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Involvement of the p97-Ufd1-Npl4 Complex in the Regulated Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors *

Involvement of the p97-Ufd1-Npl4 Complex in the Regulated Endoplasmic Reticulum-associated... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34530 –34537, October 14, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Involvement of the p97-Ufd1-Npl4 Complex in the Regulated Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors Received for publication, August 11, 2005 Published, JBC Papers in Press, August 15, 2005, DOI 10.1074/jbc.M508890200 ‡ ‡ § ‡1 Kamil J. Alzayady , Margaret M. Panning , Grant G. Kelley , and Richard J. H. Wojcikiewicz ‡ § From the Departments of Pharmacology and Medicine, SUNY Upstate Medical University, Syracuse, New York 13210-2339 Inositol 1,4,5-trisphosphate (IP ) receptors form tetrameric, IP - brane-spanning regions, a long cytosolic amino-terminal region, and a 3 3 gated channels in endoplasmic reticulum membranes that govern short cytosolic carboxyl-terminal tail (5–7). the release of Ca from this organelle. In response to activation of In response to stimulation of GPCRs that persistently elevate IP certain G protein-coupled receptors that persistently elevate IP concentration, the cellular levels of IP R1–3 are rapidly reduced, a phe- 3 3 concentration, IP receptors are ubiquitinated and degraded by the nomenon termed IP R down-regulation. This has been demonstrated in ubiquitin-proteasome pathway. IP receptor ubiquitination is many mammalian cell lines and in native tissues (3, 8–15), with the mam mediated by the ubiquitin-conjugating enzyme, Ubc7, a compo- consequence that the frequency, amplitude, and duration of elementary 2 2 nent of the endoplasmic reticulum-associated degradation path- Ca puffs are reduced (16), and global Ca signaling is restrained (8, 9, way. However, the mechanism by which ubiquitinated IP receptors 13–16). IP R down-regulation appears to be an adaptation to GPCR are transferred to the proteasome is not known. Here, we examine stimulation that protects cells against the deleterious effects of chronic this process and show in several mammalian cell types that the elevation of cytosolic Ca (17). ATPase p97 associates with IP receptors in response to hormonal IP R down-regulation is mediated by the ubiquitin-proteasome path- stimuli that induce IP receptor ubiquitination. To examine the 3 way (UPP) (10–15). Protein degradation via the UPP has two distinct functional relevance of the p97 interaction with IP receptors, we 3 steps: first, polyubiquitin chains are covalently attached to the targeted stably and specifically reduced p97 protein levels by 62  3% in protein, and second, the polyubiquitinated protein is degraded by the 26 Rat-1 fibroblasts using RNA interference. In these cells, endothelin- S proteasome (18, 19). The process by which ER resident proteins or 1-induced IP receptor degradation was markedly retarded and the 3 proteins traversing the ER are degraded is termed endoplasmic reticu- accumulation of ubiquitinated IP receptors was markedly 3 lum-associated degradation or ERAD (20–23). Two forms of ERAD enhanced. These effects were reversed by expression of exogenous appear to exist: quality control ERAD and regulated ERAD (21). Quality p97. In addition, Ufd1 and Npl4, which complex with p97, also asso- control ERAD ensures that misfolded proteins, such as mutant rhodop- ciated with IP receptors upon hormonal stimulation. We conclude sin (24) and cystic fibrosis transmembrane conductance regulator (25), that the p97-Ufd1-Npl4 complex couples ubiquitinated IP recep- or unassembled subunits of multiprotein complexes, such as T-cell tors to proteasomal degradation and, thus, plays a key role in IP 3 receptor subunits TCR (26) and CD3- (26, 27), do not accumulate in receptor processing. These data also establish that the p97-Ufd1- the ER and do not transit the secretory pathway. Regulated ERAD, on Npl4 complex mediates endoplasmic reticulum-associated degra- the other hand, accounts for the degradation of native ER proteins in dation in mammalian cells. response to various stimuli. For example, in yeast and mammalian cells, 3-hydroxy 3-methylglutaryl-CoA reductase, an ER membrane protein, is polyubiquitinated and degraded in response to sterols (28, 29). In the Activation of various G protein-coupled receptors (GPCRs) case of IP Rs, putative conformational changes associated with receptor increases phospholipase C activity and generates the second messen- activation (5–7) appear to almost instantaneously render IP Rs suscep- gers inositol 1,4,5-trisphosphate (IP ) and diacylglycerol. IP diffuses 3 3 tible to ubiquitination and degradation (30, 31). This process is medi- into the cytosol and mobilizes Ca by binding to IP receptors (IP Rs) mam 3 3 ated by Ubc7 (32), which catalyzes the ubiquitination of many present in membranes of the endoplasmic reticulum (ER). Hence, IP Rs ERAD substrates (27, 32). Furthermore, as IP Rs are unique in being play a pivotal role in converting cues conveyed by extracellular stimuli rapidly convertible from their native form into ERAD substrates, they into intracellular Ca signals (1, 2). There are three different IP R types provide a valuable system for studying the mechanism of ERAD in (IP R1, IP R2, and IP R3) expressed at various levels in different tissues 3 3 3 mammalian cells. (3, 4). Each IP R type is 2700 amino acids in length and has six mem- An intriguing feature of ERAD substrates is that they have to be translocated from the ER to reach the proteasome. While the mecha- nism involved is poorly understood, several recent studies have impli- * This work was supported by National Institutes of Health Grants DK49194 (to R. J. H. W.) cated a cytosolic ATPase, termed p97 or VCP in mammals and Cdc48p and DK56294 (to G. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked in yeast, in this retrotranslocation process (33–39). p97 is a member of “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. AAA ATPase family and is involved in a wide variety of cellular activi- To whom correspondence should be addressed: Dept. of Pharmacology, SUNY Upstate ties, including vesicular transport, homotypic membrane fusion, disso- Medical University, 750 East Adams St., Syracuse, NY 13210-2339. Tel.: 315-464-7956; Fax: 315-464-8014; E-mail: [email protected]. ciation of a ubiquitinated membrane-tethered transcription factor from The abbreviations used are: GPCR, G protein-coupled receptor; IP , inositol 1,4,5- its non-ubiquitinated binding partner, and proteasome-mediated pro- trisphosphate; IP R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; UPP, ubiquitin-proteasome pathway; ERAD, endoplasmic reticulum-associated deg- tein degradation (40–42). p97 is coupled to these various cellular activ- radation; RNAi, RNA interference; siRNA, short interfering RNA; GnRH, gonadotropin- ities by a set of cofactors or adaptor proteins (40, 42). For example, releasing hormone; ET1, endothelin-1; ALLN, N-acetyl-Leu-Leu-norleucinal; HA, hemagglutinin. VCIP135 and p47 associate with p97 to recruit it to membrane fusion 34530 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 This is an Open Access article under the CC BY license. Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing events (43–45). Likewise, heterodimers of Ufd1 and Npl4 form a com- pended in gel loading buffer (10), electrophoresed, and immunoblotted. plex with p97 and help recruit it to ubiquitinated substrates (46–48). For Rat-1 fibroblasts, cells were grown to near confluence in 15-cm In the present study, we examined whether p97 and its cofactors are diameter dishes and were serum-starved for about 15 h. Cells were then involved in IP R down-regulation. We report that p97, Ufd1, and Npl4 incubated with or without ET1 and after removing culture medium all rapidly associate with IP Rs under conditions that lead to IP R ubiq- were lysed by adding Triton X-100 lysis buffer (50 mM Tris base, 150 mM 3 3 uitination and that this association correlates with the extent to which NaCl, 1% Triton X-100, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl IP Rs are ubiquitinated. Moreover, suppression of p97 expression by flouride, 10 M leupeptin, 10 M pepstatin, 0.2 M soybean trypsin RNA interference (RNAi) impaired the regulated ERAD of IP Rs. Our inhibitor, 1 mM dithiothreitol, pH 8) directly to cell monolayers followed results indicate that the p97-Ufd1-Npl4 complex is required for the by vigorous scraping. After 30 min at 4 °C, lysates were clarified by processing of ubiquitinated IP Rs and, thus, that this complex plays a centrifugation (16,000  g for 10 min at 4 °C), and IP R1 was immuno- 3 3 key role in ERAD in mammalian cells. precipitated and processed in immunoblots as described forT3-1 cells. Levels of IP Rs and Other Proteins—For measurement of IP R down- 3 3 EXPERIMENTAL PROCEDURES regulation, serum-starved Rat-1 cells were incubated with or without ET1 and after removing culture medium were harvested by adding Tri- Materials—T3-1 mouse anterior pituitary gonadotrope cells were ton X-100 lysis buffer directly to cell monolayers followed by vigorous obtained and cultured as described (11). Rat-1 rat fibroblasts were a kind scraping. After 30 min at 4 °C, lysates were cleared by centrifugation gift from Dr. A. Cox, University of North Carolina, and were grown as (16,000  g for 10 min at 4 °C), supernatants were