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Originally published In Press as doi:10.1074/jbc.M305600200 on July 17, 2003

J. Biol. Chem., Vol. 278, Issue 40, 38238-38246, October 3, 2003
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Inositol 1,4,5-Trisphosphate Receptor Ubiquitination Is Mediated by Mammalian Ubc7, a Component of the Endoplasmic Reticulum-associated Degradation Pathway, and Is Inhibited by Chelation of Intracellular Zn2+*

Jack M. Webster {ddagger}, Swati Tiwari §, Allan M. Weissman § and Richard J. H. Wojcikiewicz {ddagger} 

From the {ddagger}Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210-2339 and the §Regulation of Protein Function Laboratory, Center for Cancer Research, NCI-Frederick, Frederick, Maryland 21702

Received for publication, May 28, 2003 , and in revised form, June 27, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
In response to activation of certain cell surface receptors, inositol 1,4,5-trisphosphate receptors (InsP3Rs), which are located in the endoplasmic reticulum, can be rapidly ubiquitinated and then degraded by the proteasome. Ubiquitination is mediated by the concerted action of ubiquitin-conjugating enzymes (Ubcs or E2s) and ubiquitin-protein ligases (E3s). In the present study we have examined the enzymology of ubiquitination of endogenous InsP3Rs in muscarinic agonist-stimulated SH-SY5Y human neuroblastoma cells, focusing our attention on two mammalian E2s, MmUbc6 and MmUbc7, that have been implicated in endoplasmic reticulum-associated degradation (ERAD) and are homologous to the yeast ERAD E2s, Ubc6p and Ubc7p. Analysis of SH-SY5Y cells stably expressing these enzymes and their dominant-negative mutants revealed that MmUbc7 mediates InsP3R ubiquitination and down-regulation, but that MmUbc6 does not. These data indicate that InsP3Rs are processed by a component of the ERAD pathway and suggest that MmUbc7 may be employed selectively to ubiquitinate proteins, like InsP3Rs, that are subject to regulated ERAD. Additional studies showed that the Zn2+ chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine blocked InsP3R ubiquitination, suggesting that a RING finger domain-containing E3 is also involved in this process. Finally, muscarinic agonist-induced InsP3R ubiquitination was seen in rat brain slices, indicating that the results obtained from SH-SY5Y cells reflect a physiological process.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Inositol 1,4,5,-trisphosphate (InsP3)1 receptors are proteins that form tetrameric ion channels in endoplasmic reticulum (ER) membranes. Upon binding of the second messenger InsP3, the channels open and Ca2+ stored in the ER is released into the cytosol (1, 2). In this way, InsP3 receptors (InsP3Rs) link cell-surface receptor-mediated InsP3 formation to increases in cytoplasmic free Ca2+ concentration ([Ca2+]i) (2).

Upon activation of certain phosphoinositidase C-linked G protein-coupled receptors (GPCRs), InsP3Rs are rapidly degraded (3–6). This InsP3R "down-regulation" has been described in a number of cell types, including SH-SY5Y human neuroblastoma cells (3, 4, 7), AR4-2J rat pancreatoma cells (5, 8), smooth muscle cells (9), WB rat liver epithelial cells (6), and {alpha}T3-1 mouse pituitary gonadotropes (10, 11), in response to stimulation with muscarinic agonists, certain secretagogues (cholecystokinin, bombesin, and pituitary adenylate cyclase-activating polypeptide), certain peptide hormones (vasopressin and bradykinin), angiotensin II, and gonadotropin-releasing hormone, respectively. Persistent elevation of InsP3 concentration (4, 8, 10, 12) and InsP3 binding (7, 11, 13) appear to be the signals that initiate InsP3R down-regulation, and this adaptive response allows chronically stimulated cells to reduce the sensitivity of their Ca2+ stores to InsP3 (3, 6, 9, 10, 14, 15). InsP3R down-regulation is mediated by the ubiquitin/proteasome pathway (6, 8, 9, 11) and is a specific response, because the levels of other ER and signaling proteins remain unaffected (4–6, 9, 16).

To be degraded by the ubiquitin/proteasome pathway (17), the substrate destined for destruction is covalently modified by the attachment of a polyubiquitin chain in a multistep process. First, in an ATP-dependent reaction, ubiquitin is covalently bound to a cysteine residue of the ubiquitin-activating enzyme, via a thiol-ester bond. Next, ubiquitin is transferred to the "active site" cysteine residue of a ubiquitin-conjugating enzyme (Ubc or E2), again employing a thiol-ester bond. The Ubc then conjugates ubiquitin to a lysine residue of either the substrate or a previously linked ubiquitin moiety via an isopeptide bond, forming a polyubiquitin chain. The specific recognition of substrates to be degraded as well as the transfer of ubiquitin from Ubc to substrate is facilitated by a ubiquitin-protein ligase (E3), of which there are three major families. The HECT ligases (HECT-E3s), which contain a domain homologous to the E6-AP carboxyl terminus, form an intermediate thiol-ester bond with ubiquitin (17). In contrast, the RING ligases (RING-E3s), whose activity is dependent upon a Zn2+-binding RING finger domain (17, 18), and the U-box ligases (U-box-E3s) (19), appear to facilitate the transfer of ubiquitin directly from Ubc to substrate. Finally, the 20 S core of the 26 S proteasome complex degrades the substrate after recognition of the polyubiquitin chain by the 19 S cap structure (17).

