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Charité-Universitätsmedizin Berlin, Bereich Molekulare Pharmakologie und Zellbiologie, Berlin, Germany
Received August 15, 2007; accepted November 26, 2007
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Abstract |
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; Aridor and Hannan, 2002). In general, there are two fates for these retained proteins: either they are degraded or they accumulate. Protein accumulation in the endoplasmic reticulum (ER) may lead to an unfolded protein response, ER overload response, or both (Zhang and Kaufman, 2006). Other proteins may accumulate in the cytosol, which eventually leads to the formation of intracellular aggregates or aggresomes (Kopito, 2000), but ultimately, in both cases, the cell is damaged and apoptosis is induced.
The intracellular retention of the V2 vasopressin receptor (V2R) causes X-linked nephrogenic diabetes insipidus (NDI). More than 170 different mutations of the AVPR2 gene, each of them causing NDI, have been described in the literature (http://www.medicine.mcgill.ca/nephros). The V2R is normally expressed at the plasma membrane of renal epithelial cells of the collecting duct, and it regulates vasopressin (AVP)-mediated water reabsorption in the kidney. The majority of NDI-causing mutations produce receptors that are retained in different intracellular compartments, such as the ER, ER/Golgi intermediate compartment (ERGIC) (Hermosilla et al., 2004), and Golgi apparatus. A few NDI-causing mutants are expressed at the cell surface, but they fail to get activated by AVP (Robben et al., 2006). Patients with NDI suffer from polyuria, polydipsia, nocturia, and failure to thrive.
Disease-causing V2R mutants that are retained in different compartments of the secretory pathway are good models to elucidate the interactions between the quality control system and degradation pathways. Intracellular degradation of membrane proteins is carried out by lysosomes, the ubiquitin/proteasome system, or both, depending on the intracellular localization of the protein. Proteins localized at the plasma membrane are usually degraded in lysosomes, such as the wild-type V2R (Bouley et al., 2005). ER-retained proteins are targeted to the ER-associated degradation (ERAD) pathway. ER-retained misfolded membrane proteins are polyubiquitinylated by ubiquitin ligases (gp78 and Hrd1), and they are pulled out of the ER membrane by AAA-ATPases [p97/valosin-containing protein (p97/VCP)] and ATPases of the 19S proteasome (Meusser et al., 2005; Wahlman et al., 2007) through channel proteins, such as Sec61
, Derlin-1, -2, or -3, or a combination. They are deglycosylated, deubiquitinylated, and unfolded before being degraded by the 26S proteasome (Werner et al., 1996; Hampton, 2002). G protein-coupled receptors (GPCR), as the 
opioid receptor, luteinizing hormone receptor, and some disease-causing V2R mutants, have been associated with proteasomal degradation in mammalian cells (Petäjä-Repo et al., 2001; Pietilä et al., 2005; Robben et al., 2005); however, their degradation mechanisms are not fully understood. Furthermore, how receptor proteins are degraded that are neither retained in the ER nor localized at the cell membrane, such as V2R mutants that are retained in the ERGIC (V226E) and Golgi apparatus (G201D), remains unknown.
In yeast, three distinct ERAD pathways (ERAD-L for misfolded ER-luminal domains; ERAD-M, for misfolded intramembrane domains; and ERAD-C, for misfolded cytosolic domains of substrate proteins) have been proposed previously (Carvalho et al., 2006). ERAD-L and ERAD-M require the ubiquitin ligase Hrd1 and its cofactor Hrd3, but not Doa10 as in the ERAD-C pathway. Der1 (Derlin-1, -2, and -3 in mammals) is required in the ERAD-L pathway. All ERAD pathways require the ATPase complex of Ubx2, Npl4, Ufd1, and Cdc48 (p97/VCP in mammals). So far, it is unknown whether these distinct pathways also operate in mammalian cells. In the present work, we analyzed the degradation pathways of the wild-type V2R and of three NDI-causing V2R mutants with misfolded cytosolic (L62P), luminal (G201D), and intramembrane (V226E) domains that are retained in different intracellular compartments, namely, the ER, ERGIC, and Golgi apparatus, respectively. The wild-type receptor and all intracellular retained mutants are polyubiquitinylated and interact with the ERAD components Derlin-1, the AAA-ATPase p97/VCP, and the 26S proteasome regulatory subunit 7 (Rpt1/S7), regardless of the location of their misfolded domains. Our results show that substrates with different misfolded domains are degraded by a Derlin-1, p97/VCP-mediated ERAD pathway in HEK923 cells.
