Mutations in the Vasopressin V2 Receptor Gene in Families with Nephrogenic Diabetes Insipidus and Functional Expression of the Q-2 Mutant

Title: Mutations in the Vasopressin V2 Receptor Gene in Families with Nephrogenic Diabetes Insipidus and Functional Expression of the Q-2 Mutant
Authors: Rosenthal, Walter; Seibold, Anita; Antaramian, Anaid; Gilbert, Stephanie; Birnbaumer, Mariel; Bichet, Daniel G.; Arthus, Marie-Francoise; Lonergan, Michele
Publisher: Cellular and Molecular Biology
Date Published: May 01, 1994
Reference Number: 104
Nephrogenic diabetes insipidus (NDI) is characterized by a resistance of the kidney towards arginine vasopressin (AVP). Following molecular cloning of the vasopressin V2 receptor, we indentified different mutations in the V2 receptor gene in families with X-linked NDI, which segregated with the disease. The Hopewell mutation (W71X) causes the disease in the largest North American NDI pedigree, with most of its members residing on Nova Scotia. Different mutations were found in three families from the Quebec area (Q-2: R137H, Q-3 R113W, Q-5: 804delG) and in the large Cannon kindred residing in Utah (L312X). In an Iranian family (O-1), another mutation was detected (A132D). Three of the six mutations (Hopewell, Cannon, Q-5) are predicted to cause the expression of a truncated V2 receptor and are therefore unlikely to function. The functional consequences of missense mutations (Q-2, Q-3, O-1) are less obvious. We therefore introduced the Q-2 mutation into wild-type cDNA. When expressed in COS.M6 or Ltk cells, the Q-2 mutant bound AVP with normal affinity. However, cells expressing the Q-2 mutant failed to respond to AVP with an increase in adenylyl cyclase activity. Thus the Q-2 mutant is unable to interact with or to activate the stimulatory G-protein Gs. The present data indicate that X-linked NDI is frequently attributable to a mutation in the V2 receptor gene. In addition, the data prove biochemically that the Q-2 mutation is the cause of NDI in the Q-2 family.

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)

Nephrogenic diabetes insipidus (NDI) is a disorder characterized by the kidney's inability to respond to the antidiuretic hormone, arginine vasopressin (AVP). Because it cannot respond to AVP, the kidney is unable to maintain body water balance through reabsorbing water and concentrating urine. Normally, AVP will bind with the vasopressin-2 receptor (V2R) that is located in the cells of the kidney collecting duct. The V2R is coupled with a stimulatory G protein, and once it binds with AVP this combination of molecules stimulates the adenylyl cyclase system. This, in turn, leads to the increase of cAMP, an important metabolic regulator. cAMP activates the enzyme known as protein kinase A which leads to water-transporting proteins called aquaporin-2 (AQP2) inserting themselves in the apex of the membranes of the principal cells of the kidney collecting duct. The cells then let much more water travel through them than usual. So a significant percentage of the body water that is being filtered through the kidney via the nephrons is reabsorbed into the kidney inner tissues through the cells of the kidney collecting duct when they are made more water permeable by the insertion of AQP2s. The water that is not reabsorbed at this point is the concentrated urine that is later excreted.

Researchers exploring the molecular basis of NDI discovered that the V2R gene was located in the same area of the X chromosome that previous analysis of X-linked NDI inheritance patterns had located as the site of NDI. This meant that mutant V2R genes could be the molecular basis for X-linked NDI. In their article, Rosenthal, et al., report their discovery of six different V2R gene mutations in North American and Iranian families with NDI.

Imagine the V2R as a beaded string. Each bead is an amino acid synthesized by a codon (a set of three nucleotide bases within the V2R gene). Most of the V2R lies within the cell membrane in seven folded clumps called the transmembrane domains 1 - 7. (The cell membrane is a thin strip of tissue that encircles the cell, separating the inside of the cell from the outside.) Part of the V2R snakes outside the cell forming three loops called extracellular loops 1, 2 and 3. Part of it snakes inside the cell forming three loops called intracellular loops 1, 2 and 3. One end of the V2R, called the amino terminus, is outside the cell with the extracellular loops; the other end, called the carboxy terminus, sits inside the cell with the intracellular loops. (Please refer to V2R illustration.)

Researchers study the relation between the structure of the V2R and its ability to carry out its function. Mutations of the V2R gene cause alterations in the structures of the V2Rs they synthesize which affect the V2Rs' ability to carry out their function. Different V2R gene mutations produce different V2R structures which affect their ability to function in different ways.

Rosenthal, et al., examined the V2R gene mutation in the Hopewell family, the largest number of related NDI patients in North America. They found their mutation, labeled W71X, resulting in a receptor that does not have anything other than its first intracellular loop, its amino terminus and its first transmembrane domain. V2Rs whose length is so seriously shortened due to mutation are referred to as truncated V2Rs.

The three other mutations they found in the other NDI families were missense mutations. Missense mutations change a codon so that it codes for a different amino acid than normal. The functional consequences of these mutations was less obvious than the others, so the authors introduced the mutation in a piece of DNA (cDNA) encoding the V2 receptor. The cDNA was introduced into cells kept in culture. These cells synthesized the mutant receptor protein and allowed them to study the functional consequences of the mutation. They found that these V2Rs could express themselves on the cell surface (though in far less numbers than a normal V2R) and they could bind with AVP, but they could not stimulate adenylyl cyclase activity. The researchers speculate that this failure to increase adenylyl cyclase was because either the V2Rs could not interact with the G protein or activate it. Their research did, however, provide biochemical proof that this mutation was the cause of NDI in the family that carried it.

The authors reasoned that the mutation also hampered the ability of the V2Rs to express on the cell surface either because it interferes with the transport of the V2R to or the insertion of the V2Rs in the cell membrane. The low number of V2Rs on the cell surface could also have been due to problems in their synthesis, most likely a decreased stability of the mutant RNA.