InsP3Rs are the only mammalian ER proteins for which ubiquitin/proteasome pathway-mediated degradation induced by cell-surface receptor activation has been described. The ubiquitin/proteasome pathway, however, is known to mediate the degradation of other substrates in the ER, including the cystic fibrosis transmembrane conductance regulator and the T-cell antigen receptor subunits, TCR{alpha} and CD3-{delta} (17, 20–25). The process by which these substrates are destroyed is known as ER-associated degradation (ERAD), which is primarily considered to be a "quality control" system that functions to specifically degrade misfolded proteins and unassembled subunits of multimeric complexes (24). It is possible, however, that certain functional, native ER proteins can also be degraded by ERAD, but in a regulated manner, the prototype for this being 3-hydroxy 3-methylglutaryl-coenzyme A reductase (HMGR) (26–28). Recent studies indicate that ubiquitination and degradation of HMGR is initiated by metabolites of the mevalonate pathway (27–30), perhaps after a "structural transition" that gives HMGR characteristics of a misfolded protein (27, 28). With regard to the Ubcs that mediate ERAD, the enzymes Ubc6p and Ubc7p have often been implicated in this process in yeast, with Ubc7p playing the dominant role in regulated degradation of HMGR (24, 26, 31–35). Likewise, in studies with exogenously expressed substrates in mammalian cells, a mammalian homologue of Ubc6p, hereafter termed mamUbc6, has been implicated in the ERAD of cystic fibrosis transmembrane conductance regulator and TCR{alpha} (23), and a mammalian homologue of Ubc7p, hereafter termed mamUbc7, has been implicated in the ERAD of TCR{alpha} and CD3-{delta} (22). With regard to the endogenous expression of these Ubcs, mamUbc7 is expressed ubiquitously in all human tissues examined, including brain (36), and although mamUbc6 distribution has not been studied, a chick homologue of Ubc6p is expressed in all avian tissues, again including brain (37).

In the present study we have examined whether the ERAD-linked Ubcs, mamUbc6 and mamUbc7, mediate GPCR-initiated ubiquitination and down-regulation of endogenous InsP3Rs in SH-SY5Y human neuroblastoma cells. We accomplished this by creating SH-SY5Y cell lines stably expressing murine homologues of Ubc6p and Ubc7p (MmUbc6 and MmUbc7), and their respective "dominant-negative" (DN) mutants, in which the active site cysteine residues have been mutated to serine (22, 38). Our results indicate that mamUbc7 mediates InsP3R ubiquitination and down-regulation and thus suggests that some of the same machinery that is responsible for the ERAD of misfolded and misassembled proteins also degrades InsP3Rs. Additionally, we provide evidence for the involvement of a RING-E3 in InsP3R ubiquitination and down-regulation, and demonstrate that muscarinic receptor activation can induce InsP3R ubiquitination in rat cerebral cortex.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Plasmids—cDNAs encoding HA-MmUbc6, HA-MmUbc6DN(C94S), myc-MmUbc7, and myc-MmUbc7DN(C89S) (GenBankTM accession nos. AF296656 [GenBank] and AF296657 [GenBank] ), with the hemagglutinin (HA) epitope (YPYDVPDYA) and the myc epitope (EQKLISEEDLN) tags at the NH2 termini (22), were ligated into the EcoR1/SalI sites of the multiple cloning region of the pIRES2-EGFP plasmid (Clontech). The amino acid sequences of the MmUbcs used in this study are each ~60% identical to yeast Ubc6p and Ubc7p (22). MmUbc6 is ~95% identical to its human homologue (22), and MmUbc7 is 100% identical to its human homologue (accession no. NM_003343 [GenBank] ).

Cell Culture and Transfection—SH-SY5Y human neuroblastoma cells were grown in monolayers as described (7). Cells were subcultured with 10 mM Hepes, 155 mM NaCl, 1 mM EDTA, pH 7.4 (HBS-E), and medium was routinely changed every other day. Cells (~75% confluent) were transfected with 1 µg of DNA and 8 µl of FuGENE 6 (Roche Applied Science) according to the instructions from the manufacturer. Cells were then subcultured and grown in the presence of 500 µg/ml G418. GFP-expressing colonies were identified by fluorescence microscopy of living cells and selected using sterile cloning cylinders. A second round of clonal selection followed to ensure that each clonal cell line was independently derived from a single transfected cell. Stock cultures were maintained in 250 µg/ml G418, whereas cells to be used in experiments were cultured in the absence of G418.

Antibodies—CT1 was produced from a peptide corresponding to the COOH-terminal 19 residues of the rat type I InsP3R and is specific for type I InsP3Rs (5). Anti-c-myc epitope clone 9E10 ({alpha}-myc) and anti-ubiquitin clone Ubi-1 ({alpha}-Ub) were from Zymed Laboratories Inc.; anti-HA epitope clone HA.11 ({alpha}-HA) was from Babco; rabbit polyclonal anti-transaldolase ({alpha}-tal) was a kind gift from Drs. Andras Perl and Katalin Banki (39); and anti-human p53 clone DO-1 ({alpha}-p53) was from Santa Cruz.

Cell Fractionation—Cells were harvested in HBS-E, centrifuged at ~700 x g for 3 min, and resuspended in 1 ml of 10 mM Tris base, 1 mM EGTA, pH 7.4 (hypotonic buffer) containing protease inhibitors (0.2 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, 0.2 µM soybean trypsin inhibitor) and 1 mM dithiothreitol (DTT). Cells were then disrupted by sonication and centrifuged at 100,000 x g for 1 h. Supernatant was collected and represented the cytosolic fraction. The pellet was resuspended in 1 ml of hypotonic buffer and represented the membrane fraction. Membrane fraction protein (10 µg) and an equivalent volume of the cytosolic fraction were then electrophoresed and processed in immunoblots.

Confocal Immunofluorescence Microscopy—Cells were grown on 8-chamber glass slides, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min, and then washed three times in PBS. Fixed cells were then incubated in blocking solution (10% goat serum, 4% bovine serum albumin in PBS) containing 0.01% digitonin for 1 h, and then incubated overnight in blocking solution containing primary antibody ({alpha}-myc or {alpha}-HA) at 4 °C. Next, cells were washed six times in PBS and incubated for 2 h in blocking solution containing rhodamine-conjugated goat anti-mouse secondary antibody. Cells were then washed six times, and coverslips were mounted with anti-fade mount (Molecular Probes). Images were obtained with a Bio-Rad MRC-1024 confocal microscope using a 60x oil immersion objective. The 568-nm line and the 488-nm line of the krypton/argon laser were used to excite rhodamine and GFP, respectively. Emission was collected between 589 and 621 nm for rhodamine and between 506 and 538 nm for GFP.

