Angiotensin II-induced Down-regulation of Inositol Trisphosphate Receptors in WB Rat Liver Epithelial Cells
EVIDENCE FOR INVOLVEMENT OF THE PROTEASOME PATHWAY*

(Received for publication, November 20, 1996, and in revised form, January 22, 1997)

Shaila Bokkala and Suresh K. Joseph Dagger

From the Department of Pathology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Chronic stimulation of WB rat liver epithelial cells by angiotensin II (Ang II) resulted in the down-regulation of both type I and type III myo-inositol 1,4,5-trisphosphate receptors (IP3Rs). Stimulation with vasopressin, bradykinin, epidermal growth factor, or 12-O-tetradecanoylphorbol-13-acetate was without effect. Ang II-induced down-regulation of IP3Rs could be detected within 2 h and resulted in an inhibition of IP3-induced Ca2+ release from permeabilized cells. IP3R down-regulation was reversible, and both homo- and heterooligomers of IP3Rs were equally susceptible to Ang II-induced degradation. Chloroquine and NH4Cl increased the basal levels of IP3Rs by 2-fold, suggesting that the basal turnover of IP3Rs occurs via a lysosomal pathway. However, Ang II-induced degradation of IP3R was not affected by these inhibitors, suggesting that stimulated degradation of IP3Rs occurs via a non-lysosomal pathway. The cysteine protease and proteasomal inhibitor N-acetyl-Leu-Leu-norleucinal completely prevented Ang II-mediated down-regulation of IP3Rs, whereas the structural analog N-acetyl-Leu-Leu-methioninal was without effect. Lactacystin, a highly specific proteasome inhibitor, also blocked Ang II-mediated IP3R degradation. Stimulation with Ang II increased the amount of IP3R immunoprecipitated by anti-ubiquitin antibodies. We conclude that Ang II-stimulated IP3R degradation involves enhanced ubiquitination of the protein and degradation by the proteasome pathway.


INTRODUCTION

Stimulation of cell surface receptors results in the formation of the second messenger myo-inositol 1,4,5-trisphosphate (IP3)1 via the activation of phospholipase C (1). IP3 mobilizes intracellular calcium by binding to a family of receptors (IP3Rs) that act as ligand-gated calcium channels (2, 3). IP3Rs are tetramers, and full-length sequences of at least three different isoforms have been identified by molecular cloning (reviewed in Ref. 4). Both homo- and heterotetramers are found in cells expressing more than one isoform (5-8).

The acute regulation of IP3Rs occurs primarily through feedback effects of cytosolic Ca2+ and/or by phosphorylation of the receptor (reviewed in Refs. 4 and 9). However, prolonged exposure of cells to agonists has also been shown to alter the expression of IP3R protein levels. For example, a decreased expression of IP3R isoforms occurs in response to chronic stimulation of muscarinic receptors in SH-SY5Y human neuroblastoma and cerebellum granule cells (10, 11), and cholecystokinin and bombesin receptors in AR4-2J pancreotoma cells (12). Down-regulation of IP3Rs has also been observed in Xenopus oocytes having a sustained activation of phospholipase C as a result of expressing a constitutively active mutant of Galpha q (13). Based on metabolic labeling studies, it has been concluded that the decreased expression of IP3Rs caused by carbachol treatment of SHY-SY5Y cells is due to enhanced degradation of IP3R protein rather than an inhibition of receptor biosynthesis (10). Down-regulation of IP3Rs has been suggested to be a possible mechanism of heterologous desensitization of responses to Ca2+-mobilizing hormones (12, 13). Examples of increased expression of IP3R protein have also been reported. The type I IP3R isoform was elevated in human promyelocytic leukemic HL-60 cells treated with retinoic acid and 1,25-dihydroxyvitamin D3 (14, 15). This effect was shown to be due to enhanced transcription of the type I IP3R gene (14, 15). Recently, large increases in the expression of the type III isoform were reported to accompany apoptosis induced in B and T lymphocytes (16).