collected, estimated monolayers in Dulbecco’s modified Eagle’s medium supplemented with for protein content, and equal amounts of protein were immunoblotted 10% fetal bovine serum. Cells were fed every other day and subcultured with anti-IP R1 and anti-IP R3. Essentially, identical methods were 3 3 every 3–5 days using 0.25% trypsin, 1 mM EDTA. Antibodies used were: used to monitor the levels of ubiquitin conjugates, p27 and p53, in cell rabbit polyclonal anti-IP R1 (11), mouse monoclonal anti-p97 lysates. (Research Diagnostics Inc.), rabbit polyclonal anti-Npl4 (a kind gift Subcellular Fractionation—Rat-1 cells were harvested by scraping from Dr. Akira Kakizuka, Kyoto University, Kyoto, Japan), rabbit poly- into 155 mM NaCl, 10 mM HEPES, 2 mM EDTA, pH 7.4, pelleted by clonal anti-Sec61 (a kind gift from Dr. T. Rapoport, Harvard Medical centrifugation (1000  g for 6 min at 4 °C), resuspended in homogeni- School, Boston, MA), rabbit polyclonal anti-calnexin (Stressgen Bio- zation buffer (10 mM Tris base, 1 mM EGTA, 0.2 mM phenylmethylsul- technologies Corp.), mouse monoclonal anti-p27 (Santa Cruz Biotech- fonyl flouride, 10 M leupeptin, 10 M pepstatin, 0.2 M soybean trypsin nology Inc.), mouse monoclonal anti-p53 (Calbiochem), mouse mono- inhibitor, 1 mM dithiothreitol, pH 7.4), and disrupted with 40 strokes of clonal anti-ubiquitin (Zymed Laboratories Inc.), mouse monoclonal a Dounce homogenizer. The homogenate was centrifuged (1100 g for anti-HSP90, anti-IP R3, anti-Ufd1 and anti-Ufd2 (BD Transduction 6 min at 4 °C) to pellet nuclei, and the supernatants were re-centrifuged Laboratories), mouse monoclonal anti-hemagglutinin (HA) epitope (100,000  g for 1h at 4 °C). The supernatants from this step were (Covance), and horseradish peroxidase-conjugated secondary antibod- designated as cytosolic fractions, while pellets and nuclei were then ies (Sigma). SDS, Triton X-100, Igepal CA-630, protease inhibitors, solubilized with Triton X-100 lysis buffer and re-centrifuged (16,000 gonadotropin-releasing hormone (GnRH), cycloheximide, and poly- g for 10 min at 4 °C) to obtain membrane and nuclear fractions. Super- brene (hexamethrine bromide) were purchased from Sigma; endo- natants were estimated for protein content and equivalent amounts of thelin-1 (ET1) was from Calbiochem; T4 ligase, BglII, and HindIII TM each fraction were then immunoblotted with anti-ubiquitin. were obtained from New England Biolabs; Precision Plus Protein RNAi Design and Generation of Stable Cell Lines—The pSUPER.retro Standards and dithiothreitol were from Bio-Rad; G418 was from vector (49, 50) was used to introduce short interfering RNA (siRNA) Cellgro; Protein A-Sepharose CL-4B was from Amersham Bio- targeting p97 into Rat-1 fibroblasts. Two 64-base complementary oli- sciences; N-acetyl-Leu-Leu-norleucinal (ALLN) was from Alexis; bort- gonucleotides (forward, 5-gatccccgtaggctatgatgacatcgttcaagagacgat- ezomib (PS-341) was obtained from Millennium Pharmaceuticals Inc. gtcatcatagcctactttttggaaa-3 and reverse, 5-agcttttccaaaaagtaggctatg- (Cambridge, MA); and puromycin was from Clontech. atgacatcgtc-tcttgaacgatgtcatcatagcctacggg-3) were synthesized to Electrophoresis, Immunoblotting, and Quantitation—Samples were contain a 19-nucleotide sequence (bold) corresponding to nucleotides resolved by SDS-polyacrylamide gel electrophoresis and transferred to TM 812–830 of Rattus norvegicus p97 mRNA (GenBank accession num- nitrocellulose essentially as described (10), probed with relevant pri- ber NM_053864), separated from the reverse complement of the same mary antibodies, followed by horseradish peroxidase-conjugated sec- 19 nucleotides by a 9-nucleotide spacer. The annealed product contains ondary antibodies, and immunoreactivity was visualized with Pierce chemiluminescence reagents. Immunoreactivity was quantitated using 5 and 3 overhangs compatible with BglII and HindIII restriction sites, respectively, and was ligated into pSUPER.retro digested with BglII and a Genegnome Imager (Syngene Bio Imaging). Data shown are mean HindIII, generating pSUPER.retro.p97. In parallel, three different con- S.E. or representative of 3 independent experiments. IP R1 Immunoprecipitation—For T3-1 gonadotropes, cells were trol vectors encoding random siRNA (pSUPER.retro.ran1–3) were con- structed using a 19-nucleotide sequence with no known homology to grown to near confluence in 15-cm diameter dishes and were incubated any of the known rat mRNAs. Correct ligation into pSUPER.retro was with or without GnRH. Cells were then harvested by vigorous scraping and pipetting in culture medium, pelleted by centrifugation (1000  g confirmed by restriction digestion and sequencing. To generate infec- tious retroviral stock, pSUPER.retro.p97 or pSUPER.retro.ran1–3 were for 6 min at 4 °C), and solubilized in Igepal CA-630 lysis buffer (50 mM transfected together with pVPack-Eco, which encodes viral envelope, Tris-HCl, 120 mM NaCl, 0.5% Igepal CA-630 (v/v), 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, 10 M pepstatin, 0.2 and pVPack-GP, which encodes Gag-pol (Stratagene), into 293T cells M soybean trypsin inhibitor, 1 mM dithiothreitol, pH 8) for 30 min at using the calcium-phosphate method (51). 72 h posttransfection, cul- 4 °C. Lysates were clarified by centrifugation (16,000  g for 10 min at ture medium was removed and passed through a 0.45-m filter (Nal- 4 °C), and IP R1 was immunoprecipitated by incubating with anti-IP R1 gene). Virus-containing media were aliquoted and frozen at 80 °C 3 3 for 1 h followed by Protein A-Sepharose CL-4B for 3 h. Immunocom- until use. To transduce Rat-1 cells, cells were seeded in a 12-well plate, plexes were washed thoroughly with Igepal CA-630 lysis buffer, resus- and after 24 h, viruses were added along with 8 g/ml polybrene, and OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34531 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 1. The p97-Ufd1-Npl4 complex associ- ates with IP R1 in stimulated cells. A, T3-1 cells were stimulated with GnRH (0.1 M) for the times indicated without (lanes 1– 4) or with 1-h preincu- bation with 1 M bortezomib (lane 5). Cells were then harvested and lysed, and IP R1 was immuno- precipitated with anti-IP R1. Samples were then electrophoresed and immunoblotted with anti- ubiquitin, anti-IP R1, anti-p97, anti-Ufd1, anti- Npl4, and anti-Ufd2. B, Rat-1 cells were incubated for times indicated with 10 nM ET1 without (lanes 1–3) or with 1h preincubation with 1 M bort- ezomib (lanes 4 – 8). Cells were then harvested and lysed, and IP R1 was immunoprecipitated with anti-IP R1. Samples were then electrophoresed and immunoblotted with anti-ubiquitin and anti- IP R1. C, Rat-1 cells were incubated with 100 nM ET1 for the times indicated without or with 1h pre- incubation with 1 M bortezomib. Cells were then harvested, lysates were prepared, and IP R1 immunoreactivity was assessed, quantitated, and expressed as percentage of immunoreactivity at time 0. D and E, Rat-1 cells were incubated with 10 nM ET1 for the times indicated without or with 1M thapsigargin. Cells were then lysed, IP R1 was immunoprecipitated with anti-IP R1, and samples were immunoblotted as described for A. Proteins migrated as follows: polyubiquitinated IP R1 at 275–380 kDa, IP R1 at 260 kDa, p97 at 97 kDa, Ufd1 at 40 kDa, Npl4 at 60 kDa, and Ufd2 at 146 kDa. cells were incubated for8hat37 °C, followed by a change of medium. Measurement of Ca Mobilization—Serum-starved Rat-1 cells were After 48 h, transduced cells were selected with 2.5 g/ml puromycin harvested in and washed with 155 mM NaCl, 10 mM HEPES, 2 mM and after several passages were maintained in 1 g/ml puromycin. EDTA, pH 7.4, resuspended in Krebs-HEPES buffer (30) and then incu- Expression of Exogenous p97—cDNA encoding full-length, amino- bated with 10 M Fura2-AM at 37 °C for 1 h. Cells were centrifuged at terminal HA-tagged mouse p97 (HA-p97) was a kind gift from Dr. 500  g for 2 min, washed twice, incubated again at 37 °C for 30 min, Masaki Matsumoto, Kyushu University, Fukuoka, Japan. To render the again washed twice, resuspended in 2 ml of Krebs-HEPES buffer, placed mRNA encoded by this plasmid refractory to the siRNA targeting in a cuvette, and excited at 340 and 380 nm. Fluorescence emission was endogenous p97, five silent mutations were introduced into the targeted recorded using a computerized LS-50B flourimeter (PerkinElmer Life TM region using the QuikChange kit (Stratagene) and were confirmed by Sciences). 0.1% Triton X-100 and 10 mM EGTA were used to determine sequencing. p97 cells (80% confluent) were transfected with 1 gof the maximum and minimum fluorescence values, respectively. 340/380 either pcDNA3 (vector) or HA-p97 plasmid using 8 l of CytoPure- nm emission ratios were used to calculate intracellular Ca as TM huv (Qbiogene) according to the manufacturer’s instructions. After described (32). 