Preparation of Lysates for Immunoblots—Cells were grown to confluence and were stimulated, harvested in HBS-E, centrifuged (700 x g for 3 min), and lysed in 150 mM NaCl, 50 mM Tris base, 1 mM EDTA, 1% Triton X-100, protease inhibitors, 1 mM DTT, pH 8.0 (lysis buffer), for 20 min at 4 °C. Lysates were then centrifuged (16,000 x g for 10 min), and supernatants were collected, electrophoresed, and processed in immunoblots with CT1, {alpha}-HA, {alpha}-myc, {alpha}-Ub, or {alpha}-p53. For ubiquitin thiol-ester analysis, cells were lysed in non-reducing lysis buffer and electrophoresed under non-reducing conditions (in the absence of DTT).

InsP3 Receptor Immunoprecipitation and Detection of Ubiquitination—Supernatants obtained from centrifuged lysates were collected, protein concentrations were equalized, and equal volumes of these supernatants were incubated for 4 h at 4 °C with CT1 and protein A-Sepharose CL-4B beads. Immunoprecipitated InsP3Rs were then electrophoresed and processed in immunoblots, first with {alpha}-Ub to assess polyubiquitin content, and then reprobed with CT1 to determine InsP3 receptor levels. When comparing between different clonal cell lines, the amount of polyubiquitin immunoreactivity associated with InsP3Rs was defined as the ratio of {alpha}-Ub immunoreactivity to CT1 immunoreactivity ({alpha}-Ub/CT1). This accounted for minor differences in InsP3R content between samples. This procedure accurately reflected ubiquitination, because the {alpha}-Ub/CT1 ratio remained constant when a range of amounts of ubiquitinated InsP3Rs from untransfected SH-SY5Y cells were analyzed (data not shown).

Ubiquitination in Rat Cerebral Cortex—Rat cerebral cortex slices (350 x 350 µm) were prepared and incubated as described (40). Slices were collected by centrifugation (700 x g for 2 min) and homogenized in lysis buffer, lysates were centrifuged (16,000 x g for 10 min), and InsP3Rs were immunoprecipitated with CT1, probed in immunoblots with {alpha}-Ub, and reprobed with CT1.

Measurement of InsP3 Formation and Ca2+ Mobilization—SH-SY5Y cells were grown to confluence, harvested in HBS-E, washed, and resuspended in Krebs-Hepes buffer as described (7). For InsP3 measurements, triplicate 100-µl aliquots of cell suspensions were incubated without or with carbachol and incubations were terminated by the addition of 100 µl of 1 M trichloroacetic acid. InsP3 mass was then determined using a radioreceptor assay as previously described (41). For [Ca2+]i measurements, cell suspensions were incubated with 10 µM Fura2-AM for 60 min at 37 °C and then excited at 340 and 380 nm, and emission intensity at 510 nm was recorded with a computerized LS-50B luminescence spectrometer (PerkinElmer Life Sciences). [Ca2+]i was calculated as described (42) using 0.1% Triton X-100 and 10 mM EGTA as calibrating agents.

Materials—Cell culture materials were obtained from Invitrogen; G418 was from CellGro; T4 DNA ligase, EcoRI, and XhoI were from New England Biolabs; peroxidase-conjugated antibodies, Triton X-100, protease inhibitors, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), carbachol, and atropine were from Sigma; protein A-Sepharose CL-4B was from Amersham Life Sciences; DTT and SDS-PAGE gel supplies were from Bio-Rad; and N-acetyl-Leu-Leu-norleucinal (ALLN) was from Alexis.

Miscellaneous—Data shown are representative of at least two independent experiments. Quantitation of immunoreactivity was accomplished using a GeneGnome Imager and GeneTools software from Syngene. Combined quantitated data from immunoblots are mean ± S.E. of results from >=3 independent experiments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of SH-SY5Y Cell Lines Stably Expressing Exogenous MmUbcs—The cDNAs encoding epitope-tagged MmUbc6 and MmUbc7 and their DN mutants (22) were ligated into the pIRES2-EGFP vector, SH-SY5Y cells were transfected with the resulting plasmids, and G418-resistant, GFP-expressing clones were isolated. Expression of exogenous MmUbc6 and MmUbc6DN (HA-tagged), and MmUbc7 and MmUbc7DN (myc-tagged) was confirmed in immunoblots with {alpha}-HA and {alpha}-myc, which identified, respectively, ~31-kDa proteins (Fig. 1A, upper panel, lanes 4 and 6) and ~19-kDa proteins (Fig. 1B, upper panel, lanes 3–6). The clonal cell lines depicted in Fig. 1 were those that expressed the highest levels of MmUbcs; these and other independently derived cell lines expressing equivalent amounts of MmUbcs were used throughout the course of this study. GFP and exogenous MmUbc expression remained stable throughout, as determined by periodic fluorescence microscopy and immunoblot analysis, respectively (data not shown).



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  		FIG. 1.
Expression and membrane association of exogenous MmUbc6 (A) and MmUbc7 (B) in stably transfected SH-SY5Y cells. Untransfected cells and cells stably expressing MmUbc6, MmUbc6DN, MmUbc7, and MmUbc7DN were harvested, and membrane (M) and cytosolic (C) fractions were separated by homogenization and centrifugation. Samples were separated by SDS-PAGE in 12% (upper and middle panels) or 5% (lower panels) gels and probed in immunoblots (IB) for exogenous MmUbcs with {alpha}-HA or {alpha}-myc (upper panels), transaldolase with {alpha}-tal (middle panels), and type I InsP3R with CT1 (lower panels).