To further study the mechanisms regulating biosynthesis and degradation of the IP3R protein, we have examined the effect of chronic agonist stimulation of WB cells. This nontransformed, cultured rat liver epithelial cell line (17) was used as an experimental model because these cells contain high levels of both type I and type III IP3R isoforms (6, 18). In the present study we report that both isoforms are down-regulated when WB cells are chronically stimulated by Ang II. We have characterized this response and present evidence to suggest that the Ang II-mediated degradation of IP3Rs involves activation of a ubiquitin-proteasome pathway.


EXPERIMENTAL PROCEDURES

Materials

Angiotensin II, epidermal growth factor, TPA, bradykinin and vasopressin, heparin-agarose type II-S, ALLN, and ALLM were from Sigma. Fura-2AM was purchased from Molecular Probes, Inc. (Eugene, OR). Richter's modified minimal essential medium was from Irvine Scientific Co. (Santa Ana, CA). Inositol-free Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. myo-[2-3H]Inositol was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Losartan was a kind gift from Dr. Steven Fluharty (University of Pennsylvania) and was also obtained from Dupont Merck Pharmaceutical Co. (Wilmington, DE). Lactacystin was purchased from Dr. E. J. Corey (Harvard University, Cambridge, MA). True-Count scintillant was purchased from IN/US Corp. (Fairfield, NJ).

Antibodies

The isoform-specific antibodies used in these studies were raised against unique C-terminal sequences of IP3 receptors. The type I antibody was raised to amino acids 2731-2749 of the rat type I IP3R and has previously been characterized (19). The type III polyclonal antibody was raised against the C-terminal peptide corresponding to residues 2657-2670 of the rat type III IP3R. The peptide was synthesized with an additional N-terminal cysteine, which was used for conjugation to keyhole limpet hemocyanin (20). The antibody was raised in rabbits by Cocalico Biologicals (Reamstown, PA) and was affinity-purified using the peptide coupled to Ultralink iodoacetyl beads as described by the manufacturer (Pierce). Results were confirmed using commercially available type I IP3R Ab (Affinity BioReagents, Inc., Golden, CO) and monoclonal type III IP3R Ab (Transduction Laboratories, Lexington, KY). Calnexin antibody was raised against a peptide comprising the C-terminal 19 amino acids of canine calnexin and was a kind gift from Dr. Ari Helenius (Yale School of Medicine). Connexin-43 antibody was a generous gift from Dr. Alan Lau (Cancer Research Center, University of Hawaii). Ikappa Balpha antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Affinity-purified polyclonal ubiquitin antibody was a generous gift from Dr. Arthur L. Haas (Medical College of Wisconsin) and was also purchased from Dako Corp. (Carpinteria, CA).

Cell Culture and Treatments

Early passage WB cells were kindly given by Dr. Robert Whitson (Beckman Research Institute of the City of Hope, Duarte, CA) and were used between passages 14-20. The cells were grown in 100-mm dishes in Richter's minimal essential medium containing 5% fetal bovine serum. At approximately 70% confluence, the cells were serum-deprived for 72 h and then treated with agonists as described in the text. At the end of the treatment period, the medium was aspirated and the plates were washed twice in ice-cold phosphate-buffered saline. The cells were scraped into 350 µl of a buffer containing 150 mM NaCl; 50 mM Tris-HCl (pH 7.8); 1% Triton X-100 (w/v); 1 mM EDTA; 0.5 mM phenylmethylsulfonyl fluoride; and 5 µg/ml each of aprotinin, soybean trypsin inhibitor, and leupeptin (WB solubilization buffer). Insoluble material was removed by centrifugation for 10 min at 25,000 × g, and the soluble lysate was quenched in SDS-PAGE sample buffer (50 mM Tris (pH 6.8), 1% SDS, 0.01% bromphenol blue, 0.14 M beta -mercaptoethanol and 5% glycerol). Twenty-five µg of protein was loaded in duplicate on 5% SDS-PAGE, and the separated polypeptides were transferred to nitrocellulose. Repeated immunoblotting of the same nitrocellulose sheet was carried out after treating blots for 30 min at 60 °C in a stripping buffer containing 65 mM Tris-HCl (pH 6.8), 2% SDS, and 100 mM beta -mercaptoethanol.

