Reconstitution of Mutant V2 Vasopressin Receptors by Adenovirus-mediated Gene Transfer

Line
Title: Reconstitution of Mutant V2 Vasopressin Receptors by Adenovirus-mediated Gene Transfer
Authors: Schoneberg, Torsten; Sandig, Volker; Wess, Jurgen; Gudermann, Thomas; Schultz, Prof. Dr. Med. Gunter
Publisher: Journal of Clinical Investigation
Date Published: September 15, 1997
Reference Number: 153
Line
Abstract • Article • Translation
Recent studies with transfected COS-7 cells have shown that functionally inactive mutant V2 vasopressin receptors (occurring in patients with nephrogenic diabetes insipidus) can be functionally rescued by coexpression of a carboxy-terminal V2 receptor fragment (V2-tail) spanning the region where various mutations occur [Schoneberg, T., J. Yun, D. Wenkert, and J. Wess. 1996. EMBO (Eur. Mol. Biol. Organ.) J. 15:1283-1291]. In this study, we set out to characterize the underlying molecular mechanism. Using a coimmunoprecipitation strategy and a newly developed sandwich ELISA system, a direct and highly specific interaction between the mutant V2 vasopressin receptor proteins and the V2-tail polypeptide was demonstrated. To study the potential therapeutic usefulness of these findings, Chinese hamster ovary (CHO) cell lines stably expressing low levels of functionally inactive mutant V2 vasopressin receptors were created and infected with a recombinant adenovirus carrying the V2-tail gene fragment. After adenovirus infection, vasopressin gained the ability to stimulate cAMP formation with high potency and efficacy in all CHO cell clones studied. Moreover, adenovirus-mediated gene transfer also proved to be a highly efficient method for achieving expression of the V2-tail fragment (as well as the wild-type V2 receptor) in Madin-Darby canine kidney tubular cells. Taken together, these studies clarify the molecular mechansims by which receptor fragments can restore function of mutationally inactivated G protein-coupled receptors and suggest that adenovirus-mediated expression of receptor fragments may lead to novel strategies for the treatment of a variety of human diseases.
The publisher has not granted permission to reproduce this article on our website. You may, however, read this article at the Journal of Clinical Investigation website. In order to view this document, you will need Acrobat Reader. If you do not already have Acrobat Reader or need to upgrade, click here.
To return to this page, use your "back" key.

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)

G Protein-coupled receptors (GPCRs) form one of the largest protein families in nature. They live within some cell membranes, that thin band that separates the inside of the cell from the outside. Their primary function is to bind with a specific hormone or neurotransmitter and transmit its message to the molecular structure (the effector) that will begin to carry the hormone's message out. If you think of a GPCR as a string, most of it sits within the cell membrane in seven distinct clumps called transmembrane helices. Part of it snakes outside the cell forming three curves called extracellular loops. Part of it curves inside the cell forming three curves called the intracellular loops. The beginning of the string is outside the cell; the tail is inside the cell. (Look at diagram of GPCR.)

Mutations may occur at any point in a GPCR, and it is now known that mutations in GPCRs can result in human disease. Nephrogenic diabetes insipidus (NDI) is such a disease. It is characterized by the kidney's inability to concentrate urine, and its primary symptoms include chronic, excessive thirst and urination. The most common form of inherited NDI is called X-linked NDI, and though females carry the mutated gene responsible for the disorder, it is males who, for the most part, manifest it. In this form of NDI, the vasopressin receptor (V2R), a member of the GPCR family, cannot bind to the antidiuretic hormone, vasopressin (VP). The result of this is that the message of vasopressin, which is to tell the kidney to begin the process of concentrating urine, is not delivered to the effector, resulting in a water balance disorder manifesting as NDI.

Working in the laboratory with cell cultures, Schoneberg, et al., discovered that they could get certain V2Rs, which due to mutations had lost their ability to function, to work again. They did this by finding the portion of the V2R where the mutations were, which in this case was in the tail end of the V2R. In fact the nature of the mutation was such that it resulted in the tail of the V2R being cut short (truncated). Then they took non-mutated tails of other V2Rs and mixed them with a virus that would carry them to where the mutated V2Rs were in the cell. The researchers found that when both the mutated V2Rs and the non-mutated V2R fragments were present together (co-expressed), then the mutated V2R was able to perform a significant amount of its function. This is something it could not do alone in its mutated state. What the authors wanted to know through their current research was the underlying molecular mechanism that allowed this restoration of function to occur.

They found that the non-mutated V2R fragment, called the V2-tail, directly interacted with the mutated V2R. That the V2-tail would associate directly with the mutant V2R, but not with other classes of GPCRs containing similar mutations (truncated tails) showed the highly specific nature of this interaction.

Finding this co-expression strategy worked in the specific cell line they used in the laboratory (the COS cell line), the researchers wanted to test its therapeutic potential by carrying out the strategy on select animal cell lines. Toward that end, they used Chinese hamster ovary (CHO) cell lines that had low levels of functionally inactive mutant V2 receptors (again, with truncated tails). They infected these cells with a virus carrying the healthy V2-tail gene fragment. The researchers found that the CHO cells that were thus infected showed a marked increase in their ability to generate an important metabolic regulator called cAMP9, a function dependent on the V2R being able to bind with vasopressin (VP). This indicated that the V2Rs in the infected cells had much of their ability to function restored. This was because in their mutated state they could not bind with VP, therefore the cAMP generation process could not be initiated.

Next the researchers wanted to know if their co-expression strategy could be used in the cells of kidney tubes because in diseases like NDI, V2Rs, mutated or not, live in select cells in the kidneys. They used a cell line of dog kidney (the little tubes that help filter water and other bodily fluids). They found that after the cells were infected with the V2-tail fragments and incubated, the cells demonstrated a 9.4 fold increase in cAMP levels, again indicating that the mutated V2Rs' ability to function properly had been restored. This strategy, then, holds therapeutic potential and should be further tested and explored.