 

Localization of Exogenous MmUbcs—To show that the exogenous MmUbcs were appropriately localized in SH-SY5Y cells, membrane and cytosol fractions were prepared from control and transfected cells. The efficiency of fractionation was established using the type I InsP3R as a marker for membranes, and transaldolase (an entirely cytosolic enzyme of the pentose-phosphate pathway) (39) as a marker for cytosol. These markers were segregated very effectively, indicating that cytosol was not contaminating the membrane fraction (Fig. 1, A and B, middle panels) and vice versa (Fig. 1, A and B, lower panels). HA-MmUbc6 and HA-MmUbc6DN were found solely in the membrane fraction (Fig. 1A, upper panel, lanes 4 and 6) consistent with mamUbc6 having a single-pass COOH-terminal transmembrane domain (22, 23, 25, 43, 44). In contrast, myc-MmUbc7 and myc-MmUbc7DN were found predominantly in the cytosol, but also at significant levels in the membrane fraction (Fig. 1B, upper panel, lanes 3–6), consistent with the existence of an ER-associated protein that docks a proportion of cellular mamUbc7 to the ER membrane (22). This docking protein has been identified as Cue1p in yeast (34) and gp78, an E3 with a Cue1-like domain, in mammalian cells (25).

When examined by confocal immunofluorescence microscopy (Fig. 2), HA-MmUbc6 (panel B), HA-MmUbc6DN (panel C), myc-MmUbc7 (panel H), and myc-MmUbc7DN (panel I) exhibited a diffuse perinuclear expression pattern. In contrast, GFP was present in both the cytosol and nucleus (Fig. 2, panels E, F, K, and L). Taken together with data from Fig. 1, the distribution of MmUbc6 and MmUbc6DN is indicative of an ER membrane localization, whereas that of MmUbc7 and MmUbc7DN appears to reflect a predominantly cytoplasmic localization, with a significant proportion associated with the ER membrane. These findings are consistent with the localization of exogenous MmUbcs in transiently transfected HEK cells (22) and also indicate that stable expression of the MmUbcs does not cause gross changes in ER morphology.



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  		FIG. 2.
Confocal immunofluorescence microscopy of exogenous MmUbc6 and MmUbc7 in stably transfected SH-SY5Y cells. Untransfected cells and cells stably expressing MmUbc6, MmUbc6DN, MmUbc7, and MmUbc7DN were fixed onto glass slides and stained with {alpha}-HA (A–F) or {alpha}-myc (G–L) and rhodamine-conjugated secondary antibody. Panels A–C and G–I show expression of the epitope-tagged MmUbcs in each cell line, and panels D–F and J–L show expression of GFP in the same cells.

 

MmUbc6 and MmUbc7 Expressed Exogenously in SH-SY5Y Cells Are Functional Ubiquitin-conjugating Enzymes—To confirm that exogenous MmUbc6 and MmUbc7 were functional in SH-SY5Y cells, we examined whether they formed ubiquitin thiol-ester-conjugated intermediates by subjecting cell lysates to SDS-PAGE under reducing and non-reducing conditions. Fig. 3A shows that, in the absence of the reducing agent DTT, an {alpha}-HA immunoreactive band, ~5 kDa larger than HA-MmUbc6, was detected (lane 5), and that this band (HA-MmUbc6~ubiquitin) was not detected in the presence of DTT (lane 2) or in HA-MmUbc6DN-expressing cells (lane 6). Likewise, Fig. 3B (upper panel, lanes 2 and 5, depicts a DTT-sensitive, {alpha}-myc immunoreactive band (myc-MmUbc7~ubiquitin), ~12 kDa larger than myc-MmUbc7,2 which was not seen in myc-MmUbc7DN-expressing cells (lane 6). Given that MmUbc6DN and MmUbc7DN lack active site cysteine residues and thus cannot form thiol-ester bonds with ubiquitin, these data strongly indicate that MmUbc6 and MmUbc7 do indeed form ubiquitin thiol-ester-conjugated intermediates.3 To confirm this, duplicate immunoblots were probed with {alpha}-Ub. This revealed that the band designated myc-MmUbc7~ubiquitin (Fig. 3B, upper panel, lane 5) was indeed {alpha}-Ub-immunoreactive and thus truly contained ubiquitin (Fig. 3B, lower panel, lane 5), and remarkably, that exogenous MmUbc7 was the most abundant ubiquitin thiol-ester-conjugated Ubc in this cell line. In contrast, the faint band designated HA-MmUbc6~ubiquitin (Fig. 3A, lane 5) could not be detected with {alpha}-Ub (data not shown), indicating that the amount of ubiquitin thiol-ester-conjugated exogenous MmUbc was much less than that in MmUbc7-expressing cells. This most likely reflects the fact that a smaller proportion of MmUbc6 was ubiquitin thiol-ester-conjugated than MmUbc7 (Fig. 3, compare A (lane 5) with B (upper panel, lane 5)).



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  		FIG. 3.
Ubiquitin thiol-ester conjugation to exogenous MmUbc6 (A) and MmUbc7 (B) in intact SH-SY5Y cells. Untransfected cells and exogenous MmUbc-expressing cells were harvested, lysed, and electrophoresed in 12% gels under reducing conditions (+DTT, lanes 1–3) or non-reducing conditions (–DTT, lanes 4–6). Immunoblots were then probed with {alpha}-HA (A), {alpha}-myc (B, upper panel), or {alpha}-Ub (B, lower panel). Reduced and non-reduced samples were separated on different gels to ensure that non-reduced samples were not exposed to DTT. Thiol-ester-conjugated MmUbcs are designated with the suffix ~ubiquitin, and the asterisk marks a minor band thought to be myc-MmUbc7 covalently linked to a ubiquitin moiety via an isopeptide bond (see Footnote 3).