Measurement of Ca2+ Mobilization

Cells grown in 75-cm2 flasks were serum-deprived for 72 h and treated in the presence or absence of 100 nM Ang II for 6 h. Cells were released from the culture plate by trypsin treatment and pelleted by centrifugation (5 min at 100 × g). The cell pellet was washed and resuspended in 120 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 20 mM HEPES/Tris (pH 7.4). IP3-induced calcium release was measured with a Ca2+-sensitive mini-electrode using experimental conditions as described previously, except that a final incubation volume of 0.2 ml was used (21).

Measurement of IP3

WB cells grown in 100-mm dishes were labeled with 5 µCi/ml myo-[3H]inositol for 24 h in inositol-free Dulbecco's modified Eagle's medium. At the end of the labeling period the medium was removed and the cells were stimulated with 100 nM Ang II for 30 s or 6 h. Where present, losartan was added together with Ang II or 5.5 h after Ang II stimulation. After treatment, the cells were washed with phosphate-buffered saline and lysed with 1 ml of 10% trichloroacetic acid. The lysates were centrifuged and the supernatants were extracted five times with 2 volumes of water-saturated diethyl ether and neutralized by the addition of 0.6 M NaHCO3 and 0.3 M EDTA. The samples were applied to 1-ml Bio-Rad AG 1-X8 anion exchange resin columns (formate form, 200-400 mesh), and inositol phosphates were separated by stepwise elution with ammonium formate buffers as described previously (22). Radioactivity of 4-ml column fractions was measured by liquid scintillation counting in 4 ml of True-Count scintillation fluid.

Immunoprecipitation of IP3R Isoforms from WB Cell Extracts

After treatment, the medium was removed and the plates were washed twice with ice-cold phosphate-buffered saline. The cells were scraped into WB solubilization buffer, and insoluble material was removed by centrifugation for 10 min at 25,000 × g. All extracts were precleared with 25 µl of a 50% (v/v) slurry of Staphylococcus aureus cell wall (Pansorbin, Calbiochem). Isoform-specific IP3R Abs were added together with protein A-Sepharose, and samples were rotated overnight at 4 °C. Immunocomplexes were washed three times in WB solubilization buffer, analyzed by immunoblotting after SDS-PAGE, and transferred to nitrocellulose. For immunoprecipitations using ubiquitin antibodies, IP3Rs in the lysates were partially purified by heparin-agarose chromatography (23). Lysates prepared from control and Ang II-treated cells were centrifuged at 25,000 × g and applied to 1-ml heparin-agarose columns (type IIS, Sigma). The column was washed with 8 ml of WB solubilization buffer, and IP3Rs were eluted with 3 ml of WB solubilization buffer containing 0.5 M NaCl (23). Control and Ang II-treated eluates were then assayed for protein, and equal amounts of protein were used for immunoprecipitations. Eluates from the two columns were split into two aliquots, and each aliquot was immunoprecipitated with type I IP3R Ab (Affinity BioReagents) or ubiquitin Ab (Dako Corp.). Immunocomplexes were processed as described above. For this series of experiments, commercially available type I IP3R Ab (Affinity Bioreagents) and ubiquitin Ab (Dako Corp.) were used for immunoprecipitation, because a lower background was obtained in subsequent ubiquitin Ab immunoblots. However, similar experimental results were obtained with C-terminal type I IP3R Ab or polyclonal ubiquitin Ab (kindly given by Dr. A. Haas).


