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Association of Calnexin With Wild Type and Mutant AVPR2 that Cause Nephrogenic Diabetes Insipidus

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Title: Association of Calnexin With Wild Type and Mutant AVPR2 that Cause Nephrogenic Diabetes Insipidus
Authors: Morello, Jean-Pierre; Salahpour, Ali; Petaja-Repo, Ulla; Laperriere, Andre; Lonergan, Michele; Arthus, Marie-Francoise; Nabi, Ivan R.; Bichet, Daniel G.; Bouvier, Michel
Publisher: Biochemistry
Date Published: June 12, 2001
Reference Number: 511
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Abstract • Translation
Over 155 mutations within the V2 vasopressin receptor (AVPR2) gene are responsible for nephrogenic diabetes insipidus (NDI). The expression and subcellular distribution of four of these was investigated in transfected cells. These include a point mutation in the seventh transmembrane domain (S315R), a frameshift mutation in the third intracellular loop (804delG), and two nonsense mutations that code for AVPR2 truncated within the first cytoplasmic loop (W71X) and in the proximal portion of the carboxyl tail (R337X). RT-PCR revealed that mRNA was produced for all mutant receptor constructs. However, no receptor protein, as assessed by Western blot analysis, was detected for 804delG. The S315R was properly processed through the Golgi and targeted to the plasma membrane but lacked any detectable AVP binding or signaling. Thus, this mutation induces a conformational change that is compatible with endoplasmic reticulum (ER) export but dramatically affects hormone recognition. In contrast, the W71X and R337X AVPR2 were retained inside the cell as determined by immunofluorescence. Confocal microscopy revealed that they were both retained in the ER. To determine if calnexin could be involved, its interaction with the AVPR2 was assessed. Sequential coimmunoprecipitation demonstrated that calnexin associated with the precursor forms of both wild-type (WT) and mutant receptors in agreement with its general role in protein folding. Moreover, its association with the ER-retained R337X mutant was found to be longer than with the WT receptor suggesting that this molecular chaperone also plays a role in quality control and ER retention of misfolded G protein-coupled receptors.

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)

Calnexin is a subcellular molecular structure that works in the endoplasmic reticulum (ER), the quality control center of the cell. Misshapen proteins are not allowed out of the ER and are instead directed to that part of the cell that breaks down malformed proteins. Calnexin is believed to have a two-fold function in the ER:
  1. to help synthesizing proteins form their final shape, and
  2. to assist in quality control by directing proteins with improper or incomplete shapes to that part of the cell which will break them down.
Morello, et al., sought to determine if calnexin is involved in keeping malformed vasopressin 2 receptors (V2Rs) in the ER. To do this, they worked both with four V2R mutants known to be linked to NDI and with normal V2Rs. The researchers found that both the precursor forms of the normal and mutant V2Rs interacted with calnexin. The fact that calnexin was associated with the precursor and not the mature forms of the V2Rs is consistent with its hypothesized role as a helper to protein folding in the ER.

Calnexin is known to undergo a prolonged association with other misshapen proteins, and this is thought to play an important role in the misshapen proteins being retained in the ER. The researchers investigated as to whether calnexin played such a role in V2R mutations. They further studied the interaction between the mutant R337 V2R and calnexin. They found there was a longer interaction between calnexin and R337 than there was between calnexin and normal V2Rs. This suggests that calnexin plays a role in quality control and ER retention of misshapen V2Rs. The researchers propose that the molecular mechanisms involved in the cell's quality control system could lead to new therapeutic avenues for treating NDI.