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Materials and Methods |
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-32P]ATP (29.6 TBq/mmol), and [35S]EasyTag protein labeling mix (43.48 TBq/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). Lipofectamine 2000 was purchased from Invitrogen (Karlsruhe, Germany), and FuGENE 6 was from Roche Diagnostics (Mannheim, Germany). Restriction enzymes were from New England Biolabs (Schwalbach, Germany). Trypsin (sequencing grade) was obtained from Promega (Madison, WI). PMSF, deoxycholic acid sodium salt, Roti-Load 1, and SDS were from Roth (Karlsruhe, Germany). Dulbecco's modified Eagle's medium (without methionine and cysteine), benzamidine, chloroquine diphosphate salt, protein A-Sepharose, protein G-agarose, rabbit polyclonal -Derlin-1 antibody, mouse monoclonal -actin antibody, rabbit polyclonal -FLAG antibody, mouse monoclonal -FLAG M2 antibody, and -FLAG M2 affinity gel were from Sigma (Munich, Germany). Aprotinin (Trasylol), Tween 20, trypsin inhibitor (STI), and the chemiluminescence detection kit for horseradish peroxidase (POD) were from Applichem (Darmstadt, Germany). The POD-conjugated goat -rabbit and goat -mouse antibodies were purchased from Dianova (Hamburg, Germany). The rabbit -p97/VCP antibody and the monoclonal rabbit -GAPDH antibody were from Cell Signaling Technology Inc. (Danvers, MA). The rabbit -19S proteasome subunit Rpt1 antibody was from Biomol GmbH (Hamburg, Germany). The rabbit -calnexin antibody and the monoclonal mouse anti-multiubiquitin antibody were from Assay Designs, Inc. (Ann Arbor, MI). The polyclonal goat -Derlin-1 antibody was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). LysoTracker Red was from Invitrogen. MG 132 was purchased from Calbiochem (Darmstadt, Germany). Plasmid pV2R.DNA3.1, coding for the untagged V2R, and plasmid pEU367.GFP, encoding a C-terminally green fluorescent protein (GFP)-tagged V2R, have been described previously (Oksche et al., 1998; Schülein et al., 1998). The pEGFP-N1 vector was from Clontech Laboratories (Heidelberg, Germany). The QuikChange site-directed mutagenesis kit was purchased from Stratagene (La Jolla, CA), and the Qproteome cell compartment kit was from QIAGEN GmbH (Hilden, Germany). HEK293 cells were purchased from DSMZ (Hannover, Germany).
Plasmid Constructions. Plasmid pFLAG.V2R encoding the V2R with a FLAG tag fused to the N terminus was made via polymerase chain reaction with a BamHI/FLAG insertion primer (5'-CGGGATCCCGGCCACCATGGACTATAAGGACGATGACGATAAGATGCTCATGGCGTCCACTTCCGC-3') and the commercially available pcDNA3.1/BGH reverse sequencing primer from Invitrogen. For this reaction, pV2R.DNA3.1 was used as a template. The polymerase chain reaction product, containing the FLAG.V2R cDNA and an N-terminally inserted BamHI site, was cloned into the pcDNA3.1 vector using the introduced BamHI site and XbaI site. Plasmids pFLAG.L62P, pFLAG.V226E, and pFLAG.G201D were constructed with the help of the QuikChange site-directed mutagenesis kit (Stratagene; primer sequences available on request); plasmid pFLAG.V2R was used as template. Plasmids pL62P.GFP, pV226E.GFP, and pG201D.GFP encoding the receptor mutants L62P, V226E, and G201D, tagged with GFP at their C termini (residue Lys367), have been described previously (Krause et al., 2000). Standard DNA preparations and cloning techniques were carried out according to the handbooks of Sambrook and Russell (2001). The nucleotide sequences of the DNA fragments were verified by sequencing, using the BigDye Terminator kit (Applied Biosystems).
Cell Culture. HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) from Sigma, supplemented with 10% (v/v) heat-inactivated fetal bovine serum, 20 mM glutamine, 100 IU penicillin, and 100 µg/ml streptomycin in a 5% CO2 atmosphere at 37°C. Stably expressing cell lines were generated through transfection with FuGENE 6 (Roche Diagnostics) or Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. Clones were selected with 400 µg/ml G-418 (Geneticin; Calbiochem). The culture medium for the stably expressing cell clones was additionally supplemented with 400 µg/ml G-418.
Adenylyl Cyclase Assay. The adenylyl cyclase assays were carried out with nuclei-free crude membranes of stably transfected HEK293 cells as described previously for stably transfected mouse Ltk- cells (Schülein et al., 1996).