RESULTS

Specificity of Agonist Effects on IP3 Receptor Down-regulation in WB Cells

WB rat liver epithelial cells contain predominantly the type I and type III isoforms of IP3Rs (6). The effect of chronic stimulation of WB cells for 6 h with 100 nM Ang II, 10 nM EGF, 50 nM TPA, 100 nM bradykinin, or 100 nM vasopressin on the levels of both these receptor isoforms is shown in Fig. 1A. It has been previously reported that Ang II, EGF, and vasopressin can elevate cytosolic free Ca2+ concentration and activate tyrosine kinases in WB cells (24, 25). Bradykinin also increased cytosolic free Ca2+ in WB cells (data not shown). When WB cells were treated for 6 h with the above agonists, only Ang II specifically down-regulated IP3Rs, whereas EGF, TPA, bradykinin, and vasopressin were ineffective (Fig. 1A). Fig. 1, B and C, shows the cumulative data assembled from densitometric scans of several experiments. Ang II down-regulated the type I and type III isoforms to 12 and 20% of the control levels, respectively (Fig. 1, B and C). Type I IP3R levels were not significantly altered by any of the other agonists. However, the other agonists caused a small reduction in type III IP3R expression, with the effect of TPA being statistically significant (p < 0.01). To further assess the specificity of down-regulation, calnexin was studied as a control protein, because, like the IP3R, it is an integral membrane protein of the endoplasmic reticulum (26). Calnexin functions as a molecular chaperone to several newly synthesized proteins participating in protein folding and retention in the endoplasmic reticulum. None of the agonists altered the levels of calnexin in WB cells (Fig. 1A). A prominent band at approximately 100 kDa, consistently observed in type I IP3R Ab immunoblots of total cell lysates, was also not altered by agonist treatment (data not shown).2 The data indicate that the effects of Ang II are specific and do not reflect a general down-regulation of integral membrane proteins of the ER.


Fig. 1. Effect of Ang II and other agonists on the levels of type I and type III IP3Rs. Panel A, WB cells were serum-deprived for 72 h and treated with 100 nM Ang II (AII), 10 nM EGF, 50 nM TPA, 100 nM bradykinin (BDK), or 100 nM vasopressin (VP) for 6 h. Twenty-five µg of lysates were processed in duplicate on 5% SDS-PAGE, and the blot was successively immunoblotted with antibodies specific to type I IP3R, type III IP3R, and calnexin. Panels B and C show densitometric quantitation of immunoreactive protein expressed relative to the control. The data are the mean ± S.E. from three different experiments. In each experiment multiple exposures of immunoblots were analyzed. Significant differences (Ang II, p < 0.001; TPA, p <0.01) from control are marked (asterisks).
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Additional characterization of the effects of Ang II with respect to the time course and dose response is shown in Fig. 2. WB cells were treated for various times with 100 nM Ang II, and the lysates were processed for immunoblotting with IP3R isoform-specific Abs (Fig. 2A). Densitometric analysis of data from several experiments indicates that down-regulation of both IP3R isoforms can be detected at 2 h, with peak inhibition being observed between 6 and 15 h. Incubation of the cells with Ang II for longer periods resulted in partial recovery of both IP3R isoforms (Fig. 2B). Fig. 2C shows that Ang II-induced down-regulation occurs in a dose-dependent manner with half-maximal down-regulation, requiring approximately 0.1 nM Ang II for both IP3R isoforms.


Fig. 2. Time course and dose response of IP3R down-regulation in response to Ang II. Panel A, cells were serum-deprived for 72 h and treated with 100 nM Ang II for the indicated times. Lysates were processed for immunoblotting with IP3R isoform-specific Abs. Panel B, experiments as shown in panel A were quantitated by densitometric scanning (open circle , type I; bullet , type III). The data are the mean ± S.E. from three independent experiments. Panel C, down-regulation of IP3R isoforms at different concentrations of Ang II was measured at 6 h. Values are expressed as percentage of control (open circle , type I; bullet , type III). The data are the mean ± S.E. from three different experiments.
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Functional Effects of Ang II on IP3R-mediated Ca2+ Release

