Pharmacochaperones Post-Translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-Type and Mutant Vasopressin V2 Receptors

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Title: Pharmacochaperones Post-Translationally Enhance Cell Surface Expression by Increasing Conformational Stability of Wild-Type and Mutant Vasopressin V2 Receptors
Authors: Wuller, Stefan; Wiesner, Burkhard; Loffler, Anja; Furkert, Jens; Krause, Gerd; Hermosilla, Ricardo; Schaefer, Michael; Schulein, Ralf; Rosenthal, Walter; Oksche, Alexander
Publisher: Journal of Biological Chemistry
Date Published: August 19, 2004
Reference Number: 660
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Abstract • Article • Translation
Some membrane-permeable antagonists restore cell surface expression of misfolded receptors retained in the endoplasmic reticulum (ER) and are therefore termed pharmacochaperones. Whether pharmacochaperones increase protein stability, thereby preventing rapid degradation, or assist folding via direct receptor interactions or interfere with quality control components remains elusive. We now show that the cell surface expression and function (binding of the agonist) of the mainly ER-retained wild-type murine vasopressin V2 receptor.GFP fusion protein (mV2R.GFP) is restored by the vasopressin receptor antagonists SR49059 and SR121463B with EC50 values similar to their KD values. This effect was preserved when protein synthesis was abolished. In addition, SR121463B rescued eight mutant human V2Rs (hV2Rs; three are responsible for nephrogenic diabetes insipidus) characterized by amino acid exchanges at the C-terminal end of transmembrane helix TMI and TMVII. In contrast, mutants with amino acid exchanges at the interface of TM II and IV were not rescued by either antagonist. The mechanisms involved in successful rescue of cell surface delivery are explained in a 3D homology model of the antagonist-bound hV2R.
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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 hormone, arginine vasopressin (AVP) initiates the kidney?s water balancing and urine concentrating activity by binding to the vasopressin 2 receptor (V2R). A majority of the cases of congenital NDI are caused by the V2R being either unable or unavailable to bind to AVP due to mutations in the V2R gene.

Most of the V2R gene mutations (there are over 150 of them) result in mutant V2Rs that are unable to transit from a compartment in the cell called the endoplasmic reticulum (ER). They are misfolded (subtly or grossly, depending on the V2R gene mutation blueprints from which the V2R are synthesized) and therefore can?t pass the quality control mechanisms in the ER. Retained in the ER, the mutant V2Rs are then dismantled by the cell. It is important to note that a significant number of these V2R mutants might be able to bind AVP if they were able to escape the ER. If there were some way of rescuing them from the ER, they could bind with AVP, possibly allowing the kidney to function normally.

An antagonist is a substance that tends to nullify the action of an agonist (stimulating substance). For example, an AVP antagonist would bind to the V2R, leaving no place on the V2R for AVP to bind. However, some antagonists are able to restore a mutated receptor?s ability to reach that area of the cell where it must be if it is to perform its function. It does this by helping the mutated receptor escape the ER. This class of antagonists is called pharmacochaperones.

How they are able to do this is not certain. They may act on the mutant protein by making the protein more stable and therefore more resistant to the cell?s dismantling machinery. They may prevent the mutant proteins from becoming significantly misshapen by interacting with them in the early stages of their synthesis to ensure they will be properly shaped as a mature protein. Or they may interfere with the ER?s quality control mechanism.

Wuller, et al., executed a series of experiments using laboratory cell cultures, in which mouse V2Rs (which are predominantly retained within the ER as immature proteins) or human mutant V2Rs were synthesized in order to help clarify the mechanisms whereby pharmacochaperones rescue ER retained V2Rs. They found that vasopressin receptor antagonists SR49059 and SR121463B restored the ability of the mouse V2R to leave the ER, travel to the cell membrane and there, perform its functions. SR121463B was also able to rescue eight human mutant V2Rs.

The research team was able to distinguish the differences in amino acid placement within the studied human V2R mutant that either enabled or prevented successful rescue by the antagonist. Further, the team was able to clarify that the antagonist achieved its rescue by increasing the stability of the shape of the mutant V2Rs by directly interacting with them in their status as a mature protein. That is, after they had been fully synthesized.

Mutants which can be rescued by SR121463B only have amino acid replacements (i.e., a different amino acid than is normally present at a specific location in the sequence of amino acid residues that make up the V2R) in the terminal (facing the cytosol) parts of the protein segments called transmembrane 1 and 7 (see diagram).