Total Cell Lysis. HEK293 cells stably expressing V2R constructs were grown to confluence on a 60-mm culture dish. Cells were washed two times with ice-cold PBS, and then they were lysed in 500 µl of radioimmunoprecipitation assay buffer; the method has been described previously (Gazit et al., 1999). After determination of protein concentration, 30 µg of the total amount of proteins was used for SDS-PAGE.
Immunoprecipitation of FLAG-Tagged V2Rs and Coimmunoprecipitation. HEK293 cells stably expressing V2R constructs were grown to confluence on a 60-mm culture dish. Cells were washed two times with ice-cold PBS, lysed in 1 ml of ice-cold lysis buffer (1% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 150 mM NaCl, 1 mM Na-EDTA, 0.5 mM PMSF, 10 µg/ml STI, 1 µg/ml aprotinin, and 0.5 mM benzamidine, pH 8.0), and then transferred to microfuge tubes. The insoluble fraction of one 60-mm culture dish was removed by centrifugation (20 min; 4°C; 18,620g), and FLAG-tagged proteins were isolated from the supernatant with an immune complex of 5 mg of protein A-Sepharose beads and 5 µg of -FLAG Sigma M2 antibodies at 4°C overnight. Beads were centrifuged (1 min; 4°C; 13,680g), and washed three times with buffer A (0.5% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, 500 mM NaCl, 1 mM Na-EDTA, pH 8.0) and once with buffer B (0.5% Triton X-100, 0.1% SDS, 50 mM Tris-HCl, and 1 mM Na-EDTA, pH 7.4). Bound proteins were eluted by 5-min boiling (95°C) in 40 µl of 2x Roti-Load 1 sample buffer. They were separated by SDS-PAGE. For coimmunoprecipitation experiments, FLAG-tagged proteins were isolated with 40 µl of -FLAG M2 Affinity gel (Sigma) (same preparation as described above) or with an immune complex of 5 mg of protein G-agarose beads and 2 µg of polyclonal -Derlin-1 antibodies (Santa Cruz Biotechnology, Inc.) at 4°C overnight.
Immunoblots. Proteins were separated by SDS-PAGE (10 or 15% acrylamide), and then they were blotted onto nitrocellulose membranes as described previously (Kyhse-Andersen, 1984). Membranes were blocked for 1 h with PBS (140 mM NaCl, 2.7 mM KCl, and 10 mM KH2PO4, pH 7.4) supplemented with 0.1% Tween 20 and 5% low fat milk powder. For detection of the V2R, membranes were incubated with a mouse monoclonal -FLAG M2 antibody (1:1500 in PBS with 0.1% Tween 20 and 2% low fat milk powder) for 1 h at RT. Membranes were washed three times (15 min each) with wash buffer (PBS supplemented with 0.1% Tween 20). Blots were incubated with a POD-conjugated -mouse antibody (1:10,000) in PBS supplemented with 0.1% Tween 20 and 2% low fat milk powder for 45 min at RT. Membranes were washed again three times with wash buffer (15 min each). Finally, membranes were incubated in ECL system for 3 min, and they were developed with a luminescence X-Omat film (Eastman Kodak, Rochester, NY). For coimmunoprecipitation experiments, polyclonal -p97/VCP antibody (Cell Signaling Technology Inc.), polyclonal rabbit -Derlin-1 (Sigma), polyclonal -19S regulator, ATPase subunit Rpt1 antibody (Biomol GmbH) (all at 1:1000 dilution), or mouse monoclonal -FLAG M2 antibody (Sigma) (1:1500 dilution) was used.
Ubiquitinylation Assay. Stably transfected HEK293 cells were treated with 20 µM MG 132 in serum-free medium for 16 h. Lysis of cells expressing FLAG-tagged receptors was carried out, the insoluble fraction was removed by centrifugation (20 min; 4°C; 18,620g), and polyubiquitinylated proteins were isolated, as described in immunoprecipitation of FLAG-tagged V2Rs with 40 µl of -FLAG M2 affinity gel (Sigma). After the washing steps, the proteins were boiled in 40 µl of 2x Roti-Load 1 sample buffer, separated by SDS-PAGE (10% acrylamide), and blotted onto nitrocellulose membranes. For detection, a mouse monoclonal anti-multiubiquitin antibody (Assay Design) (1:400) and a POD-conjugated -mouse IgG (1:10,000) were used. The receptor expression was analyzed with a mouse monoclonal -FLAG M2 antibody (Sigma) (1:1500) and a POD-conjugated -mouse IgG (1:10,000). Immunoreactive bands were developed with ECL.