To determine whether down-regulation of IP3Rs modifies intracellular Ca2+ mobilization, we measured IP3-induced Ca2+ release in permeabilized control and Ang II-treated WB cells (Fig. 3). ATP was added to initiate the sequestration of Ca2+ by the endoplasmic reticulum, and Ca2+ release in response to added IP3 was measured when Ca2+ uptake had reached a steady state. IP3-mediated Ca2+ release from Ang II-treated cells was decreased to 50-60% of the control when measured with subsaturating concentrations of 45 and 150 nM IP3, respectively (Fig. 3A). No difference between control and Ang II-pretreated cells was observed when Ca2+ release was measured with a saturating concentration of 10 µM IP3 (Fig. 3B). The relatively modest decrease in IP3-induced Ca2+ mobilization induced by Ang II pretreatment compared with the large drop in immunoreactive IP3Rs may be indicative of a substantial IP3 receptor reserve in WB cells. A marked variation in the degree of attenuation of Ca2+ signaling has been observed after carbachol-mediated IP3R down-regulation in SH-SY5Y, cerebellar granule, or Chinese hamster ovary cells (10, 27).


Fig. 3. Effect of Ang II pretreatment on IP3-mediated Ca2+ release. Panel A, WB cells grown in flasks were treated with Ang II for 6 h. At the end of the incubation the cells were trypsinized, washed, and resuspended in a "cytosol" buffer (see "Experimental Procedures"). Cells (3 mg/ml) were permeabilized with saponin, and IP3-induced Ca2+ release was measured with a Ca2+-sensitive mini-electrode (trace a, control; trace b, Ang II-pretreated cells). Panel B, conditions were the same as in panel A, except a saturating concentration of 10 µM IP3 was added.
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Reversibility of IP3R Down-regulation and Dependence on the Duration of Ang II Exposure

To determine whether down-regulation of IP3Rs is reversible, WB cells were treated with 100 nM Ang II for 6 h and type I IP3R levels were measured at different times after Ang II was removed. Fig. 4 shows that the immunoreactive type I IP3R had returned to control levels within 18 h after removal of Ang II. A partial reversal of the down-regulation response was seen even with continuous exposure to Ang II (Fig. 4, last two lanes; Fig. 2B). However, a more complete and rapid recovery was observed when Ang II was removed from the incubation medium. To determine the shortest period of Ang II exposure required to induce IP3R down-regulation, the experiments shown in Fig. 5 were carried out. WB cells were treated with Ang II for 15, 30, 60, or 90 min and type I IP3R levels were measured 4 h after the removal of Ang II. A short exposure of cells to Ang II for 30 min was sufficient to initiate IP3R down-regulation when measured 4 h later. Indeed, IP3R down-regulation caused by a transient 1-h exposure to Ang II was comparable to that observed in the continuous presence of Ang II for 4 h (Fig. 5, last two lanes). These data show that the sequence of events committing the cell to IP3R down-regulation is initiated by Ang II within a 1-h exposure and that a continuous exposure to the agonist is not required.


Fig. 4. Time course of Ang II reversibility. WB cells were serum-deprived and incubated in the presence or absence of 100 nM Ang II for 6 h. Ang II was then withdrawn and recovery of type I IP3R levels was monitored at the times indicated. For comparison, the last two lanes were incubated with Ang II continuously for 24 h.
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Fig. 5. Down-regulation of IP3R after a short exposure to Ang II and removal. WB cells were incubated in the presence or absence of 100 nM Ang II for the times indicated. Ang II was then withdrawn, and type I IP3R was measured after 4 h. For comparison, the last two lanes were exposed to Ang II continuously for 4 h.
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Effect of Ang II on IP3 Levels

It has been suggested that the ability of an agonist to maintain a prolonged elevation of IP3 levels in a cell is a causative factor in initiating degradation of IP3 receptors (10). To see whether Ang II causes a sustained increase in IP3 levels, WB cells were labeled with myo-[3H]inositol and IP3 measurements were made 30 s or 6 h after Ang II stimulation (Table I). When WB cells were stimulated with Ang II for 30 s, a 3.3-fold increase in IP3 was obtained. This increase was effectively blocked by 1.5 µM losartan, an antagonist of the type I Ang II receptor (28). A 1.6-fold elevation of IP3 could still be detected 6 h after Ang II stimulation. Addition of losartan at 5.5 h returned IP3 to basal levels, indicating that the occupied Ang II receptor was continuously activating phospholipase C over prolonged periods in WB cells. By contrast, the changes in IP3 levels after vasopressin or EGF stimulation were below the detection limit of our assays at 30 s or 6 h (data not shown).