48 h, cells were subcultured and grown in the presence of 500 g/ml RESULTS G418. Sterile cloning cylinders were used to select G418-resistant clones, which were then screened for HA-p97 expression. Five clones Dynamic Association of the p97-Ufd1-Npl4 Complex with IP Rs— expressing high levels of HA-p97 and four vector-transfected clones Initially, we used T3-1 mouse anterior pituitary gonadotropes to were used to analyze IP R ubiquitination and down-regulation. investigate mechanisms involved in GPCR-initiated IP R down-reg- 3 3 34532 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 2. RNAi of p97 in Rat-1 fibroblasts. A, sequence and predicted secondary structure of the short hairpin RNA that yields the siRNA that targets p97 mRNA. B, Rat-1 cells expressing three different control siRNA sequences (lanes 1–3)or p97 siRNA (lanes 4 – 6) were harvested and lysed, and equal amounts of cell protein were electro- phoresed and immunoblotted with anti-p97, anti- HSP90, anti-Ufd2, anti-calnexin, anti-Ufd1, and anti-Sec61. Proteins detected migrated as fol- lows: p97 at 97 kDa, HSP90 at 90 kDa, Ufd2 at 146 kDa, calnexin at 90kDa, Ufd1 at 40 kDa, and Sec61 at 14 kDa. The histogram shows combined quan- titated immunoreactivity from three independent experiments with * indicating significant differ- ences (p  0.05) from ran cell values by Welch’s unpaired t test. C, ran and p97 cells were stimu- lated with 100 nM ET1, and cytosolic free Ca con- centration was recorded. Data shown are mean S.E. of seven independent experiments; any differ- ences between ran and p97 cells were not signifi- cant (p  0.2 by unpaired t test). ulation. These cells express phospholipase C-linked GnRH recep- the U-box protein Ufd2 (52–54) associated with ubiquitinated IP R1. tors, and GnRH induces rapid and robust ubiquitination and down- Intriguingly, the amount of p97-Ufd1-Npl4 and Ufd2 that co-precip- regulation of IP R1, the predominant IP R in this cell type (11). Fig. itated with IP R1 in the presence of bortezomib, when the accumu- 3 3 3 1A shows that GnRH-induced IP R1 ubiquitination peaked at 5 lation of ubiquitinated IP R1 was greatly enhanced (Fig. 1A, lane 5), 3 3 min and thereafter declined as IP R1 was degraded (lanes 1– 4). As is was less than that seen maximally in its absence (Fig. 1A, lane 2). This typical for UPP substrates, inclusion of proteasome inhibitor (bort- likely reflects the fact that bortezomib causes generalized accumu- ezomib) caused the accumulation of ubiquitinated IP R1 and lation of ubiquitin-protein conjugates in T3-1 cells and other cells blocked IP R1 down-regulation (lane 5). Probing for co-immuno- (55) and that under such circumstances, the p97-Ufd1-Npl4 com- precipitating proteins revealed that p97, Ufd1, and Npl4 all associ- plex is deployed to process these conjugates rather than ubiquiti- ated with IP R1 after GnRH stimulation in a manner that correlated nated IP3R1. with the levels of ubiquitinated species (Fig. 1A, lanes 2– 4), indicat- To extend our analysis to other cell types, we examined Rat-1 fibro- ing that the p97-Ufd1-Npl4 complex is recruited to participate in the blasts, since our preliminary experiments indicated that RNAi was fea- processing of ubiquitinated IP R1. Additionally, Fig. 1A shows that sible and efficient in this cell type. Rat-1 cells were found to express OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34533 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing approximately equal amounts of IP R1 and IP R3 and very little IP R2, 3 3 3 and ET1, a potent IP -forming ligand (56), caused IP R1 ubiquitination 3 3 (Fig. 1B) and down-regulation (Fig. 1C). In the absence of bortezomib, IP R1 ubiquitination peaked at 20 min and thereafter declined as IP Rs were degraded (Fig. 1B, lanes 2 and 3), whereas in the presence of bortezomib, ubiquitinated IP R1 accumulated (Fig. 1B, lanes 5–8), and IP R1 down-regulation was blocked (Fig. 1C). Stimulation with ET1 also caused the association of p97, Ufd1, and Ufd2 with IP R1 (Fig. 1D), indicating that the same interactions that occur in GnRH-stimulated T3-1 cells also occur in ET1-stimulated Rat-1 cells. However, we were unable to detect Npl4 co-immunoprecipitation, most likely because of the insensitivity of the Npl4 antibody. Fig. 1E shows that depletion of ER 2 2 Ca with the Ca -ATPase inhibitor thapsigargin (11) blocked both IP R1 ubiquitination and co-precipitation of p97 and Ufd1 in Rat-1 cells. Taken together, these data indicate that in T3-1 and Rat-1 cells the p97-Ufd1-Npl4 complex and Ufd2 are recruited to and participate in the processing of ubiquitinated IP Rs. Specific Inhibition of p97 Expression by RNAi—To investigate the role of p97 in IP R1 processing, we used the pSUPER.retro vector (49, 50) to stably express siRNA targeting p97 mRNA in Rat-1 cells. The p97 siRNA chosen concurred with that used in a recent study to inhibit p97 expression in HeLa cells using chemically synthesized siRNA (57). pSU- PER.retro directs the synthesis of short hairpin RNAs (Fig. 2A) that can be processed by the Dicer enzymatic machinery into active siRNAs (49, 50, 58). We established three cell lines harboring p97 siRNA (p97-1, p97-2 and p97-3, henceforth referred to as p97 cells) and three control cell lines harboring random siRNAs (ran-1, ran-2, and ran-3, henceforth referred to as ran cells). Expression of p97 was reduced by 62  3% in p97 cells (Fig. 2B, lanes 4–6) as compared with ran cells (Fig. 2B, lanes 1–3), or unmodified Rat-1 cells, and this “knockdown” was specific, since the levels of Ufd1, Ufd2, and HSP90 (cytosolic proteins), and Sec61 and calnexin (ER membrane proteins) were unaffected (Fig. 2B). In addition, p97 knockdown did not alter IP R levels (Fig. 4, A and E). The reduction in p97 expression was maintained for 8 passages, and p97 cell growth and morphology were identical to that of ran or unmod- ified Rat-1 cells. p97 expression was not reduced further by re-trans- duction of p97 cells, indicating that 62% knockdown was the maxi- mum possible using this technique. Since we planned to examine ET1- induced IP R ubiquitination and down-regulation in these cells, we also examined whether signaling down-stream of the ET1 receptor was affected by p97 knockdown. Fig. 2C shows this was not the case, since ET1-induced Ca release was not significantly different in ran and p97 cells. Taken together, these data show that stable and specific inhibition of p97 expression is possible in Rat-1 cells and that this does not affect overall cell function. Furthermore, the three p97 cell lines behaved iden- tically, as did the three ran cell lines, and thus, we used them inter- changeably for subsequent experiments. FIGURE 3. p97 knockdown does not perturb the UPP. A, ran and p97 cells were har- We also examined whether p97 knockdown had general effects on the vested, homogenized, and fractionated to prepare cytosol, membrane, and nuclear frac- UPP. ran and p97 cells were disrupted and the steady state level of tions, as well as total cell lysates. Equal amounts of protein from each fraction were then ubiquitin-protein conjugates in different subcellular fractions was electrophoresed and immunoblotted with anti-ubiquitin. B, ran and p97 cells were incu- bated with 25 g/ml cycloheximide for the times indicated, and equal amounts of cell examined (Fig. 3A). However, no differences were detected between ran lysates were electrophoresed and immunoblotted with anti-ubiquitin. C, ran and p97 and p97 cells, indicating that the UPP was not perturbed. Likewise, the cells were incubated without or with 20 g/ml ALLN for 4 h, after which ALLN was removed, and cells were washed and allowed to recover for the times indicated. Equal rate of degradation of ubiquitin-protein conjugates, measured in the amounts of cell protein were then electrophoresed and immunoblotted with anti-p53 presence of cycloheximide, a protein synthesis inhibitor, was the same and anti-p27. in ran and p97 cells (Fig. 3B), showing that the bulk degradation of ubiquitinated proteins was unaffected by p97 knockdown. Further- 63), showed that their accumulation in the presence of ALLN, a protea- more, analysis of p27, a cyclin-dependent kinase inhibitor (59), and p53, some inhibitor, and their degradation following ALLN withdrawal were a tumor suppressor (60), both of which are degraded by the UPP (61– the same in both ran and p97 cells (Fig. 3C). Overall, these data show that 62% knockdown of p97 does not affect general protein turnover K. J. Alzayady and R. J. H. Wojcikiewicz, unpublished data. by the UPP. 34534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 4. p97 is required for IP R processing. A, ran and p97 cells were incubated with 10 nM ET1 for the times indicated. IP R1 was then immuno- precipitated with anti-IP R1 and was electro- phoresed and immunoblotted with anti-ubiquitin and anti-IP R1. B, quantitated ubiquitin immuno- reactivity associated with IP R1 expressed as a per- centage of that associated with IP R1 in ET1-stim- ulated ran cells at 20 min; * denotes p 0.05, when comparing ran and p97 cells at each time point, by Welch’s unpaired t test. C and D, ran and p97 cells were stimulated with 10 nM ET1 for 60 min in the presence of 1 M bortezomib. IP R1 was then immunoprecipitated, processed, and quantitated as described for A and B. E and F, ran and p97 cells were incubated with 10 nM ET1 for the times indi- cated. Cells were then harvested and lysed, and equal amounts of cell protein were electrophore- sed and immunoblotted with anti-IP R1 and anti- IP R3. Quantitated immunoreactivity of IP R1 3 3 (squares) and IP R3 (triangles) are expressed as percentage of immunoreactivity at time 0; * denotes p  0.05, when comparing ran and p97 cells at each time point, by unpaired t test. G, p97 cells stably transfected with either empty vector or HA-p97 and ran cells were harvested and lysed, and equal amounts of cell protein were electro- phoresed and immunoblotted with anti-HA (upper panel), anti-p97 (middle panel), or anti-Ufd2 (lower panel). H and I, p97 cells stably transfected with either empty vector (open circles) or HA-p97 (filled circles) and ran cells (open squares) were stimu- lated and analyzed as in B and F, respectively; * denotes significant differences (p 0.05) from ran cell values. Effects of p97 Knockdown on IP R1 Processing—Fig. 4A, lanes 1–3, Rat-1 cells (Fig. 1C), showing that this process was unaffected by stable shows that ET1-induced IP R1 ubiquitination in ran cells was kinetically expression of control siRNA. However, down-regulation of IP R1 and 3 3 and quantitatively similar to that seen in unmodified Rat-1 cells (Fig. IP R3 was significantly inhibited in p97 cells (Fig. 4, E, lanes 4–6, and F). 