Visualization of GFP-Tagged Receptors by Confocal Laser-Scanning Microscopy in Living HEK293 Cells. Stably transfected HEK293 expressing the wild-type and mutant V2Rs tagged with GFP were grown for 24 to 48 h in a 35-mm culture dish containing a poly-L-lysine-coated coverslip. Cells were incubated with 150 nM LysoTracker Red for 1 h, and for inhibition of degradation, they were additionally incubated with 100 µM chloroquine for 3 h, washed once with PBS, and transferred immediately into a heating chamber (details on request). Cells were covered with 1 ml of PBS, and GFP fluorescence was visualized on a 510 inverted laser-scanning microscope (Carl Zeiss, Jena, Germany) (optical section: <0.9 µm; 
exc, 488 nm; long pass filter, 505 nm). GFP and Lyso-Tracker fluorescence signals were recorded (optical section: <0.9 µm; multitrack mode; GFP, exc, 488 nm; band-pass filter, 494-548 nm; LysoTracker Red, exc, 543 nm; long pass filter, 560 nm), and an overlay of both signals was computed.
Pulse-Chase Assay. Stably transfected HEK293 cells were grown in 100-mm culture dishes until confluence. Cells were starved in 5 ml of serum-free DMEM without methionine and cysteine (Sigma) for 16 h. The cells were washed twice with PBS, treated with trypsin/EDTA, and collected in a 15-ml Falcon tube (Falcon; BD Biosciences Discovery Labware, Bedford, MA). Cells were washed with PBS two more times (5 min; RT; 1000g). Metabolic labeling with 220 µCi of [35S]EasyTag protein labeling mix in 500 µl of medium lacking methionine and cysteine was performed for 45 min. The labeling process was stopped with 4 ml of DMEM supplemented with unlabeled methionine (2.3 mM) and cysteine (0.75 mM). To indicated times at which cells were resuspended, 1 ml of suspension was harvested for each sample; the sample was then mixed with 750 µl of ice-cold DMEM supplemented with protease inhibitors [0.5 mM PMSF, 10 µg/ml STI (trypsin inhibitor), 1 µg/ml aprotinin, and 0.5 mM benzamidine], collected by a centrifugation step (5 min; 4°C; 1000g), and lysed with 500 µl of ice-cold lysis buffer. The FLAG-tagged receptors were immunoprecipitated as described above. After separation by SDS-PAGE (10% acrylamide), the gels were dried and then exposed to X-ray film. Lysosomal and proteasomal activity was inhibited with 100 µM chloroquine and 40 µMMG 132, respectively, supplemented in all buffers from the labeling step until the end of the chase.
Protein Identification by Mass Spectrometry. Proteins of HEK293 cells treated with and without MG 132 were immunoprecipitated, prepared as described above, and subjected to SDS-PAGE. Sample preparation, in-gel digestion, peptide extraction, and spectra acquisition were performed as described previously (Czupalla et al., 2006). In brief, proteins were stained with Coomassie Blue, excised from the gel, and subjected to in-gel digestion by trypsin. NanoLC-MS/MS experiments were performed on a quadruple orthogonal acceleration time-of-flight mass spectrometer Q-Tof Ultima (Micromass, Manchester, UK). A Micromass CapLC liquid-chromatography system with a capillary column (PepMap C18; 3 µm; 150 mm x 75 µm i.d.; Dionex, Idstein, Germany) was used to deliver the peptide solution to the nanoelectrospray source (PicoTip spray capillaries; New Objective, Woburn, MA). For protein identification, data were acquired in a data-dependent mode (survey scanning) using one MS scan followed by MS/MS scans of the most abundant peak. The processed MS/MS spectra and MASCOT server version 1.9 (Matrix Science Ltd., London, UK) were used to search in-house against the SwissProt protein database (210906 and 234112 sequences).
Subcellular Fractionation. Proteins of different cellular compartments were obtained with the Qproteome cell compartment kit (QIAGEN GmbH). Cytosolic and membrane fractions were isolated according to the manufacturer's protocol. Equal amounts of proteins were used for SDS-PAGE. The purity of the fractions was controlled by detection of calnexin and GAPDH, concurrently the proteins served as a loading control. Detection of receptor proteins was carried out with a monoclonal -FLAG M2 antibody (Sigma) and a POD-conjugated -mouse IgG. Immunoreactive bands were developed with the ECL system.
