Derlin-1 and p97/Valosin-Containing Protein Mediate the Endoplasmic Reticulum-Associated Degradation of Human V2 Vasopressin Receptors

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 complexglycosylated 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/valosincontaining 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 diseasecausing V2R mutants with different misfolded domains in a mammalian system.

The Sequence after the Signal Peptide of the G Protein-Coupled Endothelin B Receptor Is Required for Efficient Translocon Gating at the Endoplasmic Reticulum Membrane

“…Although the K D and EC 50 values of the mutant ET B ⌬27.GFP are similar to those of the wild type, indicating that the mutant receptor is correctly folded, the deletion may nevertheless lead to a subtle change in receptor conformation, which may be recognized by the quality control system of the ER and/or other components of the early secretory pathway. In this case, the mutant receptor would be initially retained in the early secretory pathway and finally subjected to proteolysis by the ERAD (Schwieger et al, 2008). Such a behavior of mutant ET B ⌬27.GFP may explain the decreased number of receptors at the plasma membrane.…”

“…Such a behavior of mutant ET B ⌬27.GFP may explain the decreased number of receptors at the plasma membrane. GPCR retention in the early secretory pathway and subsequent degradation by the ERAD is accompanied by a decrease in the ratio of complex and high mannose-glycosylated receptors (Schü lein et al, 1998;Robben et al, 2005;Schwieger et al, 2008); the complex-glycosylated forms represent the mature receptors, and the high mannose forms represent the immature forms present in the early secretory pathway.…”

“…4, a and Retention of a mutant receptor by the quality control system and subsequent degradation by the ERAD is also accompanied by a steady-state accumulation of the receptor in the early secretory pathway, which is detectable microscopically (e.g., Schü lein et al, 1998; Robben et al, 2005;Schwieger et al, 2008). To confirm the results that ET B ⌬27.GFP is not subjected to a more stringent quality control in the early secretory pathway, we analyzed the subcellular location of the GFP signals of the receptor by confocal LSM and assessed their colocalization with the plasma membrane dye Trypan blue (Fig.…”

“…Mutation of these motifs leads to misfolded forms that are retained intracellularly by the quality control system of the early secretory pathway. These proteins are finally subjected to proteolysis by the ER-associated degradation pathway (ERAD) (Schwieger et al, 2008).…”

Role of Derlin-1 Protein in Proteostasis Regulation of ATP-sensitive Potassium Channels

“…Our results suggest that at least in the experimental system employed in this study, Derlin-1 is involved in degradation of proteins with folding lesions in all three domains, although we recognize the possibility that the mutations we studied here may cause misfolding of protein domains other than where the mutations are located. It is worth noting that a recent study of the human V2 vasopressin receptors has also reported association of mutants with different misfolded domains with Derlin1⅐p97, although the effect of Derlin-1 levels on mutant V2 receptor expression was not evaluated (47). Curiously, although a clear increase in the immature SUR1 was observed in all three mutants, only A116P was able to exit the ER and reach the cell surface upon Derlin-1 knockdown, despite the fact that A116P had overall lower protein levels than C26S and ⌬F1388.…”