Association of calnexin with wild type and mutant AVPR2 that causes nephrogenic diabetes insipidus.
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.
Morello
J P
JP
Département de biochimie and Le groupe de recherche sur le système nerveux autonome, Université de Montréal, Montréal, Quebec, Canada.
Salahpour
A
A
Petäjä-Repo
U E
UE
Laperrière
A
A
Lonergan
M
M
Arthus
M F
MF
Nabi
I R
IR
Bichet
D G
DG
Bouvier
M
M
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D037281:Calnexin; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000235:genetics; D018500:Diabetes Insipidus, Nephrogenic / Q000209:etiology / Q000235:genetics* / Q000378:metabolism*; D018390:Gene Targeting; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding / Q000235:genetics; D014176:Protein Biosynthesis; D017510:Protein Folding; D011869:Radioligand Assay; D017483:Receptors, Vasopressin / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D002135:Calcium-Binding Proteins; D017483:Receptors, Vasopressin; D037281:Calnexin