Molecular Aspects of Vasopressin Receptor Function
| Title: | Molecular Aspects of Vasopressin Receptor Function |
|---|---|
| Authors: | Schoneberg, Torsten; Kostenis, Evi; Liu, Jie; Gudermann, Thomas; Wess, Jurgen |
| Publisher: | Advances in Experimental Medicine and Biology |
| Date Published: | 1998 |
| Reference Number: | 485 |
The molecular mechanisms governing the G protein coupling selectivity of different members of the vasopressin receptor family were studied by using a combined molecular genetic/biochemical approach. While the V1a and V1b vasopressin receptors are selectively linked to G proteins of the Gq/11 class, the V2 vasopressin receptor is preferentially coupled to Gs. Systematic functional analysis of V1a/V2 hybrid receptors showed that the second intracellular loop of the V1a receptor is required and sufficient for efficient coupling to Gq/11, whereas the third intracellular loop of the V2 receptor is required and sufficient for coupling to Gs. By using a strategy involving the coexpression of the wild type V1a receptor with chimeric G protein alpha s/alpha q subunits, two C-terminal alpha q/11 residues were identified that are critical for proper receptor recognition. We previously demonstrated -in transiently transfected COS-7 cells- that selected mutant V2 vasopressin receptors (all of which have been identified in X-linked nephrogenic diabetes insipidus patients) containing inactivating mutations in the C-terminal third of the receptor protein (including missense, frameshift, or nonsense mutations) can be functionally rescued by coexpression with a C-terminal V2 receptor fragment (V2-tail) spanning the region where the various mutations occur. co-immunoprecipitation experiments and a newly developed sandwich ELISA revealed that the V2-tail polypeptide directly interacts with the mutant V2 receptors thus creating a functional receptor protein. To study the potential therapeutic usefulness of these findings, CHO cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors (E242stop, Y280C, and W284stop) were created and infected with a recombinant adenovirus coding for the V2-tail polypeptide. Following adenovirus infection, arginine vasopressin (AVP) gained the ability to stimulate cAMP formation in all CHO cell clones studied. Adenovirus-mediated gene transfer also proved to be a highly efficient method to achieve expression of the V2-tail fragment (as well as of the wild type V2 vasopressin receptor) in MDCK renal tubular cells. We therefore speculate that the targeted expression of receptor fragments in vivo may represent a novel strategy in the treatment of human diseases caused by inactivating mutations in distinct G protein-coupled receptors.
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)
- They normally cannot bond with the antidiuretic hormone, arginine vasopressin (AVP).
- They often are unable to stimulate the Gs adenyly cyclase system.
- They often are unable to even get to the proper location of the cell membrane where they must be if they are to perform their job.
Researchers have a clear idea of what a normal V2R looks like, and they know over 60 different types of V2 gene mutations that result in XNDI. They also know the locations of many of the structural defects on mutant V2R that result in V2R functional impairments.
A normal V2R protein is a string of 371 amino acids. Much of this string is bunched in seven distinct clumps called transmembrane helices one through seven (TM 1 - 7). Part of the V2R snakes outside the cell membrane into the extracellular environment to form three curves called extracellular loops 1 - 3. Part of the V2R snakes inside the cell to form three curves called intracellular loops 1 - 3. One end of the protein, called the amino terminus, is located outside the cell with extracellular loops 1 - 3. The other end, called the carboxy terminus, is inside the cell with the intracellular loops. (You can look at a diagram of V2R here.)
Schoneberg, et al. demonstrated that the V2R's third intracellular loop is required and sufficient for coupling the Gs protein. In other words,the V2R requires an intact third intracellular loop if it is to be able to couple with the Gs protein, an important part of its job. The authors further demonstrated that certain mutant V2R whose inactivating mutations were located on the V2-tail (the section of the V2R beginning just before the midpoint of the third intracellular loop and extending to the end of the carboxy terminus) could actually be made to work again when they were expressed in a laboratory cell culture along with just the V2-tail segment of a normal V2R.
Tests revealed that the healthy V2-tail segments interacted directly with the mutant V2Rs to create a functional V2R. The 'rescue' V2-tail directly associate with the different mutant V2R and had no significant effect on receptors other than the V2R. In short, the rescue V2-tails recognized and interacted with mutant V2Rs with a high degree of specificity. These tests were performed in a laboratory cell culture in which the V2Rs were introduced and were able to express for a short time. To test whether the findings derived from these tests could have any therapeutic value in living systems. The next step was for the authors to perform these tests in cell cultures in which the V2Rs could be more stably expressed.
Schoneberg, et al., were able to do this using a recombinant adenovirus to introduce the rescue V2-tail and normal V2Rs into cells. They created cell cultures that could stably express low levels of mutant V2Rs incapable of performing their function. It is important to note that they were able to do this with several different mutant V2Rs. Nonetheless, all of these mutants were restored to nearly full function when the researchers introduced functional V-2 tail fragments into the cell culture. Finally, the authors demonstrated that V2R and the rescue V2-tails could be introduced into kidney cells and once introduced, distribute themselves into the right positions within the cell. To do this, they successfully expressed the rescue V2-tails and V2Rs into a kidney cell line generated from dogs. This cell line shares many functional characteristics with the human collecting duct cells, the area where V2Rs are expressed in human beings.
The authors speculate that introducing selected V2-tails into targeted areas of NDI patients could be a promising treatment approach.