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) (Fig. 2, Supplemental Fig. 2). We first determined whether lysosomes are involved in the degradation of all mutants. To this end, the presence of the mutants in lysosomes was assessed in living HEK293 cells by confocal laser-scanning microscopy. Cell clones stably expressing the V2R mutants tagged with GFP were stained with the fluorescent lysosome marker LysoTracker Red. GFP fluorescence signals were recorded on the green channel, and the LysoTracker Red signals were recorded on the red channel; overlays were computed. L62P and V226E were diffusely localized within the cell, excluding the nucleus (Fig. 2, left). LysoTracker Red signals were detected as intracellular vesicles, frequently nearby the nucleus (Fig. 2, middle) and no colocalization with GFP signals could be identified in overlays of mutants L62P and V226E (Fig. 2, right). Mutant G201D was detected in vesicular structures and at the plasma membrane. Colocalization with LysoTracker Red signals was observed (Fig. 2, right). GFP fluorescence signals from the wild-type V2R were localized predominantly at the plasma membrane and in vesicular structures, which also colocalized with LysoTracker Red signals.
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Treatment with 100 µM chloroquine had no apparent effect on the band pattern of the wild-type V2R, indicating that its lysosomal degradation half-life is longer than 10 h. For mutant G201D, a change was only observed in the complex-glycosylated band, confirming that the mutant is degraded in lysosomes. Mutants V226E and L62P showed no change in their band pattern, demonstrating that they do not reach the lysosomal compartment.
In addition, cell clones were treated with the proteasomal inhibitor MG 132, and bands representing the nonglycosylated and core-glycosylated receptors (immature receptor forms) could be stabilized in all samples (Fig. 4, right). This result shows that proteasomal activity is involved in receptor degradation of the immature receptor forms. Together, these results demonstrate that the complex-glycosylated receptors are subjected to lysosomal degradation whereas nonglycosylated and core-glycosylated receptor proteins are degraded by the proteasome. Thus, the ERGIC seems to be the last compartment in the secretory pathway that excludes membrane proteins from lysosomal degradation.
Wild-Type V2R and NDI-Causing Mutants Are Polyubiquitinylated. Mature forms of the mutant G201D and wild-type V2R were degraded in lysosomes. In contrast, the immature forms of the wild-type and mutant V2Rs were stabilized when proteasomal activity was blocked, demonstrating a participation of the proteasome in their degradation. ERAD substrates are usually polyubiquitinylated and retrotranslocated from the ER to the cytosol for proteasomal degradation. We investigated whether the NDI-causing mutants and the wild-type V2R are polyubiquitinylated and degraded via the ERAD. FLAG-tagged receptors were precipitated with an -FLAG M2 affinity gel, and ubiquitinylated receptor proteins were identified with a monoclonal -polyubiquitin antibody. An immunoreactive smear starting at 250 kDa was only detected in samples that contained receptor constructs (Fig. 5A). MG 132 treatment increased the intensity of this smear, which ranged from 250 to 60 kDa, because of ubiquitin chains of different length. The wild-type V2R and intracellular retained mutants were polyubiquitinylated, indicating that they are suitable substrates for ERAD.
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Furthermore, the precipitation efficiency and expression levels of the V2R constructs used for the immunoprecipitations were analyzed by Western blot. No major difference in the expression of FLAG-tagged constructs could be detected between cell clones subjected to immunoprecipitation (Fig. 5B). As expected, for the V2R wild-type and mutant G201D all three receptor forms, the nonglycosylated, core-glycosylated (immature), and complex-glycosylated form, could be detected after MG 132 treatment (compare Supplemental Fig. 3 with Fig. 4, right). Mutants V226E and L62P showed only immature receptor forms. Bands with an apparent molecular mass of 
160 and 80 kDa are presumably multimers of immature receptor proteins (Fig. 5B, *). The apparent smear detected between 200 and 80 kDa represents polyubiquitinylated forms of the V2R.
Wild-Type V2R and NDI-Causing Mutants Interact with ERAD Components. If the immature wild-type V2R and its mutants are degraded via the ERAD, canonical ERAD components that interact with polyubiquitinylated proteins should be identified after precipitation of V2Rs from HEK293 cells. Stably transfected cells expressing the FLAG-tagged wild-type V2R were treated with and without MG 132 overnight, and receptors were immunoprecipitated and separated by SDS-PAGE. Corresponding slices from Coomassie-stained gels of MG 132-treated and untreated samples were excised (for details, see Supplemental Fig. 4). Coprecipitated proteins were in-gel digested with trypsin and identified by NanoLC-tandem mass spectrometry. In samples of untreated cells, only the V2R and Ig 
-1 chain of precipitating antibodies were detected. In samples of MG 132-treated cells, several proteins related to cell stress and protein degradation were identified additionally: immunoglobulin heavy chain-binding protein, the 26S proteasome regulatory subunit 7 (Rpt1/S7), the proteasome 26S non-ATPase subunit 2 (Rpn1/S2), elongation factor 1-1, 90-kDa heat shock protein- (HSP 90-/HSP 86) and 90-kDA heat shock protein-β (HSP 90-β/HSP 84) (Table 1). Both identified proteasomal proteins are subunits of the 19S proteasome regulatory complex.
