2004 Global Researcher Conference Proceeding
April 09 - 11, 2004
|Conference:||2004 Global Researcher Conference|
|Title:||Differences between ER-retained vasopressin V2 receptor mutants in antagonist-mediated restoration of cell surface expression|
|Authors:||Wuller, Stefan; Wiesner, Burkhard; Krause, Gerd; Rosenthal, Walter; Oksche, Alexander|
|Institutions:||University Hospital of the RWTH Aachen, Forschunginstitut fur Molekulare Pharmakologie (FMP), Charite - Universitatsmedizin Berlin, Institut Fuer Pharmakologie|
About 50% of nephrogenic diabetes insipidus (NDI)-causing mutations in the vasopressin V2 receptor (V2R) gene code for single amino acid replacements (missense mutations). In most cases the encoded mutant V2Rs are misfolded and retained in the ER. Cell permeable antagonists can restore cell surface expression, but their mechanism of action is still not clear. Therefore we studied the effects of SR121463B and SR49059 (V2R and V1R-specific antagonists) on ER-retained V2Rs transiently expressed in HEK293 cells. In the case of the wild-type murine V2R (mV2R), which is mainly localized in the ER in untreated controls, both antagonists promoted a time - and dose - dependent restoration of the cell surface expression. SR49059-mediated restoration of cell surface expression of the mV2R was accompanied by a dramatic increase in [3H]AVP binding sites. An SR49059-mediated increase in [3H]AVP binding was also observed for primary cultured rat inner medullary collecting duct cells endogenously expressing the V2R.
For human NDI-causing mutants (hL62P, hDLAR 62-64, hH80R, hW164R, hS167T, hS167L, hC319Y, hP322S) and several in vitro mutants (hD136A, hD368K/S371X, hF328A) transiently expressed in HEK cells also predominant ER retention was observed. Interestingly, cell surface expression of hDLAR 62-64, hD136A, hS167T, hP322S, and hF328A was only restored by SR121463B. In the case of the mutants hL62P, hH80R, hW164R, hS167L, and hD368K/S371X none of the two antagonist restored cell surface expression. Only for the mutant hC319Y both antagonists promoted cell surface expression as found for the mV2R. The data show that ER-retained mutant V2Rs differ in their sensitivity to antagonist-promoted cell surface expression. It is likely that these differences can be attributed to the extent of the folding defect and/or an alteration of the binding pocket. Further studies are required to analyze the general applicability of antagonists in the treatment of NDI.
Around half of the V2R gene mutations that result in NDI lead to V2R proteins where, out of the long chain of amino acids comprising the protein, there is just one single amino acid error. The result is a misshapen protein that is retained in the endoplasmic reticulum (ER), a structure within the cell where quality control mechanisms take place.
There are molecular structures called antagonists that can bind to proteins. Certain antagonists can bind to mutant V2Rs and help them to escape from the ER to the cell membrane, though how they do it is unclear. Wüller, et al., studied two antagonists (SR121463B and SR49059) that bind to normal and mutant V2Rs. Tests with laboratory cell cultures revealed that both antagonists are able to help retained V2Rs to the cell surface, and that for certain V2Rs a significant increase in AVP binding sites occurred.
The researchers tested the effects of the 2 antagonists on 11 human V2R mutants in laboratory cell cultures. Five of the mutants were able to escape from the ER with one of the antagonists, SR121463B, so that they could reach the cell surface. Another five of the mutants showed no effect to the treatment with either antagonist. Only one was capable of being restored to the cell surface by both antagonists. The V2R mutants differed in their sensitivity to the treatment with the antagonists, most likely due to different degrees of receptor misfolding.