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Association of calnexin with wild type and mutant AVPR2 that causes nephrogenic diabetes insipidus
J P Morello1, A Salahpour, U E Petäjä-Repo
1Département de biochimie and Le groupe de recherche sur le système nerveux autonome, Université de Montréal, Montréal, Quebec, Canada.
Abstract:
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.
Insights
Mutations in the V2 vasopressin receptor (AVPR2) gene cause nephrogenic diabetes insipidus (NDI). Some AVPR2 mutations lead to misfolded proteins retained in the endoplasmic reticulum, impacting receptor function.
Area of Science:
- Genetics and Molecular Biology
- Cell Biology
- Endocrinology
Background:
- Over 155 mutations in the V2 vasopressin receptor (AVPR2) gene are linked to nephrogenic diabetes insipidus (NDI).
- Understanding how these mutations affect AVPR2 expression and function is crucial for NDI research.
Purpose of the Study:
- To investigate the expression and subcellular localization of four distinct AVPR2 mutations.
- To elucidate the molecular mechanisms underlying AVPR2 dysfunction in NDI.
Main Methods:
- Reverse transcription polymerase chain reaction (RT-PCR) to assess mRNA levels.
- Western blot analysis to detect receptor protein expression.
- Immunofluorescence and confocal microscopy to determine subcellular localization.
- Co-immunoprecipitation to study protein interactions, including with calnexin.
Main Results:
- All four mutant AVPR2 constructs produced detectable mRNA.
- The 804delG mutation resulted in no detectable receptor protein.
- The S315R mutation allowed ER export and plasma membrane targeting but abolished AVP binding.
- W71X and R337X mutations caused AVPR2 retention within the endoplasmic reticulum.
- Calnexin associated with both wild-type and mutant AVPR2, with prolonged interaction observed for the R337X mutant, suggesting a role in quality control.
Conclusions:
- AVPR2 mutations can lead to absent protein, impaired hormone binding, or ER retention.
- Calnexin plays a role in the quality control and ER retention of misfolded AVPR2 mutants.
- These findings provide insights into the molecular basis of NDI caused by AVPR2 gene mutations.