1B), indicating that the expression of control siRNA does not affect IP R Together with the enhanced accumulation of ubiquitinated IP R1 in 3 3 processing. In contrast, in p97 cells (Fig. 4A, lanes 4–6), ET1-induced p97 cells, these data indicate that p97 plays a key role in processing accumulation of ubiquitinated IP R1s was significantly enhanced such IP R1 once it has been ubiquitinated. 3 3 that at both 20 and 60 min, the amount of ubiquitinated IP R1s in Exogenous p97 Reverses the Effects of Endogenous p97 Knockdown—To con- p97 cells was approximately twice that seen in ran cells (Fig. 4B). To firm that the defect in IP R processing in p97 cells was truly due to p97 knock- determine whether this effect was due to an increase in the rate of down, we expressed exogenous HA-p97 in p97 cells. Expression of HA-p97 IP R1 ubiquitination or a reduction in the rate of ubiquitinated IP R1 was confirmed in immunoblots with anti-HA (Fig. 4G, upper panel) and anti- 3 3 processing, ran and p97 cells were incubated with ET1 in the pres- p97 (Fig. 4G, middle panel) and raised total p97 immunoreactivity in the five ence of bortezomib to block degradation of ubiquitinated IP R1. celllineschosenforanalysisto885%ofthatseeninrancells.Thegrowthand Under these conditions, IP R1 was ubiquitinated equally in ran and morphology of HA-p97-expressing cells was identical to ran cells, and the p97 cells (Fig. 3, C and D), indicating that the rate of IP R1 ubiquiti- expression level of Ufd2, a representative control protein, was not affected by nation was not enhanced in p97 cells. Thus, the marked accumula- HA-p97 expression (Fig. 4G, lower panel). Importantly, the expression of exog- tion of ubiquitinated IP R1 in p97 cells was due to reduced process- enous p97 restored IP R1 ubiquitination and down-regulation to a level not 3 3 ing of ubiquitinated IP R1. significantly different from that seen in ran cells (Fig. 4, H and I). These data Fig. 4, E and F, show the effect of p97 knockdown on ET1-induced demonstrate that expression of exogenous p97 can overcome the effects of IP R down-regulation. Both IP R1 and IP R3 were down-regulated in endogenous p97 knockdown and confirm that p97 plays a role in IP R 3 3 3 3 ran cells (Fig. 4, E, lanes 1–3, and F) similarly to that seen in unmodified processing. OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34535 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing DISCUSSION cessed. This processing may require a large proportion of the available p97-Ufd1-Npl4 complexes and knocking down p97 will reduce their The ATPase p97 forms a complex with heterodimers of Ufd1 and abundance and thus slow IP R processing. Npl4 (46, 47) and plays a role in the UPP (34, 36–39). Each member of It is intriguing that the maximum p97 knockdown we could obtain the p97-Ufd1-Npl4 complex can bind ubiquitin (35, 46, 47), and with was only62%. This limit may result from a reduction in the viability of regard to ERAD, it has been proposed that p97 initially binds a non- cells in which p97 knockdown was 62%; these cells would not prolif- ubiquitinated segment of ERAD substrates en route from the ER and erate well and would not be well represented in the cell lines we gener- then, in concert with Ufd1 and Npl4, binds polyubiquitin chains as they ated. In this regard, other studies in which endogenous p97 has been are attached to the substrate (35). Additionally, it is thought that p97 interfered with have noted that cell function was perturbed. In neuronal uses ATP hydrolysis to translocate ubiquitinated proteins from the ER PC12 cells overexpressing dominant negative p97, there was general- membrane to the proteasome (34–39). However, these conclusions ized accumulation of ubiquitin-protein conjugates in nuclear and mem- about p97 function are based on data from a limited repertoire of ERAD brane fractions, cytoplasmic vacuolization, ER stress and expansion, substrates often expressed in model systems, and it remains unclear and cell death (64). In another study, transient transfection of HeLa cells what role p97 and its cofactor play in the ERAD of endogenously with synthetic siRNAs to knockdown p97 mRNA by80% caused accu- expressed proteins in mammalian cells. mulation of ubiquitin-protein conjugates, extensive cytoplasmic vacu- In the present study, we provide two lines of evidence that implicate olization, ER expansion, impaired cell proliferation, and apoptosis (57). the p97-Ufd1-Npl4 complex in the regulated ERAD of endogenously It is important to note that the studies described herein are the first in expressed IP Rs. First, it interacted with IP R1 in cells stimulated with 3 3 mammalian cells in which inhibition of endogenous p97 activity has GPCR agonists that induced IP R1 ubiquitination. This interaction was been demonstrated to affect the processing of an ERAD substrate, in the widespread, since it was detected in a variety of cell types, including absence of possible confounding effects from UPP perturbation. Fur- T3-1 mouse gonadotropes stimulated with GnRH (Fig. 1A), Rat-1 thermore, our studies addressed the regulation of endogenous IP Rs by fibroblasts stimulated with ET1 (Fig. 1D), and SH-SY5Y human neuro- endogenous signaling pathways, as opposed to overexpressed proteins blastoma cells stimulated with carbachol, a muscarinic agonist. Fur- activated by non-physiological means. This focus on proteins expressed thermore, the amount of co-precipitating p97-Ufd1-Npl4 complex cor- and activated normally, and the lack of UPP perturbation resulting from related closely with the amount of ubiquitinated IP R1. Interestingly, p97 knockdown means that the results obtained should accurately our data also showed that Ufd2 interacted with ubiquitinated IP R1 in reflect the role of p97 under physiological conditions. parallel with the pattern seen for the p97-Ufd1-Npl4 complex. Ufd2 is a If p97 participates in driving ubiquitinated IP Rs from the ER mem- U-box-containing protein that has been described as a multiubiquitin brane, what else mediates this retrotranslocation? It has recently been chain assembly factor, E4 (52), or as a ubiquitin-protein ligase, E3 (53, shown that the ER membrane protein derlin-1 interacts with ERAD 54). Ufd2 and its homologues interact with ubiquitin-protein conju- substrates (65) and might constitute part of the retrotranslocation com- gates via the U-box domain, a stretch of70 amino acids that is present plex (65). It has also been shown that derlin-1 interacts with VIMP, an in U-box proteins from yeast to man (53). It is also known to interact ER membrane protein that serves as receptor for p97 (66). Thus, VIMP with p97 (53). We are currently investigating the functional relevance of may recruit p97 to ER membranes and bring it to the vicinity of the the interaction of Ufd2 with IP Rs. retrotranslocation complex. Therefore, based on our findings and those The second line of evidence is that p97 knockdown in Rat-1 fibro- of others, we propose the following model. In response to GPCR stim- blasts caused a marked increase in the accumulation of ubiquitinated ulation, IP is liberated and binds to IP Rs, inducing conformational 3 3 IP R1 in ET1-stimulated cells, with concomitant inhibition of IP R 3 3 mam changes that render them susceptible to ubiquitination by Ubc7. down-regulation. These effects could be attributed to inhibition of the Simultaneously, the Ufd1-Npl4 heterodimer and p97, which is tethered degradation of ubiquitinated IP Rs and were not due to perturbation of to the ER membrane by VIMP, interact with ubiquitinated IP Rs and signaling induced by ET1 receptor activation, an increase in basal IP R transport them through the retrotranslocation complex to the protea- levels or alteration of other cell processes. Furthermore, the specificity some. Support for this model comes from preliminary experiments of the effects of p97 knockdown was confirmed by experiments showing showing that derlin-1 co-precipitates with IP R1 in GnRH-stimulated that expression of exogenous HA-p97 in p97 cells restored the degra- T3-1 cells. dation of ubiquitinated IP Rs. Thus, it appears that p97 (and by exten- In summary, the current study provides evidence that the p97-Ufd1- sion, the p97-Ufd1-Npl4 complex) plays a key role in IP R processing Npl4 complex plays a key role in post-ubiquitination but pre-proteaso- after ubiquitination has occurred. The role of the complex is most likely mal processing of IP Rs in response to GPCR activation. These findings in the recognition and translocation of ubiquitinated IP Rs from the ER both further our understanding of the mechanism IP R down-regula- membrane. tion and how ERAD substrates are processed in mammalian cells. 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Involvement of the p97-Ufd1-Npl4 Complex in the Regulated Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors *

