Functional Rescue of the Constitutively Internalized V2 Vasopressin Receptor Mutant R137H by the Pharmacological Chaperone Action of SR49059

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Title: Functional Rescue of the Constitutively Internalized V2 Vasopressin Receptor Mutant R137H by the Pharmacological Chaperone Action of SR49059
Authors: Bernier, Virginie; Lagace, Monique; Lonergan, Michele; Arthus, Marie-Francoise; Bichet, Daniel G.; Bouvier, Michel
Publisher: Molecular Endocrinology
Date Published: May 27, 2004
Reference Number: 652
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In most cases, nephrogenic diabetes insipidus results from mutations in the V2 vasopressin receptor gene that cause intracellular retention of improperly folded receptors. We previously reported that cell permeable V2 vasopressin receptor antagonists act as pharmacological chaperones that rescue folding, trafficking and function of several V2 vasopressin receptor mutants. More recently, the vasopressin antagonist, SR49059, was found to be therapeutically active in nephrogenic diabetes insipidus patients. Three of the patients with positive responses harbored the mutation R137H, previously reported to lead to constitutive endocytosis. This raises the possibility that instead of acting as a pharmacological chaperone by favoring proper maturation of the receptors, SR49059 could mediate its action on R137H V2 vasopressin receptor by preventing its endocytosis. Here we report that the betaarrestin-mediated constitutive endocytosis of R137H V2 vasopressin receptor is not affected by SR49059, indicating that the functional rescue observed does not result from a stabilization of the receptor at the cell surface. Moreover, metabolic labeling revealed that R137H V2 vasopressin receptor is also poorly processed to the mature form. SR49059 treatment significantly improved its maturation and cell surface targeting, indicating that the functional rescue of R137H V2 vasopressin receptors results from the pharmacological chaperone action of the antagonist.
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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)

The majority of the vasopressin 2 receptor (V2R) gene mutations that result in NDI produce V2R proteins that are improperly folded. That is, the shape of the protein is different than normal. Because of this, the mutant V2Rs are retained in the endoplasmic reticulum (ER), a quality control system inside the cell. As they are retained in the ER because they are not properly folded, the mutant V2Rs are unable to travel to the cell membrane, which is where they need to be to perform their function of binding with the hormone, arginine vasopressin (AVP).

In biology, an antagonist is a substance that tends to nullify the action of another substance. It often does this by filling the position or space that the original substance normally does. For example, a V2R antagonist will bind with AVP, thereby depriving the V2R of the opportunity to do so. The V2R antagonist will bind with AVP, but will not allow the molecular sequence that occurs when V2R binds with AVP. Bernier, et al., had previously reported that certain V2R antagonists were actually capable of acting as a type of pharmacological chaperone that could restore specific V2R mutants? ability to bind with AVP. They did this by helping fold the developing mutant V2Rs into a more normally shaped V2R. This allowed them to exit the ER and travel to the cell membrane.

The vasopressin antagonist, SR49059, was found to have a significant therapeutic effect on the NDI patients on whom it was tested. This was indicated by the patients? increased water absorption and reduced urine output. The tested NDI patients had different V2R gene mutations, most known to produce V2R proteins that were misfolded and retained in the ER.

However, three patients bore a mutated V2R gene that produced the mutant V2R protein, R137H. Though the patients showed the same improvement as the others, there was some question as to whether SR49059 acted as a pharmacological chaperone because R137H had been reported to act differently than other V2R mutants. Specifically, R137H acts in a manner that leads a process called constitutive endocytosis. This means that the V2R protein can?t be stabilized on the cell membrane and is returned to the cell interior before it can do its job of binding with AVP. Because the return to the cell interior is constantly triggered without reliance on a hormonal signal to do so (i.e., the return to the cell interior is constitutive), the fact that SR49059 is therapeutic for patients with the R137H mutant may indicate that SR49059 may produce its therapeutic affect by preventing the mutant V2R from inappropriately returning to the cell interior. If, in fact, SR49059 did stabilize the mutant V2R at the cell membrane, it would mean it could exert its therapeutic effects in two ways: as a pharmaceutical chaperone and as an agent that prevented constitutive endocytosis by stabilizing the R137H mutant at the cell membrane.

Bernier, et al., designed a series of experiments to test whether SR49059 exerted its therapeutic effect on R137H by preventing its constitutive endocytosis. Their research indicated that SR49059?s therapeutic effect did not result from an ability to stabilize the R137H V2R on the cell membrane surface. R137H is misshapen and does not get to the cell membrane. R137H cell cultures treated with SR49059 showed that it helped R137H achieve a more normal shape and improved its ability to reach the cell surface. Hence, even in this case, SR49059 acted as a pharmacological chaperone to affect its therapeutic action in R137H.