Abstract
The endoplasmic reticulum-associated degradation (ERAD), the main quality control pathway of the cell, is crucial for the elimination of unfolded or misfolded proteins. Several diseases are associated with the retention of misfolded proteins in the early secretory pathway. Among them is X-linked nephrogenic diabetes insipidus, caused by mutations in the gene encoding the V2 vasopressin receptor (V2R). We studied the degradation pathways of three intracellularly retained V2R mutants with different misfolded domains in human embryonic kidney 293 cells. At steady state, the wild-type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, whereas core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the nonglycosylated and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild-type receptor were found to be polyubiquitinylated. Nonglycosylated and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Distinct Derlin-1-dependent and -independent ERAD pathways have been proposed for proteins with different misfolded domains (cytosolic, extracellular, and membrane) in yeast. Here, we show for the first time that V2R mutants with different misfolded domains are able to coprecipitate the ERAD components p97/valosin-containing protein, Derlin-1 and the 26S proteasome regulatory subunit 7. Our results demonstrate the presence of a Derlin-1-mediated ERAD pathway degrading wild-type and disease-causing V2R mutants with different misfolded domains in a mammalian system.
Footnotes
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This work was supported by Deutsche Forschungsgemeinschaft grants DFG HE4468-1/1 and -1/2.
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ABBREVIATIONS: ER, endoplasmic reticulum; V2R, human V2 vasopressin receptor; NDI, nephrogenic diabetes insipidus; AVP, 8-arginine vasopressin; ERGIC, ER/Golgi intermediate compartment; ERAD, endoplasmic reticulum-associated degradation (-C, -L, and -M, misfolded cytosolic, luminal, and intramembrane domain, respectively); p97/VCP, valosin-containing protein; GPCR, G protein-coupled receptor; Der, Derlin; HEK, human embryonic kidney; PMSF, phenylmethylsulfonyl fluoride; STI, soybean trypsin inhibitor; POD, horseradish peroxidase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MG 132, N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal; GFP, green fluorescent protein; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; RT, room temperature; ECL, enhanced chemiluminescence; MS/MS, tandem mass spectrometry; HSP, heat shock protein; Rpt1/S7, 26S protease regulatory subunit 7; Rpn1/S2, proteasome 26S non-ATPase subunit 2; Rpn10/S5a, 26S protease regulatory subunit 5a; RP, regulatory particle.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received August 15, 2007.
- Accepted November 26, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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