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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 280, NO. 41, pp. 34530 –34537, October 14, 2005 © 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Involvement of the p97-Ufd1-Npl4 Complex in the Regulated Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors Received for publication, August 11, 2005 Published, JBC Papers in Press, August 15, 2005, DOI 10.1074/jbc.M508890200 ‡ ‡ § ‡1 Kamil J. Alzayady , Margaret M. Panning , Grant G. Kelley , and Richard J. H. Wojcikiewicz ‡ § From the Departments of Pharmacology and Medicine, SUNY Upstate Medical University, Syracuse, New York 13210-2339 Inositol 1,4,5-trisphosphate (IP ) receptors form tetrameric, IP - brane-spanning regions, a long cytosolic amino-terminal region, and a 3 3 gated channels in endoplasmic reticulum membranes that govern short cytosolic carboxyl-terminal tail (5–7). the release of Ca from this organelle. In response to activation of In response to stimulation of GPCRs that persistently elevate IP certain G protein-coupled receptors that persistently elevate IP concentration, the cellular levels of IP R1–3 are rapidly reduced, a phe- 3 3 concentration, IP receptors are ubiquitinated and degraded by the nomenon termed IP R down-regulation. This has been demonstrated in ubiquitin-proteasome pathway. IP receptor ubiquitination is many mammalian cell lines and in native tissues (3, 8–15), with the mam mediated by the ubiquitin-conjugating enzyme, Ubc7, a compo- consequence that the frequency, amplitude, and duration of elementary 2 2 nent of the endoplasmic reticulum-associated degradation path- Ca puffs are reduced (16), and global Ca signaling is restrained (8, 9, way. However, the mechanism by which ubiquitinated IP receptors 13–16). IP R down-regulation appears to be an adaptation to GPCR are transferred to the proteasome is not known. Here, we examine stimulation that protects cells against the deleterious effects of chronic this process and show in several mammalian cell types that the elevation of cytosolic Ca (17). ATPase p97 associates with IP receptors in response to hormonal IP R down-regulation is mediated by the ubiquitin-proteasome path- stimuli that induce IP receptor ubiquitination. To examine the 3 way (UPP) (10–15). Protein degradation via the UPP has two distinct functional relevance of the p97 interaction with IP receptors, we 3 steps: first, polyubiquitin chains are covalently attached to the targeted stably and specifically reduced p97 protein levels by 62  3% in protein, and second, the polyubiquitinated protein is degraded by the 26 Rat-1 fibroblasts using RNA interference. In these cells, endothelin- S proteasome (18, 19). The process by which ER resident proteins or 1-induced IP receptor degradation was markedly retarded and the 3 proteins traversing the ER are degraded is termed endoplasmic reticu- accumulation of ubiquitinated IP receptors was markedly 3 lum-associated degradation or ERAD (20–23). Two forms of ERAD enhanced. These effects were reversed by expression of exogenous appear to exist: quality control ERAD and regulated ERAD (21). Quality p97. In addition, Ufd1 and Npl4, which complex with p97, also asso- control ERAD ensures that misfolded proteins, such as mutant rhodop- ciated with IP receptors upon hormonal stimulation. We conclude sin (24) and cystic fibrosis transmembrane conductance regulator (25), that the p97-Ufd1-Npl4 complex couples ubiquitinated IP recep- or unassembled subunits of multiprotein complexes, such as T-cell tors to proteasomal degradation and, thus, plays a key role in IP 3 receptor subunits TCR (26) and CD3- (26, 27), do not accumulate in receptor processing. These data also establish that the p97-Ufd1- the ER and do not transit the secretory pathway. Regulated ERAD, on Npl4 complex mediates endoplasmic reticulum-associated degra- the other hand, accounts for the degradation of native ER proteins in dation in mammalian cells. response to various stimuli. For example, in yeast and mammalian cells, 3-hydroxy 3-methylglutaryl-CoA reductase, an ER membrane protein, is polyubiquitinated and degraded in response to sterols (28, 29). In the Activation of various G protein-coupled receptors (GPCRs) case of IP Rs, putative conformational changes associated with receptor increases phospholipase C activity and generates the second messen- activation (5–7) appear to almost instantaneously render IP Rs suscep- gers inositol 1,4,5-trisphosphate (IP ) and diacylglycerol. IP diffuses 3 3 tible to ubiquitination and degradation (30, 31). This process is medi- into the cytosol and mobilizes Ca by binding to IP receptors (IP Rs) mam 3 3 ated by Ubc7 (32), which catalyzes the ubiquitination of many present in membranes of the endoplasmic reticulum (ER). Hence, IP Rs ERAD substrates (27, 32). Furthermore, as IP Rs are unique in being play a pivotal role in converting cues conveyed by extracellular stimuli rapidly convertible from their native form into ERAD substrates, they into intracellular Ca signals (1, 2). There are three different IP R types provide a valuable system for studying the mechanism of ERAD in (IP R1, IP R2, and IP R3) expressed at various levels in different tissues 3 3 3 mammalian cells. (3, 4). Each IP R type is 2700 amino acids in length and has six mem- An intriguing feature of ERAD substrates is that they have to be translocated from the ER to reach the proteasome. While the mecha- nism involved is poorly understood, several recent studies have impli- * This work was supported by National Institutes of Health Grants DK49194 (to R. J. H. W.) cated a cytosolic ATPase, termed p97 or VCP in mammals and Cdc48p and DK56294 (to G. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked in yeast, in this retrotranslocation process (33–39). p97 is a member of “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. AAA ATPase family and is involved in a wide variety of cellular activi- To whom correspondence should be addressed: Dept. of Pharmacology, SUNY Upstate ties, including vesicular transport, homotypic membrane fusion, disso- Medical University, 750 East Adams St., Syracuse, NY 13210-2339. Tel.: 315-464-7956; Fax: 315-464-8014; E-mail: [email protected]. ciation of a ubiquitinated membrane-tethered transcription factor from The abbreviations used are: GPCR, G protein-coupled receptor; IP , inositol 1,4,5- its non-ubiquitinated binding partner, and proteasome-mediated pro- trisphosphate; IP R, inositol 1,4,5-trisphosphate receptor; ER, endoplasmic reticulum; UPP, ubiquitin-proteasome pathway; ERAD, endoplasmic reticulum-associated deg- tein degradation (40–42). p97 is coupled to these various cellular activ- radation; RNAi, RNA interference; siRNA, short interfering RNA; GnRH, gonadotropin- ities by a set of cofactors or adaptor proteins (40, 42). For example, releasing hormone; ET1, endothelin-1; ALLN, N-acetyl-Leu-Leu-norleucinal; HA, hemagglutinin. VCIP135 and p47 associate with p97 to recruit it to membrane fusion 34530 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 This is an Open Access article under the CC BY license. Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing events (43–45). Likewise, heterodimers of Ufd1 and Npl4 form a com- pended in gel loading buffer (10), electrophoresed, and immunoblotted. plex with p97 and help recruit it to ubiquitinated substrates (46–48). For Rat-1 fibroblasts, cells were grown to near confluence in 15-cm In the present study, we examined whether p97 and its cofactors are diameter dishes and were serum-starved for about 15 h. Cells were then involved in IP R down-regulation. We report that p97, Ufd1, and Npl4 incubated with or without ET1 and after removing culture medium all rapidly associate with IP Rs under conditions that lead to IP R ubiq- were lysed by adding Triton X-100 lysis buffer (50 mM Tris base, 150 mM 3 3 uitination and that this association correlates with the extent to which NaCl, 1% Triton X-100, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl IP Rs are ubiquitinated. Moreover, suppression of p97 expression by flouride, 10 M leupeptin, 10 M pepstatin, 0.2 M soybean trypsin RNA interference (RNAi) impaired the regulated ERAD of IP Rs. Our inhibitor, 1 mM dithiothreitol, pH 8) directly to cell monolayers followed results indicate that the p97-Ufd1-Npl4 complex is required for the by vigorous scraping. After 30 min at 4 °C, lysates were clarified by processing of ubiquitinated IP Rs and, thus, that this complex plays a centrifugation (16,000  g for 10 min at 4 °C), and IP R1 was immuno- 3 3 key role in ERAD in mammalian cells. precipitated and processed in immunoblots as described forT3-1 cells. Levels of IP Rs and Other Proteins—For measurement of IP R down- 3 3 EXPERIMENTAL PROCEDURES regulation, serum-starved Rat-1 cells were incubated with or without ET1 and after removing culture medium were harvested by adding Tri- Materials—T3-1 mouse anterior pituitary gonadotrope cells were ton X-100 lysis buffer directly to cell monolayers followed by vigorous obtained and cultured as described (11). Rat-1 rat fibroblasts were a kind scraping. After 30 min at 4 °C, lysates were cleared by centrifugation gift from Dr. A. Cox, University of North Carolina, and were grown as (16,000  g for 10 min at 4 °C), supernatants were collected, estimated monolayers in Dulbecco’s modified Eagle’s medium supplemented with for protein content, and equal amounts of protein were immunoblotted 