Table I. Effect of Ang II treatment on IP3 levels

WB cells were labeled with 5 µCi/ml of myo-[3H]inositol for 24 h and treated with Ang II in the presence or absence of 1.5 µM losartan for the times indicated. IP3 was extracted and measured as described under "Experimental Procedures." Basal IP3 levels were found to be 576.4 ± 85.1 cpm/mg protein. The data are the mean ± S.E. from four independent experiments, with each measurement made in duplicate. WB cells were labeled with 5 µCi/ml of myo-[3H]inositol for 24 h and treated with Ang II in the presence or absence of 1.5 µM losartan for the times indicated. IP3 was extracted and measured as described under "Experimental Procedures." Basal IP3 levels were found to be 576.4 ± 85.1 cpm/mg protein. The data are the mean ± S.E. from four independent experiments, with each measurement made in duplicate.
Additions Time of treatment [3H]IP3 levels (% control)

Ang II 30 s 333  ± 27
Ang II + losartan 30 s 138  ± 18
Ang II 6 h 158  ± 15
Ang II + losartan 6 h 110  ± 15

Are Hetero-oligomers of IP3Rs Down-regulated by Ang II?

A pool of type I and type III IP3R isoforms exists as heterotetrameric complexes in WB cells (6). It has been shown that antibodies specific to one IP3R isoform can coimmunoprecipitate additional isoforms (6, 12). The isoform specificity of the IP3R antibodies has been studied in detail, and it has been established that the coimmunoprecipitation occurs as a consequence of the association of receptor isoforms and not as a result of antibody cross-reactivity (6). To determine whether Ang II down-regulates hetero-oligomers, WB cells were treated in the presence or absence of 100 nM Ang II for 6 h and the lysates were immunoprecipitated with either type I- or type III-specific Abs (Fig. 6). Each immunoprecipitate was successively immunoblotted with type I and type III IP3R Abs. Both type I and type III IP3R isoforms were decreased by Ang II treatment in lysate samples that were immunoprecipitated and immunoblotted with the same antibody (Fig. 6, A, lanes 1 and 2, and B, lanes 3 and 4). This fraction of IP3Rs represents both homo- and hetero-oligomers. Hetero-oligomers represented by the population of type III IP3Rs coprecipitated by type I Ab (Fig. 6B, lanes 1 and 2) or type I IP3Rs coprecipitated by type III Ab (Fig. 6A, lanes 3 and 4) were also reduced by Ang II treatment. A quantitative assessment of the proportion of hetero-oligomers present in the presence or absence of Ang II is not possible from these experiments because of the inherent difficulties of comparing immunoblots carried out with two different antibodies. Nevertheless, it is clear that IP3Rs present as homo- or hetero-oligomers are both susceptible to down-regulation upon exposure of WB cells to Ang II.


Fig. 6. Down-regulation of hetero-oligomers by Ang II. WB cells were serum-deprived for 72 h followed by incubation in the presence (lanes 2 and 4) or absence (lanes 1 and 3) of 100 nM Ang II for 2 h. The cell extracts were immunoprecipitated with antibodies specific to type I (lanes 1 and 2) or type III IP3R (lanes 3 and 4) as described under "Experimental Procedures." The immunoprecipitates (ip) were sequentially immunoblotted with type I IP3R Ab (top panel) or type III IP3R Ab (bottom panel).
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Effect of Protease Inhibitors on Ang II-induced IP3R Degradation

