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

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.

Effects of Cellular, Chemical, and Pharmacological Chaperones on the Rescue of a Trafficking-defective Mutant of the ATP-binding Cassette Transporter Proteins ABCB1/ABCB4

“…The cellular chaperone calnexin is one of the chaperones involved in quality control of the ER. Previous studies have shown that a number of mutant proteins exhibit prolonged interaction with calnexin (30,31), including ABCB1 mutants (19). It has been suggested that calnexin binds the misfolded polypeptide and prevents its degradation (31).…”

N-Glycan-mediated Quality Control in the Endoplasmic Reticulum Is Required for the Expression of Correctly Folded δ-Opioid Receptors at the Cell Surface

“…The ability of h␦ORs to interact with calnexin was confirmed by sequential co-immunoprecipitation, and all three N-glycosylated receptor forms, but not the non-N-glycosylated one, were found to co-purify with calnexin. Previously, calnexin has been shown to be involved in ER retention of mutant GPCRs, like the human V2 vasopressin receptor (28), and more recently it has also been found to interact with a number of wild-type GPCRs (e.g. see Refs.…”

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

“…In brief, HEK293 cells transiently expressing GFP fusion proteins grown on 60-mm Petri dishes were incubated with sulfo-NHS-biotin in PBS (0.5 mg/ml, 30 min, 4°C). The reaction was terminated with 50 mM NH 4 Cl in PBS (10 min, 4°C). Cells were then washed three times with PBS and incubated with lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 150 mM NaCl, 1 mM Na-EDTA, pH 8.0; 1 h, 4°C).…”

“…Most likely, the amino acid exchanges result in improper folding of the mutant hV 2 Rs and subsequently prolonged association with molecular chaperones. For example, for the NDI mutant hR337X, a prolonged association with the ER-chaperone calnexin has been observed (4). Chaperone association prevents the aggregation of misfolded proteins, but also inhibits the exit of improperly folded proteins from the ER until correct folding is established.…”