of adenylyl cyclase activity promoted by AVP and the V2 vasopressin receptor agonist deamino[Cys1,D-Arg8]-vasopressin, suggesting an involvement of Gly201 in the selectivity of the receptor for different ligands. These data demonstrated that while decreased ligand-binding affinity and decreased coupling to Gs are responsible for the attenuation of response to ligand in the Asp85Asn mutant V2R, cell surface expression of the V2R is the major factor reducing cellular responses to ligand for the Gly201Asp mutant V2R.
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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)

Polyuria (chronic, excessive passing of large amounts of urine) and polydipsia (chronic, excessive thirst) are two of the most obvious symptoms of nephrogenic diabetes insipidus (NDI). The most common form of inherited NDI is X-linked NDI, where females carry the mutated vasopressin-2 receptor (V2R) gene responsible for NDI and their male offspring have the highest chance to manifest the disease.

Normally, when V2R binds with the antidiuretic hormone, arginine vasopressin (AVP), the adenylyl cyclase system is stimulated via a Gs protein to increase the production of cAMP (an important metabolic regulator), and the enzyme protein kinase A. This begins a sequence that signals a water-transporting protein called aquaporin-2 (AQP2) to travel to and insert itself into the membranes of the principal cells of the kidney collecting duct. This makes the membranes much more water permeable, which allows the kidneys to reabsorb water and concentrate urine. In X-linked NDI, the V2R is unable to bind with AVP, so the urine concentrating process cannot begin. In most males afflicted with X-linked NDI, administering synthetic AVP, such as DDAVP, does not reduce the symptoms of NDI. It is not that the males lack proper amounts of AVP; it is that their V2Rs cannot bind with it.

Sadeghi, et al., investigated the biochemical properties of the V2R of four males with X-linked NDI. These four showed a partial response to DDAVP and, while subjected to dehydration through a period of water deprivation, their kidneys were able to produce concentrated urine, something that normally doesn't happen with an X-linked NDI patient. The researchers wanted to know what, in the nature of their subjects' mutations, allowed this to occur.

Genes are constructed of four simple nucleotide bases linked by sugar and phosphate side chains. The four bases are adenine (A), cytosine (C), guanine (G), and thymine (T). Each gene will have a unique sequence of the four bases (e.g., part of one gene base sequence could look like ATAGCGCCGTAT). The bases are decoded by the cell in sets of three (e.g., ATA), called codons. Each codon manufactures an amino acid, and together all the gene's codons produce all the amino acids required to produce the protein for which the gene is responsible. Mutations in the gene can result in the gene producing a defective protein.

There are different types of mutations, and the type of mutation all four of Sadeghi's, et al., subjects had was a missense mutation. A missense mutation is one that changes a codon so that it codes for a different amino acid than normal. Since proteins are made of amino acids, changing one of the amino acids will result in an altered protein.

Three of the males had the same mutation, a G324A mutation located in the second transmembrane region. The fourth had a G673A mutation of the V2R located at the second extracellular loop. (Please look at diagram of V2R.)

The researchers placed the mutant V2Rs in cell cultures in the laboratory and compared them with similarly placed normal V2Rs. They found the G324A mutated V2R's ability to bind with AVP was six-fold less than normal. However, it still could reach its mature level of development, and it could still travel to the cell surface. This is important because the cell surface is where V2R and AVP meet in order to bind. The G324A-mutated V2R was less efficient than the normal V2R in stimulating adenylyl cyclase, which suggests this V2R was less capable of coupling to the Gs protein, a necessary step in the urine concentrating process.

The G673A-mutated V2R could only get to the cell surface 25% of the time that fully functional V2Rs could. Its ability to bind with AVP and couple with the Gs protein was only slightly less than normal V2Rs, but its ability to stimulate adenylyl cyclase activity was far less than normal.

Why do these mutations allow a partial response to DDAVP and an increased AVP level in response to water deprivation? The researchers explain that the G324A mutation can express normal levels of V2Rs in the kidneys, but not bind as well to AVP or couple as well to the Gs protein. This situation could reduce response to normal levels of naturally occurring AVP enough to cause NDI. This deficiency could be overcome by the high levels of AVP induced by water deprivation or introduced by administration of DDAVP because the mutant V2Rs are at least on the cell surface in sufficient enough quantity to be there for the elevated AVP levels. And even though the G673A mutation's ability to stimulate cAMP production is impaired, it still produces at 70% of normal V2R levels and has a sufficient capacity to bind with AVP. This leaves sufficient receptors on the cell surface to allow the principal cell of the kidney collecting ducts to respond to elevated levels of AVP or DDAVP. The biochemical functioning of these mutations reinforces the researchers' previous conclusions as to the importance of the number of V2Rs per cell in determining the ability to respond to normal levels of circulating AVP.