Previous studies on the down-regulation of type I IP3R by carbachol in SH-SY5Y cells have established that the effects of agonists are mediated by stimulation of IP3R degradation and does not involve an inhibition of biosynthesis (10). In WB cells down-regulation of IP3Rs by Ang II was not affected by pretreatment with 6-30 µg/ml cycloheximide, suggesting that the primary effect of Ang II also does not involve changes in receptor biosynthesis (data not shown). Lysosomal proteolysis and proteosomal degradation are the two major pathways of cellular protein degradation. To determine whether lysosomes are involved in the degradation of IP3Rs by Ang II, WB cells were pretreated with the lysosomal protease inhibitors chloroquine (200 µM) and NH4Cl (5 mM) for 90 min and then incubated in the presence or absence of 100 nM Ang II for 6 h. Lysates were processed for immunoblotting with type I IP3R antibody (Fig. 7A). Fig. 7B shows the densitometric scans of cumulative data from independent experiments. A 2-fold increase in the basal levels of immunoreactive type I IP3R was seen after treatment of cells with chloroquine or NH4Cl. However, Ang II was still able to induce degradation of IP3R in the presence of these inhibitors. Similar results were obtained with type III IP3R (data not shown). These results suggest that, although a component of the basal turnover of IP3Rs involves lysosomes, the degradation of IP3Rs induced by Ang II occurs via an alternate pathway.


Fig. 7. Effect of lysosomal protease inhibitors on Ang II IP3R degradation. Panel A, WB cells were pretreated with 200 µM chloroquine or 5 mM NH4Cl for 90 min and then incubated in the presence or absence of 100 nM Ang II for 6 h. Lysates were processed for immunoblotting with type I IP3R Ab. Panel B, histograms showing densitometric quantitation of type I IP3R bands (expressed as percentage of control). The data are the mean ± S.E. from three independent experiments.
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The activity of the multicatalytic 28 S proteasome has been shown to play a key role in the regulated degradation of cellular proteins and represents the major non-lysosomal protein degradation pathway present in cells (29, 30). To assess the contribution of the proteasomal pathway to the degradation of IP3Rs by Ang II, peptide-aldehyde inhibitors of proteasomal activity were used. ALLN and ALLM are inhibitors of cysteine proteases such as calpain and cathepsins (31). However, only ALLN inhibits the proteolytic activity of proteasomes, whereas its structural analogue, ALLM, is without effect (31). WB cells were pretreated with 50 µM ALLM or ALLN for 90 min followed by incubation in the presence or absence of 100 nM Ang II for 6 h. Basal levels of type I IP3R were unaffected by preincubation with ALLN (Fig. 8, A and C). However, Ang II-induced degradation of IP3R was effectively blocked by ALLN pretreatment. The half-maximal concentration of ALLN required to observe this effect was approximately 15 µM (Fig. 8B). The structural analogue, ALLM, had no effect on Ang II-induced IP3R degradation at concentrations up to 50 µM. The same pattern of effects of ALLN and ALLM was also observed for the type III isoform (Fig. 8D), with the exception that basal levels of type III IP3R were modestly elevated by ALLN pretreatment.


Fig. 8. Effect of peptide-aldehyde inhibitors on Ang II-induced IP3R degradation. Panel A, WB cells were pretreated with the inhibitors, ALLM or ALLN, for 90 min, followed by Ang II treatment for 6 h. Lysates were processed for immunoblotting with type I and type III IP3R Abs. Panel B shows a dose-response curve for the effect of ALLM and ALLN on Ang II-induced degradation of type I IP3 receptor. WB cells were pretreated with ALLM or ALLN for 90 min followed by Ang II treatment for 6 h. The samples were processed as in panel A. Panels C and D, the histograms represent densitometric quantitation of experiments as shown in panel A. The data are expressed as a percentage of IP3R levels in untreated cells and are control and mean ± S.E. from three independent experiments.
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Lactacystin, a Streptomyces metabolite, is the most potent and specific of all currently available inhibitors of proteasomal protease activity (32). In addition, this molecule is structurally very different from peptide-aldehyde protease inhibitors. Fig. 9 shows that pretreatment of WB cells for 90 min with 10 µM lactacystin does not significantly alter the basal expression of type I IP3R. However, this pretreatment was sufficient to suppress Ang II-mediated IP3R degradation. These data suggest the involvement of the proteasomal pathway in the degradation of IP3R by Ang II.


