2004 Global Researcher Conference Proceeding

April 09 - 11, 2004

Conference: 2004 Global Researcher Conference
Title: Action Mechanism of Pharmacological Chaperones Acting on the V2 Vasopressin Receptor in the Treatment of Nephrogenic Diabetes Insipidus
Authors: Bernier, Virginie; Pontier, Stephanie M.; Charest, PG; Bichet, Daniel G.; Bouvier, Michel
Institutions: Universite de Montreal, Hopital du Sacre-Coeur de Montreal
Bernier Conformational disease often results from modest mutations in proteins which are then recognized by the endoplasmic reticulum (ER) quality control system as misfolded. These misfolded proteins interact for a longer period of time with ER molecular chaperones, are ubiquitinated and degraded through the proteasome. Recently, many studies have suggested that small lipophillic molecules, termed pharmacological chaperones, can stabilize mutant proteins, facilitating their export from the ER and their trafficking to the proper site of action. In the present study, we have used a mutant form of the V2 vasopressin receptor (W164S V2R) that causes nephrogenic diabetes insipidus (NDI) to explore the action mechanism of pharmacological chaperones. Immunofluorescence microscopy revealed that the W164S V2R expressed in HEK293T cells are retained in the ER. Lowering the cell growth temperature facilitated surface trafficking indicating that the ER retention resulted from receptor misfolding. Bioluminescence Resonance Energy Transfer (BRET) studies showed that the mutant receptor could still dimerize indicating that the maturation process was interrupted downstream of dimer formation. Treatment with the pharmacological chaperone SR121463 significantly increased cell surface expression and function of W164S V2R. This rescue was accompanied by a more efficient maturation and a reduced ubiquitination of the mutant receptor, indicating that a smaller proportion was targeted to the proteasome. Pulse chase experiments also revealed that the SR121463 treatment decreased the interaction time between W164S V2R and the ER molecular chaperone, calnexin, indicative of a better folding process. Similarly, the treatment with SR121463 reduced the interaction of the mutant receptor with the ER export protein, Dopamine Receptor interacting Protein (DRiP78) suggesting a more efficient export process. Taken together, these results confirm that pharmacological chaperone act early in the ER quality control system to facilitate folding and export of mutant proteins. A better understanding of the mechanisms involved should facilitate the development of new therapeutic approaches for patients suffering of NDI and other conformational diseases.

Pharmacological chaperone is a term given to a class of molecules that can help misshapen proteins leave the endoplasmic reticulum (ER), which is a quality control system inside the cell. Bernier, et al., studied the effect of SR121463, a pharmacological chaperone, on a mutant V2R called W164S V2R. This mutant is misshapen and it is normally held in the ER and then dissolved by the cell. Thus, it never gets to the cell membrane to perform its function.

The researchers found that SR121463 significantly increased the mutant’s ability to reach the cell membrane and perform its function. The chaperone was accompanied by greater maturation and a reduced ubiquitination of the mutant. That indicates that a smaller portion of it was directed towards the enzyme that would dissolve it. The chaperone also reduced the amount of time the mutant V2R interacted with the proteins in the ER, indicating that the chaperone helped the mutant V2R to achieve a more normal shape and be released from the ER more efficiently. They conclude that SR121463 initiated its helpful action early in the ER quality control system to help the mutant V2R form a more normal shape and thus be able to leave the ER to travel to the cell membrane.