 

MmUbc7DN Expression Inhibits Carbachol-induced Ubiquitination and Down-regulation of InsP3 Receptors—SH-SY5Y cells endogenously express type I InsP3Rs (5), which are rapidly ubiquitinated and down-regulated in response to stimulation of cell-surface muscarinic receptors with agonists, such as carbachol (4, 7, 13). Thus, we next examined whether expression of the exogenous MmUbcs affected carbachol-induced InsP3R ubiquitination and down-regulation.

For analysis of InsP3R ubiquitination (Fig. 4), control and MmUbc-expressing cells were pre-incubated with ALLN for 60 min to inhibit the proteasome and then incubated without or with carbachol for 20 min to initiate InsP3R ubiquitination. Fig. 4A (lanes 1 and 2) shows that, in untransfected cells, stimulation with carbachol led to the appearance of InsP3R-associated ubiquitin immunoreactivity, indicative of the formation of a spectrum of polyubiquitinated InsP3Rs (8, 13), and that, although expression of MmUbc7 did not affect this response (lanes 3 and 4), it was markedly inhibited by expression of MmUbc7DN (lanes 5 and 6). Additionally, neither MmUbc6 nor MmUbc6DN expression affected InsP3R ubiquitination (Fig. 4A, lanes 8 and 9). As with untransfected cells (Fig. 4A, lane 1), ubiquitin immunoreactivity was not detected in the MmUbc-expressing cell lines in the absence of carbachol (data not shown). That essentially identical results were obtained from two independently-derived cell lines expressing MmUbc7 (102A and 108B) and MmUbc7DN (208F and 209C) shows that effects were not an artifact of clonal selection. Combined quantitated data (Fig. 4B) confirm that MmUbc7 expression was without effect and that MmUbc7DN expression significantly inhibited ubiquitination by ~65%. Fig. 4B also shows that ubiquitination was unaffected by stable transfection with empty vector,4 or by MmUbc6 and MmUbc6DN expression.



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  		FIG. 4.
MmUbc7DN expression inhibits carbachol-induced InsP3R ubiquitination. A, cells were incubated with 20 µg/ml ALLN for 60 min, followed by 20 min without or with 1 mM carbachol, as indicated. InsP3Rs were then immunoprecipitated with CT1, subjected to SDS-PAGE (5% gels), probed in immunoblots with {alpha}-Ub, and then reprobed with CT1. Arrows mark the position of the unmodified type I InsP3R (~260 kDa), and the square bracket identifies an {alpha}-Ub immunoreactive smear corresponding to polyubiquitinated InsP3Rs (~270–380 kDa). Shown are data from SH-SY5Y cells (lanes 1, 2, and 7) together with two independently derived MmUbc7-expressing cell lines (102A and 108B, lanes 3 and 4), two independently derived MmUbc7DN-expressing cell lines (208F and 209C, lanes 5 and 6), MmUbc6-expressing cells (lane 8), and MmUbc6DN-expressing cells (lane 9). B, combined quantitated data from three independent experiments on carbachol-stimulated control (SH-SY5Y) cells, empty vector-transfected cells, and cells stably expressing MmUbc7, MmUbc7DN, MmUbc6, and MmUbc6DN. Ubiquitin immunoreactivity associated with immunoprecipitated InsP3Rs in each cell line was normalized to carbachol-stimulated control (SH-SY5Y) cells; * denotes p < 0.05, by Welch's t test, compared with control cells.

 

For analysis of InsP3R down-regulation, control and MmUbc-expressing cells were incubated without or with carbachol for 2 h and type I InsP3R content was assessed (Fig. 5). Fig. 5A shows that type I InsP3Rs were down-regulated in response to carbachol (lanes 1 and 2); that expression of MmUbc7 (lanes 3–6), MmUbc6 (lanes 11 and 12), and MmUbc6DN (lanes 13 and 14) did not affect this process; and that MmUbc7DN expression markedly inhibited down-regulation (lanes 7–10), consistent with its effect on InsP3R ubiquitination. Combined quantitated data (Fig. 5B) confirm that, although InsP3R down-regulation was unaffected by MmUbc7 expression, it was significantly inhibited by MmUbc7DN expression, and that it was unaffected by stable transfection with empty vector,4 or by MmUbc6 and MmUbc6DN expression. Again, the two independently derived cell lines yielded essentially identical results. In total, these data show that expression of MmUbc7DN inhibits endogenous InsP3R ubiquitination and down-regulation, implicating endogenous mamUbc7 in this process.



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  		FIG. 5.
MmUbc7DN expression inhibits carbachol-induced InsP3R down-regulation. A, cells were incubated without or with 1 mM carbachol for 2 h, as indicated. Samples were then separated by SDS-PAGE (5% gels) and probed in immunoblots with CT1 to assess type I InsP3R content (arrow). Shown are data from SH-SY5Y cells (lanes 1 and 2) together with two independently derived cell lines expressing MmUbc7 (lanes 3–6), two independently derived cell lines expressing MmUbc7DN (lanes 7–10), MmUbc6-expressing cells (lanes 11 and 12), and MmUbc6DN-expressing cells (lanes 13 and 14). B, combined quantitated data from three independent experiments on control (SH-SY5Y) cells, empty vector-transfected cells, and cells stably expressing MmUbc7, MmUbc7DN, MmUbc6, and MmUbc6DN; * denotes p < 0.05, and ** denotes p < 0.01, by unpaired Student's t test, compared with control cells.

 

The effect of MmUbc7DN was specific, rather than being caused by a general inhibition of the ubiquitin/proteasome pathway, because cellular ubiquitin conjugate levels (without and with proteasome inhibition) and the degradation of the ubiquitin/proteasome pathway substrate p53 were not altered by MmUbc7DN expression or, for that matter, by the expression of any of the exogenous MmUbcs (data not shown). Thus, inhibition of ubiquitination by MmUbc7DN appears to be restricted to a small subset of proteins that includes InsP3Rs and presumably other mamUbc7 substrates. Likewise, both basal and carbachol-stimulated InsP3 formation and [Ca2+]i were unaltered by MmUbc7DN expression (data not shown). Thus, perturbation of neither InsP3 synthesis, nor InsP3R function, nor Ca2+ mobilization can account for the inhibitory effect of MmUbc7DN on InsP3R ubiquitination.