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To verify the mass spectrometry results, we first studied the endogenous expression levels of Rpt1/S7 in whole cell lysates from stable cell clones. A specific immunoreactive band running at 48 kDa was identified in all lysates (Fig. 6, top); no difference in the expression of Rpt1/S7 could be detected between cell clones. Next, blots of immunoprecipitated samples pretreated with or without MG 132 showed also a specific band with an apparent mass of 48 kDa, representing Rpt1/S7, and an unspecific band of 60 kDa. In untreated samples of mutant L62P, more Rpt1/S7 could be coprecipitated than in other samples (Fig. 6, middle, Rpt1/S7). As expected, a strong increase in the intensity of the specific immunoreactive band could be observed, when cells were treated with MG 132 (Fig. 6, bottom, Rpt1/S7). As a control for V2R precipitation levels, the same membranes were stripped and used for V2R detection (Fig. 6, V2R). In the presence of MG 132 immature V2R forms were increased, partially explaining the increased coimmunoprecipitation of Rpt1.
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Another canonical ERAD component is p97/VCP, an AAA-ATPase involved in the extraction of misfolded proteins from the ER. It binds to polyubiquitinylated ERAD substrates. Expression levels of p97/VCP were analyzed in whole cell lysates by immunoblotting, and again no differences could be detected between cell clones (Fig. 7, top, p97/VCP). Coimmunoprecipitation studies showed an immunoreactive band with an apparent size of 89 kDa in samples of whole cell lysates detected against p97/VCP. No specific signals were found in coimmunoprecipitated samples from untreated cell clones. The only exception was mutant L62P, where a specific band running at 89 kDa could be identified, indicating an interaction of this mutant with p97/VCP at steady state (Fig. 7, middle, p97/VCP). As expected, treatment with MG 132 increased the amount of coprecipitated p97/VCP significantly (Fig. 7, bottom, p97/VCP). V2R precipitation levels were assayed in the same membranes used for the coimmunoprecipitation study (Fig. 7, V2R).
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-Derlin-1 antibody (data not shown). As expected, the intensity of this band increased, when cells were treated overnight with MG 132 (Fig. 8A, middle, Derlin-1). V2R precipitation levels were verified in the same membranes used for the detection of Derlin-1 (Fig. 8A, bottom, V2R). In addition, we also precipitated Derlin-1 and detected against the V2R with a mouse monoclonal -FLAG M2 antibody. As expected, only immature forms of all mutants and the wild-type V2R were coprecipitated by Derlin-1 in a MG 132-dependent manner (Fig. 8B). Unfortunately, reverse coimmunoprecipitation studies were not successful using -Rpt1 and -p97/VCP antibodies for precipitation.
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Proteasomal inhibition leads to an accumulation of immature, polyubiquitinylated, and nonpolyubiquitinylated proteins, thereby increasing the amount of immunoprecipitated receptors and coprecipitated proteins, in Rpt1/S7, p97/VCP, and Derlin-1, confirming that these proteins bind polyubiquitinylated ERAD substrates (Wang et al., 2004) (Figs. 5, A and B, and 6, 7 and 8, V2R). Our results show that immature and/or polyubiquitinylated V2Rs interact with ERAD components, namely, Rpt1/S7, p97/VCP, and Derlin-1. However, these results do not preclude the possibility that other ERAD proteins participate in this complex.
Immature Receptor Proteins Are Retrotranslocated into the Cytosol. V2R mutants are polyubiquitinylated and interact with membrane and cytosolic components of the ERAD complex. For determination of the site of turnover, cytosolic and membrane fractions of HEK293 cells stably expressing FLAG-tagged L62P mutants and wild-type receptors were isolated. Immunoblot analysis revealed specific bands in the cytosolic and membrane fractions of MG 132-treated cells (Fig. 9). In the cytosolic fraction of L62P, a specific band of approximately 35-kDa could be detected that corresponds to the unglycosylated receptor form. In the membrane fraction, a specific band of 38 kDa could be identified, representing the core-glycosylated receptor form. For the wild-type receptor, a similar band pattern could be recognized; however, in the membrane fraction of MG 132-treated cells, two additional immunoreactive bands could be detected, one band between 53 and 45 kDa and a second band of 35 kDa. These bands represent the complex- and unglycosylated receptor forms, respectively. As expected, an increase in the intensity or emergence of bands representing the immature receptor forms was observed in cells treated with MG 132, confirming our results obtained by pulse-chase analysis. These results indicate that ERAD substrates are deglycosylated and retrotranslocated completely into the cytosol. Furthermore, glycosylated receptor forms are restricted to the membrane fraction.
