Expression Studies of Two Vasopressin V2 Receptor Gene Mutations, R202C and 804insG, in Nephrogenic Diabetes Insipidus
| Title: | Expression Studies of Two Vasopressin V2 Receptor Gene Mutations, R202C and 804insG, in Nephrogenic Diabetes Insipidus |
|---|---|
| Authors: | Tsukaguchi, Hiroyasu; Matsubara, Hiroaki; Inada, Mitsuo |
| Publisher: | Kidney International |
| Date Published: | August 01, 1995 |
| Reference Number: | 57 |
Nephrogenic diabetes insipidus (NDI) is a rare X-linked disorder associated with renal tubule resistance to arginine vasopressin (AVP). To understand the mechanisms of AVP resistance underlying this disorder, we have analyzed the vasopressin V2 receptor gene in two unrelated Japanese kindreds with NDI and expressed the mutants to characterize their functional properties. Direct sequencing revealed two V2 receptor gene mutations: a missense mutation from Arg202 to Cys in the third extracellular domain (R202C) and a single base insertion (G) in two consecutive GGG triplets (nucleotide 804 to 809) in the third cytoplasmic domain, resulting in a frame shift with premature termination at codon 258 (804insG). Transient expression study with COS-7 cells showed that R202C mutation reduced both binding affinity (15%) and capacity (30%), while 804insG mutation abolished binding ability. For further evaluation of the binding ability of the R202C mutant, we expressed the mutants in Chinese hamster ovary (CHO) cells. Although the mutant cell lines produced V2 receptor mRNA comparable levels to the wild-type receptor cell lines, R202C mutant cell lines had no binding ability. Our results suggest an introduction of a new cysteine residue in the extraceullular domain and a receptor truncation removing one third of the carboxyl terminus could impair ligand binding activity of the V2 receptor through a post-transcriptional mechanism, thereby causing AVP resistance in the NDI patients.
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)
In the kidney, V2R is located within the membrane of principal cells of the kidney collecting duct and inner medulla. The membrane is that thin strip of tissue encircling the cell, separating it from its environment. If you imagine the V2R as a beaded string, the major part of it sits inside the membrane in seven folded clumps called transmembrane domains. Part of the V2R snakes outside the cell to form three curves called extracellular loops 1, 2 and 3. Part of it snakes inside the cell to form three curves called intracellular loops 1,2 and 3. One end of the V2R, called the amino-terminus, sits outside the cell with the extracellular loops, and the other end, called the carboxy-terminus, sits inside the cell with the intracellular loops. (You can look at an example of V2R here.)
All genes are comprised of nucleotide bases linked by sugar-phosphate side chains. There are only four nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T). And the way they combine determines the gene. For example, a small part of one gene sequence could read GATCATGACCAG. The bases are decoded by the cell in sets of three (e.g. GAT) called codons. Each codon is responsible for manufacturing one amino acid. When all the codons are normal they produce the normal amino acids which make up the normal protein, in this case a normal V2R. When there is a faulty sequence in the V2R gene, it could result in a defective V2R.
Tsukaguchi, et al., analyzed the V2R gene mutations of two unrelated Japanese NDI patients. They expressed clones of their defective V2Rs in laboratory cell cultures in order to better understand how the structure of the defective V2Rs influence their ability to perform their function: binding with AVP. One patient had a missense mutation in his V2R gene. A missense mutation changes a codon (one of the sets of three nucleotide bases) so that it produces a different amino acid than normal. In this case, the missense mutation was located in the third extracellular domain and called a R202C mutation. This mutation changed the Arg202 amino acid to the amino acid, cysteine. The second patient had a frameshift mutation consisting of a single base insertion (a guanine) in two consecutive GGG codons in the third intracellular domain. This resulted in the premature termination of the V2R at codon 258 so the V2R was missing the last third of its carboxy-terminus. This mutation was labeled 804insG.
When the R202 mutant was expressed in a laboratory cell culture it lost about 70 to 85% of its binding capacity and attraction to AVP. When the 804insG truncated V2R was similarly expressed it was found to have lost all its binding activity. When the R202C mutant was expressed in a different laboratory cell culture it showed no binding capacity.
The authors speculated that the first laboratory cell culture the R202C was expressed in could produce a higher number of V2Rs at the cell level than the second cell culture. But in both cases it was clear that the R202C could not effectively bind with AVP.
The authors' study also showed the two mutants had normal mRNA levels. This indicates that the mutations disrupted the V2Rs' ability to bind to AVP after the point where the V2R gene makes a complementary sequence of itself. However, it remains unclear how the defective V2R is produced or if it reaches the cell membrane - the place where it must get to if it is to bind with AVP.
Finally, the authors note that most of the missense mutations that occur in the third extracellular domain of the V2R are substitutions that incorporate the amino acid cysteine. The human V2R has two cysteine residues (the portion of cysteine that remains after it combines with another amino acid) in the second and third extracellular domains, and these may be critical to the structural or functional integrity of the receptors. These cysteine residues form bands between the two extracellular loops which may shape the V2R so it is able to bind with AVP. So perhaps an additional cysteine loop produced by a mutation interferes with the shaping of the V2R so it cannot bind with AVP. The authors believe that as more V2R mutations are identified it will produce further insights into the V2R structure-function relationship. And this might eventually enable researchers to find the key amino acid for receptor function and thus enable science to affect an effective therapeutic strategy to treat NDI.