TPEN Inhibits InsP3 Receptor Ubiquitination and Down-regulation—RING-E3s are Zn2+-dependent enzymes (18, 45). TPEN, a Zn2+ chelator, has been shown to inhibit the activity of RING-E3s, but not HECT-E3s or U-box-E3s in vitro (19, 33, 45), and is capable of quickly entering intact cells and chelating intracellular Zn2+ (46, 47). To examine whether the E3 that mediates InsP3R ubiquitination is a RING-E3, we determined the effect of TPEN on InsP3R ubiquitination and down-regulation in SH-SY5Y cells. Fig. 6 shows that TPEN inhibited carbachol-induced InsP3R ubiquitination (lanes 1 and 2) and that this inhibition was largely reversed by excess Zn2+ (lane 3). Consistent with these data, TPEN also inhibited carbachol-induced InsP3R down-regulation in a Zn2+-reversible manner (Fig. 7A). Combined quantitated data show that TPEN significantly inhibited carbachol-induced InsP3R ubiquitination by ~75% (Fig. 6B) and InsP3R down-regulation by ~70% (Fig. 7B).



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  		FIG. 6.
TPEN inhibits carbachol-induced InsP3R ubiquitination. A, SH-SY5Y cells were incubated with 20 µg/ml ALLN for 60 min followed by 20-min incubation with 1 mM carbachol either alone, or together with TPEN (100 µM), or together with TPEN (100 µM) and ZnCl2 (200 µM), as indicated. InsP3Rs were then immunoprecipitated and separated by SDS-PAGE (5% gels), probed in immunoblots with {alpha}-Ub, and then reprobed with CT1. Arrows mark the position of unmodified type I InsP3R (~260 kDa), and the square bracket identifies polyubiquitinated InsP3Rs (~270–380 kDa). B, combined quantitated data from five independent experiments; *** denotes p < 0.001, by Welch's t test, compared with cells stimulated with carbachol alone. The square and rounded brackets delineate, respectively, the amount of inhibition of InsP3R ubiquitination that can be attributed to chelation of Zn2+, and the amount that can be attributed to antagonism of the muscarinic receptor.

 


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  		FIG. 7.
TPEN inhibits carbachol-induced InsP3R down-regulation. A, SH-SY5Y cells were incubated without or with carbachol (1 mM) for 2 h, either alone, or together with TPEN (100 µM), or together with TPEN (100 µM) and ZnCl2 (200 µM), as indicated. Cells were harvested and lysed, and samples were separated by SDS-PAGE (5% gels) and probed in immunoblots with CT1 to assess type I InsP3R content (arrow). B, combined quantitated data from four independent experiments; ** denotes p < 0.01, by unpaired Student's t test, compared with cells stimulated with carbachol alone.

 

Effects of TPEN Are the Result of Inhibition of a Zn2+-dependent Process Downstream of InsP3 Formation and InsP3R Activation—TPEN has recently been shown to be a muscarinic receptor antagonist that can inhibit carbachol-induced InsP3 formation in SH-SY5Y cells (48). That this antagonistic activity was not responsible for the effects of TPEN on InsP3R ubiquitination and down-regulation (Figs. 6 and 7) is supported by several lines of evidence. First, although TPEN retains its muscarinic antagonist activity in the presence of excess Zn2+ (48), the inhibitory effect of TPEN on InsP3R ubiquitination was almost completely reversed by excess Zn2+ (Fig. 6B). This indicates that a post-muscarinic effect (depletion of Zn2+) was responsible for the majority of the inhibitory effect of TPEN (Fig. 6B, square bracket); the residual inhibitory effect of TPEN after ZnCl2 addition (Fig. 6B, rounded bracket) may indeed be the result of antagonism of the muscarinic receptor. Second, although 100 µM TPEN strongly inhibited InsP3R ubiquitination (by ~75%), this amount of TPEN only inhibited InsP3 formation by ~35% (Fig. 8A, bar 2); this modest decrease in InsP3 formation cannot account for the robust inhibition of ubiquitination, because lower concentrations of carbachol (e.g. 10 µM) that result in even less InsP3 formation cause near-maximal ubiquitination (Fig. 8A, bar 3). Third, TPEN inhibits InsP3R ubiquitination in gonadotropin-releasing hormone-stimulated {alpha}T3-1 cells (11) and in cholecystokinin-stimulated AR4-2J cells,5 without interfering with GPCR-induced InsP3 formation (11, 48). Finally, and most importantly, TPEN had no effect on carbachol-induced Ca2+ mobilization in SH-SY5Y cells (Fig. 8B), indicating that neither the anti-muscarinic effects of TPEN nor Zn2+ chelation significantly perturbs InsP3-mediated Ca2+ signaling. In total, these data show that a Zn2+-dependent process downstream of InsP3 formation and InsP3R activation is required for InsP3R ubiquitination, and strongly implicate a RING-E3 in this process.



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  		FIG. 8.
TPEN does not affect InsP3 formation or InsP3R function. A, carbachol-induced increases in InsP3 formation (gray bars) and ubiquitin immunoreactivity associated with InsP3Rs (black bars) were normalized to control (1 mM carbachol) values. Data shown are mean ± S.E. from three independent experiments. B, carbachol (1 mM)-induced Ca2+ mobilization in the absence (solid line) and presence (dashed line) of 200 µM TPEN. Either ethanol (vehicle) or TPEN was added 2 min prior to carbachol as indicated.