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). It is accepted that integral membrane proteins expressed at the cell surface are internalized and degraded by the lysosome. In contrast, membrane proteins retained in the ER by the quality control system are retrotranslocated into the cytosol, and they are degraded by the ubiquitin/proteasome system. This particular degradation pathway is known as ERAD (Brodsky and McCracken, 1999; Hampton, 2002). In mammalian cells, the ERAD pathway is still not well characterized. In addition, the degradation pathways used by the cell when proteins are retained outside the ER are also not well defined.
In the present study, we analyzed the degradation pathways of the wild-type V2R and three intracellularly retained disease-causing mutants. These particular mutants are good tools to analyze the degradation pathways, because they are retained in different compartments of the secretory pathway (Hermosilla et al., 2004). Furthermore, the defects resulting from mutations are localized in different domains of the protein; L62P is located in a cytosolic domain (beginning of the first intracellular loop), G201D is in a luminal domain (second extracellular loop), and V226E is in a transmembrane domain (fifth helix) (Fig. 1A). The colocalization studies in living cells showed that the wild-type V2R and mutant G201D are localized in lysosomes. Furthermore, pulse-chase experiments demonstrated that the complex glycosylated (mature) forms of mutant G201D are degraded by lysosomes. Similar results have been reported for the wild-type V2R and mature mutants at steady state and after activation by AVP (Robben et al., 2004). Our results demonstrate that membrane proteins, which reach the Golgi apparatus or are expressed at the plasma membrane, are transported to the lysosomal compartment for degradation at steady state. However, it is not known whether receptors that reach the Golgi apparatus are first transported to the plasma membrane and then to lysosomes, or whether a direct route is followed, or whether both routes are operating.
All immature V2R forms (mutant and wild type) were degraded via the ERAD, regardless of their intracellular localization (Figs. 6, 7 and 8, Supplemental Fig. 2). As expected, all mutants were found to be polyubiquitinylated (Fig. 5, A and B). We could demonstrate that in the presence of a proteasome inhibitor, all nonglycosylated and core-glycosylated V2R proteins were stabilized (Figs. 4, MG 132, and 5B), and that wild-type and mutant receptors coprecipitated Derlin-1, p97/VCP, Rpt1/S7, and Rpn2/S2. These results indicate that newly synthesized misfolded or unfolded receptors are targeted to the ERAD by the quality control system. Therefore, we conclude that the ERAD is predominantly involved in the degradation of immature disease-causing V2R mutants, but it is also involved in the degradation of immature, misfolded wild-type V2Rs. The fact that immature wild-type proteins also are degraded by the ERAD has been described for the opioid (Petäjä-Repo et al., 2001), luteinizing hormone receptor (Pietilä et al., 2005), and the cystic fibrosis transmembrane regulator, and it is ascribed to inefficient folding or structural instability of the proteins (Sun et al., 2006). Presented data show that only nonglycosylated and core-glycosylated misfolded receptor proteins are degraded via ERAD and that complex-glycosylated misfolded receptor proteins are targeted to lysosomal degradation. Our results demonstrate that the ERGIC is the last compartment of quality control that participates in ERAD.
The wild-type V2R has been described to be ubiquitinylated together with β-arrestin 2, but only after agonist-induced internalization; both proteins are thought to be transported together to the lysosomal compartment for degradation (Martin et al., 2003). The ubiquitin ligases involved in the polyubiquitinylation of immature wild-type V2Rs and NDI-causing mutants are still unknown. Most probable ubiquitin ligases such as gp78/autocrine motility factor receptor (membrane-associated E3), Hrd1 (membrane-associated E3), and Ufd2 (E4 enzyme), which interact with p97/VCP and participate in the retrotranslocation of ERAD substrates (Ye, 2006), are involved in the polyubiquitinylation of immature forms of the V2R. In an attempt to determine the E3 that is responsible for the polyubiquitinylation of NDI-causing V2R mutants, immunoprecipitated samples were analyzed for the presence of Hrd1. It was not possible for us to specifically detect Hrd1 in these samples (data not shown).