10% fetal bovine serum. Cells were fed every other day and subcultured with anti-IP R1 and anti-IP R3. Essentially, identical methods were 3 3 every 3–5 days using 0.25% trypsin, 1 mM EDTA. Antibodies used were: used to monitor the levels of ubiquitin conjugates, p27 and p53, in cell rabbit polyclonal anti-IP R1 (11), mouse monoclonal anti-p97 lysates. (Research Diagnostics Inc.), rabbit polyclonal anti-Npl4 (a kind gift Subcellular Fractionation—Rat-1 cells were harvested by scraping from Dr. Akira Kakizuka, Kyoto University, Kyoto, Japan), rabbit poly- into 155 mM NaCl, 10 mM HEPES, 2 mM EDTA, pH 7.4, pelleted by clonal anti-Sec61 (a kind gift from Dr. T. Rapoport, Harvard Medical centrifugation (1000  g for 6 min at 4 °C), resuspended in homogeni- School, Boston, MA), rabbit polyclonal anti-calnexin (Stressgen Bio- zation buffer (10 mM Tris base, 1 mM EGTA, 0.2 mM phenylmethylsul- technologies Corp.), mouse monoclonal anti-p27 (Santa Cruz Biotech- fonyl flouride, 10 M leupeptin, 10 M pepstatin, 0.2 M soybean trypsin nology Inc.), mouse monoclonal anti-p53 (Calbiochem), mouse mono- inhibitor, 1 mM dithiothreitol, pH 7.4), and disrupted with 40 strokes of clonal anti-ubiquitin (Zymed Laboratories Inc.), mouse monoclonal a Dounce homogenizer. The homogenate was centrifuged (1100 g for anti-HSP90, anti-IP R3, anti-Ufd1 and anti-Ufd2 (BD Transduction 6 min at 4 °C) to pellet nuclei, and the supernatants were re-centrifuged Laboratories), mouse monoclonal anti-hemagglutinin (HA) epitope (100,000  g for 1h at 4 °C). The supernatants from this step were (Covance), and horseradish peroxidase-conjugated secondary antibod- designated as cytosolic fractions, while pellets and nuclei were then ies (Sigma). SDS, Triton X-100, Igepal CA-630, protease inhibitors, solubilized with Triton X-100 lysis buffer and re-centrifuged (16,000 gonadotropin-releasing hormone (GnRH), cycloheximide, and poly- g for 10 min at 4 °C) to obtain membrane and nuclear fractions. Super- brene (hexamethrine bromide) were purchased from Sigma; endo- natants were estimated for protein content and equivalent amounts of thelin-1 (ET1) was from Calbiochem; T4 ligase, BglII, and HindIII TM each fraction were then immunoblotted with anti-ubiquitin. were obtained from New England Biolabs; Precision Plus Protein RNAi Design and Generation of Stable Cell Lines—The pSUPER.retro Standards and dithiothreitol were from Bio-Rad; G418 was from vector (49, 50) was used to introduce short interfering RNA (siRNA) Cellgro; Protein A-Sepharose CL-4B was from Amersham Bio- targeting p97 into Rat-1 fibroblasts. Two 64-base complementary oli- sciences; N-acetyl-Leu-Leu-norleucinal (ALLN) was from Alexis; bort- gonucleotides (forward, 5-gatccccgtaggctatgatgacatcgttcaagagacgat- ezomib (PS-341) was obtained from Millennium Pharmaceuticals Inc. gtcatcatagcctactttttggaaa-3 and reverse, 5-agcttttccaaaaagtaggctatg- (Cambridge, MA); and puromycin was from Clontech. atgacatcgtc-tcttgaacgatgtcatcatagcctacggg-3) were synthesized to Electrophoresis, Immunoblotting, and Quantitation—Samples were contain a 19-nucleotide sequence (bold) corresponding to nucleotides resolved by SDS-polyacrylamide gel electrophoresis and transferred to TM 812–830 of Rattus norvegicus p97 mRNA (GenBank accession num- nitrocellulose essentially as described (10), probed with relevant pri- ber NM_053864), separated from the reverse complement of the same mary antibodies, followed by horseradish peroxidase-conjugated sec- 19 nucleotides by a 9-nucleotide spacer. The annealed product contains ondary antibodies, and immunoreactivity was visualized with Pierce chemiluminescence reagents. Immunoreactivity was quantitated using 5 and 3 overhangs compatible with BglII and HindIII restriction sites, respectively, and was ligated into pSUPER.retro digested with BglII and a Genegnome Imager (Syngene Bio Imaging). Data shown are mean HindIII, generating pSUPER.retro.p97. In parallel, three different con- S.E. or representative of 3 independent experiments. IP R1 Immunoprecipitation—For T3-1 gonadotropes, cells were trol vectors encoding random siRNA (pSUPER.retro.ran1–3) were con- structed using a 19-nucleotide sequence with no known homology to grown to near confluence in 15-cm diameter dishes and were incubated any of the known rat mRNAs. Correct ligation into pSUPER.retro was with or without GnRH. Cells were then harvested by vigorous scraping and pipetting in culture medium, pelleted by centrifugation (1000  g confirmed by restriction digestion and sequencing. To generate infec- tious retroviral stock, pSUPER.retro.p97 or pSUPER.retro.ran1–3 were for 6 min at 4 °C), and solubilized in Igepal CA-630 lysis buffer (50 mM transfected together with pVPack-Eco, which encodes viral envelope, Tris-HCl, 120 mM NaCl, 0.5% Igepal CA-630 (v/v), 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, 10 M pepstatin, 0.2 and pVPack-GP, which encodes Gag-pol (Stratagene), into 293T cells M soybean trypsin inhibitor, 1 mM dithiothreitol, pH 8) for 30 min at using the calcium-phosphate method (51). 72 h posttransfection, cul- 4 °C. Lysates were clarified by centrifugation (16,000  g for 10 min at ture medium was removed and passed through a 0.45-m filter (Nal- 4 °C), and IP R1 was immunoprecipitated by incubating with anti-IP R1 gene). Virus-containing media were aliquoted and frozen at 80 °C 3 3 for 1 h followed by Protein A-Sepharose CL-4B for 3 h. Immunocom- until use. To transduce Rat-1 cells, cells were seeded in a 12-well plate, plexes were washed thoroughly with Igepal CA-630 lysis buffer, resus- and after 24 h, viruses were added along with 8 g/ml polybrene, and OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34531 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 1. The p97-Ufd1-Npl4 complex associ- ates with IP R1 in stimulated cells. A, T3-1 cells were stimulated with GnRH (0.1 M) for the times indicated without (lanes 1– 4) or with 1-h preincu- bation with 1 M bortezomib (lane 5). Cells were then harvested and lysed, and IP R1 was immuno- precipitated with anti-IP R1. Samples were then electrophoresed and immunoblotted with anti- ubiquitin, anti-IP R1, anti-p97, anti-Ufd1, anti- Npl4, and anti-Ufd2. B, Rat-1 cells were incubated for times indicated with 10 nM ET1 without (lanes 1–3) or with 1h preincubation with 1 M bort- ezomib (lanes 4 – 8). Cells were then harvested and lysed, and IP R1 was immunoprecipitated with anti-IP R1. Samples were then electrophoresed and immunoblotted with anti-ubiquitin and anti- IP R1. C, Rat-1 cells were incubated with 100 nM ET1 for the times indicated without or with 1h pre- incubation with 1 M bortezomib. Cells were then harvested, lysates were prepared, and IP R1 immunoreactivity was assessed, quantitated, and expressed as percentage of immunoreactivity at time 0. D and E, Rat-1 cells were incubated with 10 nM ET1 for the times indicated without or with 1M thapsigargin. Cells were then lysed, IP R1 was immunoprecipitated with anti-IP R1, and samples were immunoblotted as described for A. Proteins migrated as follows: polyubiquitinated IP R1 at 275–380 kDa, IP R1 at 260 kDa, p97 at 97 kDa, Ufd1 at 40 kDa, Npl4 at 60 kDa, and Ufd2 at 146 kDa. cells were incubated for8hat37 °C, followed by a change of medium. Measurement of Ca Mobilization—Serum-starved Rat-1 cells were After 48 h, transduced cells were selected with 2.5 g/ml puromycin harvested in and washed with 155 mM NaCl, 10 mM HEPES, 2 mM and after several passages were maintained in 1 g/ml puromycin. EDTA, pH 7.4, resuspended in Krebs-HEPES buffer (30) and then incu- Expression of Exogenous p97—cDNA encoding full-length, amino- bated with 10 M Fura2-AM at 37 °C for 1 h. Cells were centrifuged at terminal HA-tagged mouse p97 (HA-p97) was a kind gift from Dr. 500  g for 2 min, washed twice, incubated again at 37 °C for 30 min, Masaki Matsumoto, Kyushu University, Fukuoka, Japan. To render the again washed twice, resuspended in 2 ml of Krebs-HEPES buffer, placed mRNA encoded by this plasmid refractory to the siRNA targeting in a cuvette, and excited at 340 and 380 nm. Fluorescence emission was endogenous p97, five silent mutations were introduced into the targeted recorded using a computerized LS-50B flourimeter (PerkinElmer Life TM region using the QuikChange kit (Stratagene) and were confirmed by Sciences). 0.1% Triton X-100 and 10 mM EGTA were used to determine sequencing. p97 cells (80% confluent) were transfected with 1 gof the maximum and minimum fluorescence values, respectively. 340/380 either pcDNA3 (vector) or HA-p97 plasmid using 8 l of CytoPure- nm emission ratios were used to calculate intracellular Ca as TM huv (Qbiogene) according to the manufacturer’s instructions. After described (32). 