 

Carbachol-induced InsP3 Receptor Ubiquitination Occurs in Neonatal Rat Cerebral Cortex—To determine whether muscarinic receptor-induced InsP3R ubiquitination occurs in a more physiologically relevant system than SH-SY5Y cells, we examined rat cerebral cortical tissue, ex vivo. Eight-day-old rat cortices were used because muscarinic agonist-induced InsP3 formation is most robust at this age (49). Fig. 9 shows that InsP3Rs are indeed ubiquitinated in response to carbachol (lanes 1 and 2). That this ubiquitination was inhibited by atropine (lane 3) demonstrates that it was the result of activation of muscarinic receptors.



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  		FIG. 9.
Activation of muscarinic receptors in neonatal rat cerebral cortex causes InsP3R ubiquitination. Cerebral cortex slices from 8-day-old rats were incubated with 20 µg/ml ALLN for 60 min followed by 30-min incubation without (lane 1) or with (lane 2) 1 mM carbachol, or with carbachol plus 20 µM atropine (lane 3). InsP3Rs were then immunoprecipitated, separated by SDS-PAGE (5% gel), probed in immunoblots with {alpha}-Ub, and then reprobed with CT1. Arrows mark the position of the unmodified type I InsP3R (~260 kDa), and the square bracket identifies an {alpha}-Ub immunoreactive smear corresponding to polyubiquitinated InsP3Rs (~270–380 kDa).

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The fact that InsP3Rs are expressed in the ER led us to examine the possibility that InsP3R ubiquitination and degradation might be mediated by Ubcs that are known to be involved in ERAD. The data we obtained provide evidence that mamUbc7 mediates signal-induced ubiquitination and down-regulation of endogenous InsP3Rs, because overexpression of a DN mutant of MmUbc7 inhibited InsP3R ubiquitination and down-regulation, whereas wild-type MmUbc7 had no effect. Our interpretation of these findings is that the catalytically inactive MmUbc7DN competes with endogenous mamUbc7 (the expression of which in SH-SY5Y cells was confirmed by RT-PCR)5 for a binding site either on the InsP3R or on the putative E3 complex that ubiquitinates InsP3Rs, and that wild-type MmUbc7 competes for the same binding site, but has no effect because it can substitute for endogenous mamUbc7 as a mediator of InsP3R ubiquitination. These conclusions are consistent with the observation that both MmUbc7 and MmUbc7DN were present at significant levels in ER membranes, and are validated by the finding that the effect of MmUbc7DN was not a consequence of a general inhibition of the ubiquitin/proteasome pathway. In contrast, although MmUbc6 was also localized to the ER, its DN mutant did not affect the ubiquitination of InsP3Rs. Clearly, given that MmUbc6 and MmUbc6DN expression was restricted exclusively to membranes, including to ER membranes, we would expect MmUbc6DN to be inhibitory if mamUbc6 was a mediator of signal-induced InsP3R ubiquitination. Although it remains a possibility that mamUbc6 could be a cooperative participant with mamUbc7 in InsP3R ubiquitination, our data plainly identify mamUbc7 as the primary mediator of this process. Attempts to co-immunoprecipitate MmUbc7 with InsP3Rs under a variety of conditions were not successful, indicating that the association between these proteins may be transient or requires a membrane environment. It is noteworthy that MmUbc7DN expression did not increase basal InsP3R levels, indicating that mamUbc7 does not mediate basal InsP3R turnover. This is consistent with the finding that the lysosomal pathway, rather than the ubiquitin/proteasome pathway, mediates basal InsP3R turnover (6).

Importantly, the data presented here were obtained from the analysis of endogenous InsP3Rs and indeed are the first to link a Ubc of the mammalian ERAD pathway to the ubiquitination and degradation of an endogenous substrate. In this regard, evidence was presented recently that mamUbc6 and mamUbc7 mediate the quality control function of ERAD when both substrates and mamUbcs were exogenously expressed in mammalian cells by transient transfection (22, 23). Although some of these data are readily interpretable, for example, that mamUbc7DN, but not mamUbc7 inhibits TCR{alpha} degradation (22), others are more difficult to explain, for example, that both mamUbc6 and mamUbc6DN inhibit the degradation of TCR{alpha} (23). This latter result may reflect unanticipated complexities associated with highly overexpressed substrates. Indeed, any ER protein may become an ERAD substrate when overexpressed at a high enough level, as indicated by observations that even InsP3Rs can be targeted by the ubiquitin/proteasome pathway in an unregulated manner when exogenously overexpressed at high levels (11). That we have implicated mamUbc7 in the regulation of endogenous InsP3Rs in response to activation of an endogenous signaling pathway argues strongly that this linkage is physiologically relevant and that processing of InsP3Rs by the ERAD pathway is a normal cellular event.

The signals that initiate the quality control function of ERAD are probably general features of misfolded and unassembled proteins, perhaps the exposure of hydrophobic regions (17, 24, 27, 35). This may also be the case for HMGR, which is degraded by ERAD in a regulated manner; in response to sterols, its transmembrane regions are thought to undergo a structural transition that may mimic misfolding, thereby directing HMGR to the ERAD pathway (27–30). Given that InsP3R ubiquitination requires InsP3 binding (7, 8, 10, 12, 13), and InsP3 binding causes a conformational change in the trans-membrane domain of the receptor, which is presumed to be required for channel opening (1), it is possible that a similar structural transition accounts for InsP3R ubiquitination. This model is supported by the finding that 2-aminoethoxydiphenyl borate (an InsP3R calcium channel blocker), which may block conformational changes that lead to channel opening, also blocks InsP3R ubiquitination (50). With the very recent determination of the tertiary structure of InsP3Rs (51–54), we may soon be able to precisely define the conformational changes that are required for InsP3R ubiquitination. In summary, InsP3R degradation, like HMGR degradation, appears to represent an example of regulated ERAD (RERAD), a process by which a signal directs a correctly folded functional protein to the ERAD pathway. It is intriguing that although both Ubc6p and Ubc7p (and their mammalian homologues) appear to mediate the quality control functions of ERAD, only Ubc7p and mamUbc7 appear to be involved in HMGR (26, 33) and InsP3R RERAD. However, this selectivity may not apply to all substrates, as RERAD of mammalian type 2 iodothyronine mono-deiodinase, when expressed in yeast, is mediated by both Ubc6p and Ubc7p (55).