In yeast, three distinct ERAD pathways have been proposed: ERAD-L, ERAD-M, and ERAD-C, which destroy proteins with misfolded luminal, intramembrane, and cytosolic domains, respectively (Carvalho et al., 2006). These pathways use the cytosolic Cdc48 ATPase complex, whose membrane recruitment is facilitated by Ubx2. However, the ERAD-L and ERAD-M pathway require the ubiquitin ligase Hrd1, additionally ERAD-L needs Der1; both proteins have mammalian homologs (Hrd1, gp78, and Derlin-1, -2, and -3) that are involved in the mammalian ERAD. ERAD-C requires the ubiquitin ligase Doa10, but not Hrd1 and Der1. Our results for the studied V2R mutants suggest that only the Derlin-1-mediated ERAD pathway is used in HEK293 cells. Regardless of their misfolded domain, either cytosolic (L62P), luminal (G201D), or membrane (V226E), all mutants precipitated Derlin-1 and p97/VCP, the mammal homolog of Cdc48. We also investigated a second mutant with a mutation within the fourth transmembrane domain (S167L) that also interacts with Derlin-1 (data not shown). Our results demonstrate that at least the putative channel protein Derlin-1 and the AAA-ATPase p97/VCP are central components of the retrotranslocation machinery of the ERAD in a mammalian system.
Sec61 and Derlin-1 (and its homologs Derlin-2 and -3) are thought to be the channel or to be located at the site of retrotranslocation of misfolded proteins from the ER to the cytosol (Ye et al., 2004, 2005; Kalies et al., 2005). It is still unclear whether the proteasome is recruited to the ER membrane by binding to the retrotranslocation channel, so that substrates are directly released into the proteasome (Kalies et al., 2005); or whether substrates are first released into the cytosol and then targeted to the proteasome (Petäjä-Repo et al., 2001; Meusser et al., 2005). It has been shown that the 26S proteasome disassembles in an ATP-dependent catalytic cycle, leading to 19S RP subcomplexes and free 26S protease regulatory subunit 5a (Babbitt et al., 2005). In contrast, 19S RPs are able to interact with Sec61 at the ER membrane (Ng et al., 2007). Wahlman et al. (2007) demonstrated that ATP, the 19S RP, the ER luminal chaperone PDI, and Derlin-1 are sufficient to retrotranslocate a soluble ERAD substrate in vitro (Wahlman et al., 2007). It is noteworthy that retrotranslocation of the substrate was ubiquitin, Sec61, p97/VCP, and Ufd1 and Npl4 cofactors independent. However, the ERAD substrate used in this study was the fluorescence-labeled 
gpf, a soluble, nonubiquitinylated protein lacking disulfide bonds and transmembrane domains, which is derivative of the yeast pro- factor mating pheromone. In our system, V2Rs were located in the ER-membrane, glycosylated, and/or polyubiquitinylated, which might explain the differences regarding the association with p97/VCP. Furthermore, we could identify deglycosylated V2R (wild type and mutant) in the cytosolic fraction after proteasomal inhibition, indicating a complete retrotranslocation of the receptors to the cytosol. Our results support the idea that 19S RP plays a central role in retrotranslocation and recognition of ERAD substrates, before their degradation by the 26S proteasome. Because retrotranslocated V2Rs seemed not to be polyubiquitinylated, the interaction with 19S RP might induce their deubiquitinylation. Therefore, we propose that subunits of the 19S RP, like Rpt1/S7, Rpn1/S2, and 26S protease regulatory subunit 5a, function as scaffolds on the ER-substrate for the 19S RP and 20S core particle, facilitating the assembly of the 26S proteasome at the substrate. Moreover, Derlin-1 is centrally implicated in the recognition, retrotranslocation, or both of the V2R, independent of the location of the misfolded domain.
Until now, no effective pharmacological treatment of NDI has been developed. A promising therapeutic strategy is the use of pharmacochaperones that rescue transport-defective receptors from the early secretory pathway to the plasma membrane (Wüller et al., 2004; Bernier et al., 2006). Another possible approach to rescue intracellularly retained proteins may be the inhibition of the ERAD, as exemplary shown for the CFTR by reducing the expression of p97/VCP or blocking the proteasome with bortezomib (Vij et al., 2006). However, this new therapeutic strategy may be only valid for NDI-causing mutants that are transport-defective but still functional.
In summary, our data reveal that the quality control system of the cell determines the degradation pathway of NDI-causing mutants of the V2R. Immature receptor proteins that reach the ERGIC are polyubiquitinylated and degraded via the ERAD; all other mature receptor proteins located beyond the ERGIC are degraded via lysosomes. Furthermore, we demonstrate that a Derlin-1- and p97/VCP-mediated ERAD pathway is responsible for the degradation of the V2R and its disease-causing mutants, regardless of the location of the misfolded domain within the receptor.
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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.
The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Ricardo Hermosilla, Leibniz-Institut für Molekulare Pharmakologie (FMP), Campus Buch, Robert-Rössle Str. 10, 13125 Berlin, Germany. E-mail: ricardo.hermosilla{at}charite.de
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