48 h, cells were subcultured and grown in the presence of 500 g/ml RESULTS G418. Sterile cloning cylinders were used to select G418-resistant clones, which were then screened for HA-p97 expression. Five clones Dynamic Association of the p97-Ufd1-Npl4 Complex with IP Rs— expressing high levels of HA-p97 and four vector-transfected clones Initially, we used T3-1 mouse anterior pituitary gonadotropes to were used to analyze IP R ubiquitination and down-regulation. investigate mechanisms involved in GPCR-initiated IP R down-reg- 3 3 34532 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 2. RNAi of p97 in Rat-1 fibroblasts. A, sequence and predicted secondary structure of the short hairpin RNA that yields the siRNA that targets p97 mRNA. B, Rat-1 cells expressing three different control siRNA sequences (lanes 1–3)or p97 siRNA (lanes 4 – 6) were harvested and lysed, and equal amounts of cell protein were electro- phoresed and immunoblotted with anti-p97, anti- HSP90, anti-Ufd2, anti-calnexin, anti-Ufd1, and anti-Sec61. Proteins detected migrated as fol- lows: p97 at 97 kDa, HSP90 at 90 kDa, Ufd2 at 146 kDa, calnexin at 90kDa, Ufd1 at 40 kDa, and Sec61 at 14 kDa. The histogram shows combined quan- titated immunoreactivity from three independent experiments with * indicating significant differ- ences (p  0.05) from ran cell values by Welch’s unpaired t test. C, ran and p97 cells were stimu- lated with 100 nM ET1, and cytosolic free Ca con- centration was recorded. Data shown are mean S.E. of seven independent experiments; any differ- ences between ran and p97 cells were not signifi- cant (p  0.2 by unpaired t test). ulation. These cells express phospholipase C-linked GnRH recep- the U-box protein Ufd2 (52–54) associated with ubiquitinated IP R1. tors, and GnRH induces rapid and robust ubiquitination and down- Intriguingly, the amount of p97-Ufd1-Npl4 and Ufd2 that co-precip- regulation of IP R1, the predominant IP R in this cell type (11). Fig. itated with IP R1 in the presence of bortezomib, when the accumu- 3 3 3 1A shows that GnRH-induced IP R1 ubiquitination peaked at 5 lation of ubiquitinated IP R1 was greatly enhanced (Fig. 1A, lane 5), 3 3 min and thereafter declined as IP R1 was degraded (lanes 1– 4). As is was less than that seen maximally in its absence (Fig. 1A, lane 2). This typical for UPP substrates, inclusion of proteasome inhibitor (bort- likely reflects the fact that bortezomib causes generalized accumu- ezomib) caused the accumulation of ubiquitinated IP R1 and lation of ubiquitin-protein conjugates in T3-1 cells and other cells blocked IP R1 down-regulation (lane 5). Probing for co-immuno- (55) and that under such circumstances, the p97-Ufd1-Npl4 com- precipitating proteins revealed that p97, Ufd1, and Npl4 all associ- plex is deployed to process these conjugates rather than ubiquiti- ated with IP R1 after GnRH stimulation in a manner that correlated nated IP3R1. with the levels of ubiquitinated species (Fig. 1A, lanes 2– 4), indicat- To extend our analysis to other cell types, we examined Rat-1 fibro- ing that the p97-Ufd1-Npl4 complex is recruited to participate in the blasts, since our preliminary experiments indicated that RNAi was fea- processing of ubiquitinated IP R1. Additionally, Fig. 1A shows that sible and efficient in this cell type. Rat-1 cells were found to express OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34533 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing approximately equal amounts of IP R1 and IP R3 and very little IP R2, 3 3 3 and ET1, a potent IP -forming ligand (56), caused IP R1 ubiquitination 3 3 (Fig. 1B) and down-regulation (Fig. 1C). In the absence of bortezomib, IP R1 ubiquitination peaked at 20 min and thereafter declined as IP Rs were degraded (Fig. 1B, lanes 2 and 3), whereas in the presence of bortezomib, ubiquitinated IP R1 accumulated (Fig. 1B, lanes 5–8), and IP R1 down-regulation was blocked (Fig. 1C). Stimulation with ET1 also caused the association of p97, Ufd1, and Ufd2 with IP R1 (Fig. 1D), indicating that the same interactions that occur in GnRH-stimulated T3-1 cells also occur in ET1-stimulated Rat-1 cells. However, we were unable to detect Npl4 co-immunoprecipitation, most likely because of the insensitivity of the Npl4 antibody. Fig. 1E shows that depletion of ER 2 2 Ca with the Ca -ATPase inhibitor thapsigargin (11) blocked both IP R1 ubiquitination and co-precipitation of p97 and Ufd1 in Rat-1 cells. Taken together, these data indicate that in T3-1 and Rat-1 cells the p97-Ufd1-Npl4 complex and Ufd2 are recruited to and participate in the processing of ubiquitinated IP Rs. Specific Inhibition of p97 Expression by RNAi—To investigate the role of p97 in IP R1 processing, we used the pSUPER.retro vector (49, 50) to stably express siRNA targeting p97 mRNA in Rat-1 cells. The p97 siRNA chosen concurred with that used in a recent study to inhibit p97 expression in HeLa cells using chemically synthesized siRNA (57). pSU- PER.retro directs the synthesis of short hairpin RNAs (Fig. 2A) that can be processed by the Dicer enzymatic machinery into active siRNAs (49, 50, 58). We established three cell lines harboring p97 siRNA (p97-1, p97-2 and p97-3, henceforth referred to as p97 cells) and three control cell lines harboring random siRNAs (ran-1, ran-2, and ran-3, henceforth referred to as ran cells). Expression of p97 was reduced by 62  3% in p97 cells (Fig. 2B, lanes 4–6) as compared with ran cells (Fig. 2B, lanes 1–3), or unmodified Rat-1 cells, and this “knockdown” was specific, since the levels of Ufd1, Ufd2, and HSP90 (cytosolic proteins), and Sec61 and calnexin (ER membrane proteins) were unaffected (Fig. 2B). In addition, p97 knockdown did not alter IP R levels (Fig. 4, A and E). The reduction in p97 expression was maintained for 8 passages, and p97 cell growth and morphology were identical to that of ran or unmod- ified Rat-1 cells. p97 expression was not reduced further by re-trans- duction of p97 cells, indicating that 62% knockdown was the maxi- mum possible using this technique. Since we planned to examine ET1- induced IP R ubiquitination and down-regulation in these cells, we also examined whether signaling down-stream of the ET1 receptor was affected by p97 knockdown. Fig. 2C shows this was not the case, since ET1-induced Ca release was not significantly different in ran and p97 cells. Taken together, these data show that stable and specific inhibition of p97 expression is possible in Rat-1 cells and that this does not affect overall cell function. Furthermore, the three p97 cell lines behaved iden- tically, as did the three ran cell lines, and thus, we used them inter- changeably for subsequent experiments. FIGURE 3. p97 knockdown does not perturb the UPP. A, ran and p97 cells were har- We also examined whether p97 knockdown had general effects on the vested, homogenized, and fractionated to prepare cytosol, membrane, and nuclear frac- UPP. ran and p97 cells were disrupted and the steady state level of tions, as well as total cell lysates. Equal amounts of protein from each fraction were then ubiquitin-protein conjugates in different subcellular fractions was electrophoresed and immunoblotted with anti-ubiquitin. B, ran and p97 cells were incu- bated with 25 g/ml cycloheximide for the times indicated, and equal amounts of cell examined (Fig. 3A). However, no differences were detected between ran lysates were electrophoresed and immunoblotted with anti-ubiquitin. C, ran and p97 and p97 cells, indicating that the UPP was not perturbed. Likewise, the cells were incubated without or with 20 g/ml ALLN for 4 h, after which ALLN was removed, and cells were washed and allowed to recover for the times indicated. Equal rate of degradation of ubiquitin-protein conjugates, measured in the amounts of cell protein were then electrophoresed and immunoblotted with anti-p53 presence of cycloheximide, a protein synthesis inhibitor, was the same and anti-p27. in ran and p97 cells (Fig. 3B), showing that the bulk degradation of ubiquitinated proteins was unaffected by p97 knockdown. Further- 63), showed that their accumulation in the presence of ALLN, a protea- more, analysis of p27, a cyclin-dependent kinase inhibitor (59), and p53, some inhibitor, and their degradation following ALLN withdrawal were a tumor suppressor (60), both of which are degraded by the UPP (61– the same in both ran and p97 cells (Fig. 3C). Overall, these data show that 62% knockdown of p97 does not affect general protein turnover K. J. Alzayady and R. J. H. Wojcikiewicz, unpublished data. by the UPP. 34534 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 280 • NUMBER 41 •OCTOBER 14, 2005 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing FIGURE 4. p97 is required for IP R processing. A, ran and p97 cells were incubated with 10 nM ET1 for the times indicated. IP R1 was then immuno- precipitated with anti-IP R1 and was electro- phoresed and immunoblotted with anti-ubiquitin and anti-IP R1. B, quantitated ubiquitin immuno- reactivity associated with IP R1 expressed as a per- centage of that associated with IP R1 in ET1-stim- ulated ran cells at 20 min; * denotes p 0.05, when comparing ran and p97 cells at each time point, by Welch’s unpaired t test. C and D, ran and p97 cells were stimulated with 10 nM ET1 for 60 min in the presence of 1 M bortezomib. IP R1 was then immunoprecipitated, processed, and quantitated as described for A and B. E and F, ran and p97 cells were incubated with 10 nM ET1 for the times indi- cated. Cells were then harvested and lysed, and equal amounts of cell protein were electrophore- sed and immunoblotted with anti-IP R1 and anti- IP R3. Quantitated immunoreactivity of IP R1 3 3 (squares) and IP R3 (triangles) are expressed as percentage of immunoreactivity at time 0; * denotes p  0.05, when comparing ran and p97 cells at each time point, by unpaired t test. G, p97 cells stably transfected with either empty vector or HA-p97 and ran cells were harvested and lysed, and equal amounts of cell protein were electro- phoresed and immunoblotted with anti-HA (upper panel), anti-p97 (middle panel), or anti-Ufd2 (lower panel). H and I, p97 cells stably transfected with either empty vector (open circles) or HA-p97 (filled circles) and ran cells (open squares) were stimu- lated and analyzed as in B and F, respectively; * denotes significant differences (p 0.05) from ran cell values. Effects of p97 Knockdown on IP R1 Processing—Fig. 4A, lanes 1–3, Rat-1 cells (Fig. 1C), showing that this process was unaffected by stable shows that ET1-induced IP R1 ubiquitination in ran cells was kinetically expression of control siRNA. However, down-regulation of IP R1 and 3 3 and quantitatively similar to that seen in unmodified Rat-1 cells (Fig. IP R3 was significantly inhibited in p97 cells (Fig. 4, E, lanes 4–6, and F). 