Received for publication August 15, 2007.
Revised November 26, 2007.
Accepted for publication November 26, 2007.

Derlin-1 and p97/VCP Mediate the ER-Associated Degradation of Human V2 Vasopressin Receptors

Abstract

The ER-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 HEK293 cells. At steady state, the wild type V2R and the complex-glycosylated mutant G201D were partially located in lysosomes, while core-glycosylated mutants L62P and V226E were excluded from this compartment. In pulse-chase experiments, proteasomal inhibition stabilized the non- and core-glycosylated forms of all studied receptors. In addition, all mutants and the wild type receptor were found to be polyubiquitinylated. Non- and core-glycosylated receptor forms were located in cytosolic and membrane fractions, respectively, confirming the deglycosylation and retrotranslocation of ERAD substrates to the cytosol. Recently, 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 co-precipitate 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.

Key words: Vasopressin/Oxytocin, Receptor synthesis/trafficking, Receptor degradation, Fluorescence techniques, Mass Spectroscopy