V2 Vasopressin Receptor Dysfunction in Nephrogenic Diabetes Insipidus Caused By Different Molecular Mechanisms
| Title: | V2 Vasopressin Receptor Dysfunction in Nephrogenic Diabetes Insipidus Caused By Different Molecular Mechanisms |
|---|---|
| Authors: | Schoneberg, Torsten; Schulz, Angela; Biebermann, Heike; Gruters, Annette; Grimm, Torsten; Hubschmann, Klaus; Filler, M.D., Ph.D., FRCPC, Guido; Gudermann, Thomas; Schultz, Prof. Dr. Med. Gunter |
| Publisher: | Human Mutation |
| Date Published: | January 01, 1998 |
| Reference Number: | 219 |
Loss-of-function mutations in the V2 vasopressin receptor (AVPR2) gene have been identified as a molecular basis for X-linked nephrogenic diabetes insipidus (NDI). Herein, we describe a novel deletion mutation at nucleotide position 102 (delG102) found in a Russian family resulting in a frameshift and a truncated receptor protein. Furthermore, we analyzed the AVPR2 gene of two other unrelated boys with NDI from our patient clientele. These patients showed previously described mutations (R137H, R181C). In-depth characterization of the three mutant AVPR2s by a combination of functional and immunological techniques permitted further insight into molecular mechanisms leading to receptor dysfunction. Premature truncation of the AVPR2 (delG102) led to a drastically reduced receptor protein expression in transfected COS-7 cells and, as expected, precluded specific AVPR2 functions. As indicated by different ELISA and binding studies, the R137H mutant was almost completely retained in the cell interior. In contrast to previous studies, the few mutant receptors in the plasma membrane displayed a low (2.3-fold above basal) but significant ability to stimulate the Gs/adenylyl cyclase system. In contrast to the latter mutation, the R181C mutant is properly delivered to the cell surface but the mutation interferes with high affinity vasopressin binding. Impaired ligand binding is reflected in an about 100-fold shift of the concentration-response curve toward higher vasopressin concentrations with only slightly reduced agonist potency.
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)
First, the AVPR2 travels from its holding place inside the CD principal cells to its work site in the basolateral membrane of the CD principal cells. There, it binds with AVP. AVP/AVPR2 then stimulates the Gs/adenylyl cyclase system. (The Gs is a stimulatory G protein to which AVPR2 is coupled. Adenylyl cyclase (AdC) is an enzyme.) The stimulated Gs/AdC system elevates cellular levels of cyclic adenosine monophosphate (cAMP). cAMP signals water transporting proteins to insert themselves in the apical membrane of the CD principal cells. This makes the apical membrane much more water permeable than usual, which is how the kidney can reabsorb the body water flowing through the CD and concentrate urine.
The AVPR2 must perform a number of functions in order for this urine concentrating sequence to occur. It must:
1. be able to travel to the basolateral membrane;
2. have an affinity for AVP;
3. be able to couple with the Gs protein.
X-linked nephrogenic diabetes insipidus (NDI) is a hereditary type of NDI. NDI patients have kidneys which can't respond to AVP. Therefore, their kidneys can't reabsorb body water flowing through the kidney CDs, and they can't concentrate urine. As a result, NDI patients experience polyuria (chronic passage of large volumes of urine) and polydipsia (chronic, excessive thirst). NDI has been identified in different ethnic groups worldwide.
X-linked NDI is caused by mutations in the AVPR2 gene, the gene that encodes for AVPR2. Over 150 different AVPR2 gene mutations associated with X-linked NDI have been identified. Each of these mutations alters the structure of its AVPR2 in a different way. Oftentimes, when the structure of an AVPR2 is altered, its ability to function is impaired. Different structural alterations impair the function of AVPR2s in different ways. The extent and severity of the functional impairment is dependent on the location and extent of the structural alteration.
Each gene is composed of a unique sequence of four nucleotide bases (adenine (A), cytosine (C), guanine (G) and thymine (T)). The gene's sequence of nucleotide bases is decoded by the cell, which reads the sequence in sets of three, which are called codons (e.g. CGC ACG TCA CGT). Each codon encodes for one amino acid. So, the cell helps synthesize the string of amino acids that comprises the protein based upon the sequence of codons within the gene. When a gene is mutated, part of its code may be out of order. For example, the codon CGC may instead be CCC. This altered sequence will produce a different amino acid than normal, and this change in the protein's structure may cause a change in its ability to function.
Schoneberg, et al., carefully evaluated the AVPR2 mutations of three unrelated X-linked NDI patients. Patient 1, an 8-year-old Russian boy, had a AVPR2 gene mutation not previously described in NDI literature. This boy's AVPR2 gene was missing a G nucleotide base in the 34th codon. This single deletion in the gene sequence resulted in the development of the boy's AVPR2s being cut short (truncated) well before they reached their normal structure. This mutation was named delG102.
The normal AVPR2 has 371 amino acids strung together like beads on a string. A portion of the AVPR2 lies within the cell membrane, that thin strip of tissue that encircles the cell, separating the cell from the environment. That portion lies in seven coiled folds called transmembrane helices 1 - 7. Part of the AVPR2 snakes outside the membrane into the outside of the cell to form three curved loops called extracellular loops 1 - 3. Part of it snakes into the interior of the cell to form three curved loops called intracellular loops 1 - 3. One end of the AVPR2, called the amino-terminus, lies outside the cell with the extracellular loops. The other end, called the carboxy-terminus, lies inside the cell with the intracellular loops. (For a visual representation of the AVPR2, please see V2R.)
Patient 2's mutation was named R137H. It had been previously identified in other NDI patients. This mutation substituted an A nucleotide for a G, resulting in an arginine amino acid in the AVPR2 where a histidine amino acid should have been. Patient 3's mutation (R181C) substituted a T nucleotide where a C should have been. This mutation has also been previously identified in NDI patients.
Having clearly delineated the structure of each mutation, the authors cloned them and introduced them into laboratory cell cultures to see how the structural alterations affected functional abilities. delG102 led to a premature truncation of the AVPR2s; they were missing all their transmembrane helices, extracellular and intracellular loops, and their carboxy-terminals. This led to impaired expression of the AVPR2 on the cell membrane. Recent studies show that AVPR2s must have at least 341 amino acids to properly travel to the basolateral membrane.
Most of these AVPR2s produced by the R137H mutant were retained within the cell inside their holding site, the endoplasmic reticulum (ER). Those few AVPR2s that did make it to the cell surface retained a significant, but not complete, ability to stimulate the Gs/AdC system. The R181C mutant AVPR2s were able to travel to the cell membrane, but once there, they did not show a high affinity with AVP, and therefore, the proper level of AVPR2/AVP binding could not take place.
The authors suggest that such detailed functional characterization of AVPR2 mutations may, at some point, offer novel therapeutic approaches in the treatment of X-linked NDI.