Because of its ability to chelate Zn2+, TPEN has been shown previously to inhibit RING-E3s in vitro; in contrast, the activities of U-box-E3s and HECT-E3s, which are Zn2+-independent, were unaffected by TPEN (19, 33, 45). Our finding that TPEN inhibits InsP3R ubiquitination indicates that it is also possible to use TPEN in intact cells to probe for RING-E3-mediated processes. In this regard, the mammalian ER membrane-bound RING-E3s, gp78 (25, 56) and HRD1 (57, 58), and another RING-E3, parkin (59), have been implicated in mammalian ERAD, as has CHIP, a U-box-E3 (60). HECT-E3s do not appear to play a role in ERAD. Thus, our findings with TPEN suggest that a RING-E3, rather than a U-box-E3, mediates InsP3R RERAD. The possibility that gp78, which docks MmUbc7 to the ER membrane (25), is the E3 in question will be addressed in future studies.

Processing of InsP3Rs by the ubiquitin/proteasome pathway has been demonstrated in a model of pancreatitis (61) and in fertilization of oocytes (12, 62). Fig. 9 shows that InsP3R ubiquitination, and thus possibly InsP3R RERAD, occurs in brain in response to muscarinic receptor activation. Although the physiological significance of this finding can only be speculated upon, it may well be important clinically when cholinergic drugs are used therapeutically. For example, in Alzheimer's disease both muscarinic agonists (63) and acetylcholine esterase inhibitors (64, 65) are employed to counter the degenerative loss of cholinergic neurotransmission (66). Currently, acetylcholine esterase inhibitors, which elevate synaptic acetylcholine levels, are the only FDA-approved treatment for Alzheimer's disease (66); results of treatment are modest, perhaps in part caused by adaptive responses to elevated acetylcholine levels (66, 67). It is quite possible that InsP3R RERAD contributes to this adaptation.

In summary, the present study provides evidence for the involvement of the ubiquitin conjugating enzyme mamUbc7 and a RING-E3 in signal-induced InsP3R ubiquitination and down-regulation, and suggests that this process is mediated by RERAD. mamUbc7 and this putative RING-E3 represent attractive targets for the development of inhibitors that could be used to gain insight into the functional significance and prevalence of RERAD, and also perhaps the ERAD of misfolded and unassembled proteins.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grant 5RO1DK49194 and by an advanced predoctoral fellowship in pharmacology/toxicology from the Pharmaceutical Research and Manufacturers of America Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Pharmacology, State University of New York Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210-2339. Tel.: 315-464-7956; Fax: 315-464-8014; E-mail: wojcikir{at}upstate.edu.

1 The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; ER, endoplasmic reticulum; InsP3R, inositol 1,4,5-trisphosphate receptor; [Ca2+]i, cytoplasmic free Ca2+ concentration; GPCR, G protein-coupled receptor; Ubc or E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; HECT-E3, ubiquitin-protein ligase containing a domain homologous to the E6-associated protein carboxyl terminus; RING-E3, RING finger domain-containing ubiquitin-protein ligase; U-box-E3, U-box domain-containing ubiquitin-protein ligase; ERAD, endoplasmic reticulum-associated degradation; HMGR, 3-hydroxy 3-methylglutarylcoenzyme A reductase; mamUbc6, mammalian homologue of yeast Ubc6p; mamUbc7, mammalian homologue of yeast Ubc7p; MmUbc6, murine homologue of yeast Ubc6p; MmUbc7, murine homologue of yeast Ubc7p; DN, dominant negative; HA, hemagglutinin; DTT, dithiothreitol; PBS, phosphate-buffered saline; TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine; ALLN, N-acetyl-Leu-Leu-norleucinal; RERAD, regulated endoplasmic reticulum-associated degradation; GFP, green fluorescent protein. Back

2 The molecular mass shifts in MmUbc6 and MmUbc7 upon ubiquitin thiol-ester conjugation (~5 and ~12 kDa, respectively) differed from the molecular mass of ubiquitin (~8.5 kDa) and also differed between the two MmUbcs. This most likely reflects a combination of reasons. First, ubiquitin is conjugated to a central region of the proteins and therefore would not be expected to slow migration in direct proportion to the increase in molecular mass. Second, the detection of thiol-ester intermediates required the use of non-reducing conditions, which results in aberrant migration of some proteins in SDS-PAGE. For example, unconjugated MmUbc7 and MmUbc7DN migrate slightly more rapidly under non-reducing conditions than reducing conditions (Fig. 3B, upper panel, lanes 2, 3, 5, and 6). Back

3 Surprisingly, Fig. 3B (upper panel, lane 2) shows that a minor, high molecular mass, {alpha}-myc-immunoreactive band (marked with an asterisk) was detected under reducing conditions. This conjugate was ~10 kDa larger than myc-MmUbc7 (lane 2) and was not seen with myc-MmUbc7DN (lane 3). A similar band was detected in HEK-293 cells transiently transfected with both myc-MmUbc7 and HA-ubiquitin, where we confirmed it to be a ubiquitin conjugate (data not shown). This band appears to represent a single ubiquitin moiety conjugated to myc-MmUbc7 via an isopeptide bond, probably as a result of autoubiquitination. Back

4 The SH-SY5Y cells stably transfected with empty vector (pIRES2-EGFP) were, like the MmUbc-expressing cells, G418-resistant and expressed GFP. Back

5 J. M. Webster and R. J. H. Wojcikiewicz, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Andras Perl and Katalin Banki for providing {alpha}-tal, and Kamil Alzayady, Matt Soulsby, and Qun Xu for valuable discussions.



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