1B), indicating that the expression of control siRNA does not affect IP R Together with the enhanced accumulation of ubiquitinated IP R1 in 3 3 processing. In contrast, in p97 cells (Fig. 4A, lanes 4–6), ET1-induced p97 cells, these data indicate that p97 plays a key role in processing accumulation of ubiquitinated IP R1s was significantly enhanced such IP R1 once it has been ubiquitinated. 3 3 that at both 20 and 60 min, the amount of ubiquitinated IP R1s in Exogenous p97 Reverses the Effects of Endogenous p97 Knockdown—To con- p97 cells was approximately twice that seen in ran cells (Fig. 4B). To firm that the defect in IP R processing in p97 cells was truly due to p97 knock- determine whether this effect was due to an increase in the rate of down, we expressed exogenous HA-p97 in p97 cells. Expression of HA-p97 IP R1 ubiquitination or a reduction in the rate of ubiquitinated IP R1 was confirmed in immunoblots with anti-HA (Fig. 4G, upper panel) and anti- 3 3 processing, ran and p97 cells were incubated with ET1 in the pres- p97 (Fig. 4G, middle panel) and raised total p97 immunoreactivity in the five ence of bortezomib to block degradation of ubiquitinated IP R1. celllineschosenforanalysisto885%ofthatseeninrancells.Thegrowthand Under these conditions, IP R1 was ubiquitinated equally in ran and morphology of HA-p97-expressing cells was identical to ran cells, and the p97 cells (Fig. 3, C and D), indicating that the rate of IP R1 ubiquiti- expression level of Ufd2, a representative control protein, was not affected by nation was not enhanced in p97 cells. Thus, the marked accumula- HA-p97 expression (Fig. 4G, lower panel). Importantly, the expression of exog- tion of ubiquitinated IP R1 in p97 cells was due to reduced process- enous p97 restored IP R1 ubiquitination and down-regulation to a level not 3 3 ing of ubiquitinated IP R1. significantly different from that seen in ran cells (Fig. 4, H and I). These data Fig. 4, E and F, show the effect of p97 knockdown on ET1-induced demonstrate that expression of exogenous p97 can overcome the effects of IP R down-regulation. Both IP R1 and IP R3 were down-regulated in endogenous p97 knockdown and confirm that p97 plays a role in IP R 3 3 3 3 ran cells (Fig. 4, E, lanes 1–3, and F) similarly to that seen in unmodified processing. OCTOBER 14, 2005• VOLUME 280 • NUMBER 41 JOURNAL OF BIOLOGICAL CHEMISTRY 34535 Role of the p97-Ufd1-Npl4 Complex in IP Receptor Processing DISCUSSION cessed. This processing may require a large proportion of the available p97-Ufd1-Npl4 complexes and knocking down p97 will reduce their The ATPase p97 forms a complex with heterodimers of Ufd1 and abundance and thus slow IP R processing. Npl4 (46, 47) and plays a role in the UPP (34, 36–39). Each member of It is intriguing that the maximum p97 knockdown we could obtain the p97-Ufd1-Npl4 complex can bind ubiquitin (35, 46, 47), and with was only62%. This limit may result from a reduction in the viability of regard to ERAD, it has been proposed that p97 initially binds a non- cells in which p97 knockdown was 62%; these cells would not prolif- ubiquitinated segment of ERAD substrates en route from the ER and erate well and would not be well represented in the cell lines we gener- then, in concert with Ufd1 and Npl4, binds polyubiquitin chains as they ated. In this regard, other studies in which endogenous p97 has been are attached to the substrate (35). Additionally, it is thought that p97 interfered with have noted that cell function was perturbed. In neuronal uses ATP hydrolysis to translocate ubiquitinated proteins from the ER PC12 cells overexpressing dominant negative p97, there was general- membrane to the proteasome (34–39). However, these conclusions ized accumulation of ubiquitin-protein conjugates in nuclear and mem- about p97 function are based on data from a limited repertoire of ERAD brane fractions, cytoplasmic vacuolization, ER stress and expansion, substrates often expressed in model systems, and it remains unclear and cell death (64). In another study, transient transfection of HeLa cells what role p97 and its cofactor play in the ERAD of endogenously with synthetic siRNAs to knockdown p97 mRNA by80% caused accu- expressed proteins in mammalian cells. mulation of ubiquitin-protein conjugates, extensive cytoplasmic vacu- In the present study, we provide two lines of evidence that implicate olization, ER expansion, impaired cell proliferation, and apoptosis (57). the p97-Ufd1-Npl4 complex in the regulated ERAD of endogenously It is important to note that the studies described herein are the first in expressed IP Rs. First, it interacted with IP R1 in cells stimulated with 3 3 mammalian cells in which inhibition of endogenous p97 activity has GPCR agonists that induced IP R1 ubiquitination. This interaction was been demonstrated to affect the processing of an ERAD substrate, in the widespread, since it was detected in a variety of cell types, including absence of possible confounding effects from UPP perturbation. Fur- T3-1 mouse gonadotropes stimulated with GnRH (Fig. 1A), Rat-1 thermore, our studies addressed the regulation of endogenous IP Rs by fibroblasts stimulated with ET1 (Fig. 1D), and SH-SY5Y human neuro- endogenous signaling pathways, as opposed to overexpressed proteins blastoma cells stimulated with carbachol, a muscarinic agonist. Fur- activated by non-physiological means. This focus on proteins expressed thermore, the amount of co-precipitating p97-Ufd1-Npl4 complex cor- and activated normally, and the lack of UPP perturbation resulting from related closely with the amount of ubiquitinated IP R1. Interestingly, p97 knockdown means that the results obtained should accurately our data also showed that Ufd2 interacted with ubiquitinated IP R1 in reflect the role of p97 under physiological conditions. parallel with the pattern seen for the p97-Ufd1-Npl4 complex. Ufd2 is a If p97 participates in driving ubiquitinated IP Rs from the ER mem- U-box-containing protein that has been described as a multiubiquitin brane, what else mediates this retrotranslocation? It has recently been chain assembly factor, E4 (52), or as a ubiquitin-protein ligase, E3 (53, shown that the ER membrane protein derlin-1 interacts with ERAD 54). Ufd2 and its homologues interact with ubiquitin-protein conju- substrates (65) and might constitute part of the retrotranslocation com- gates via the U-box domain, a stretch of70 amino acids that is present plex (65). It has also been shown that derlin-1 interacts with VIMP, an in U-box proteins from yeast to man (53). It is also known to interact ER membrane protein that serves as receptor for p97 (66). Thus, VIMP with p97 (53). We are currently investigating the functional relevance of may recruit p97 to ER membranes and bring it to the vicinity of the the interaction of Ufd2 with IP Rs. retrotranslocation complex. Therefore, based on our findings and those The second line of evidence is that p97 knockdown in Rat-1 fibro- of others, we propose the following model. In response to GPCR stim- blasts caused a marked increase in the accumulation of ubiquitinated ulation, IP is liberated and binds to IP Rs, inducing conformational 3 3 IP R1 in ET1-stimulated cells, with concomitant inhibition of IP R 3 3 mam changes that render them susceptible to ubiquitination by Ubc7. down-regulation. These effects could be attributed to inhibition of the Simultaneously, the Ufd1-Npl4 heterodimer and p97, which is tethered degradation of ubiquitinated IP Rs and were not due to perturbation of to the ER membrane by VIMP, interact with ubiquitinated IP Rs and signaling induced by ET1 receptor activation, an increase in basal IP R transport them through the retrotranslocation complex to the protea- levels or alteration of other cell processes. Furthermore, the specificity some. Support for this model comes from preliminary experiments of the effects of p97 knockdown was confirmed by experiments showing showing that derlin-1 co-precipitates with IP R1 in GnRH-stimulated that expression of exogenous HA-p97 in p97 cells restored the degra- T3-1 cells. dation of ubiquitinated IP Rs. Thus, it appears that p97 (and by exten- In summary, the current study provides evidence that the p97-Ufd1- sion, the p97-Ufd1-Npl4 complex) plays a key role in IP R processing Npl4 complex plays a key role in post-ubiquitination but pre-proteaso- after ubiquitination has occurred. The role of the complex is most likely mal processing of IP Rs in response to GPCR activation. These findings in the recognition and translocation of ubiquitinated IP Rs from the ER both further our understanding of the mechanism IP R down-regula- membrane. tion and how ERAD substrates are processed in mammalian cells. 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Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Oct 14, 2005

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