Fig. 9. Effect of lactacystin on Ang II-induced IP3R degradation. WB cells were pretreated with 10 µM lactacystin for 90 min and then incubated in the presence or absence of 100 nM Ang II for 6 h. Lysates were processed for immunoblotting with type I IP3R Ab as described in Fig. 1.
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Effect of Ang II on Other Substrates of Ubiquitin Proteasome Pathway

To determine whether a general stimulation of proteasomal degradation occurs in WB cells after Ang II stimulation, we examined the levels of two other proteins that are known to be degraded by this pathway. Connexin-43 is a gap junction protein whose basal turnover in epithelial cells is known to occur via the ubiquitin proteasome pathway (33). Ikappa Balpha is an inhibitor of the transcription factor NFkappa B. Regulated degradation of Ikappa Balpha via the ubiquitin-proteasome pathway plays a central role in the activation of gene transcription in many cells (34). Ang II stimulation of WB cells had no effect on the content of Ikappa Balpha (Fig. 10). However, chronic exposure to Ang II consistently increased connexin-43 levels under conditions where Ang II promoted IP3R degradation (Fig. 10). Although the mechanism underlying the increased expression of connexin-43 has not been further explored in this study, it is clear that Ang II-induced degradation of IP3Rs is not accompanied by an enhanced degradation of other proteasomal substrates.


Fig. 10. Effect of Ang II on connexin-43 and Ikappa Balpha . WB cells were treated with 100 nM Ang II for 6 h, and the cells were lysed and processed on a 10% SDS-PAGE and immunoblotted with type I IP3 receptor Ab, connexin-43 Ab, and Ikappa Balpha Ab successively.
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Polyubiquitination of IP3R

Because our previous data using ALLN and lactacystin suggested the involvement of the proteasome pathway, we carried out experiments to determine whether IP3Rs were tagged with ubiquitin prior to degradation. To increase the sensitivity of detection, we partially purified IP3R from the lysates on heparin-agarose columns (23) and included N-ethylmaleimide in the lysis buffer to inhibit the enzymes that deubiquitinate proteins (35). Extracts from control and Ang II-treated cells were prepared using these conditions and immunoprecipitated with anti-Ub Ab. When these immunoprecipitates were immunoblotted with Ub Ab, a series of bands were visualized, including one located at the expected position of IP3R (Fig. 11, top panel, lanes 1 and 2). The intensity of these bands was increased in lysates prepared from Ang II-treated cells. The presence of ubiquitinated IP3R in the Ub Ab immunoprecipitates was confirmed by type I IP3R Ab immunoblotting (Fig. 11, top panel, lanes 3 and 4). The same series of bands that were recognized by Ub Ab were also immunoreactive with IP3R Ab, suggesting that they may correspond to IP3Rs with a multiple number of attached polyubiquitin chains. The intensity of these bands was also increased in lysates prepared from Ang II-treated cells. When cell extracts were first immunoprecipitated with type I IP3R Ab and then immunoblotted with Ub Ab, a weak reactivity of the IP3R was observed in untreated cells (Fig. 11, lower panel, lane 1). The Ub reactivity of the IP3R was increased after Ang II treatment and included a series of Ub Ab-reactive bands of higher molecular weight (Fig. 11, lower panel, lane 2). IP3R Ab immunoblotting of the IP3R Ab immunoprecipitates indicated that the total amount of IP3R present in the cell extracts in control and Ang II-treated cells was the same, as expected in ALLN-treated cells (Fig. 11, lower panel, lanes 3 and 4). Surprisingly, the higher molecular weight series of bands was not reactive with IP3R Ab on immunoblots. The reason for this is not clear, but it may reflect differences in the relative ability of Ub Ab and IP3R Ab to recognize the polyubiquitinated species of IP3R during immunoprecipitation or immunoblotting. These experiments also suggest that the polyubiquitinated forms of the IP3R may represent only a small fraction of the total IP3R, even in Ang II-stimulated cells. Similar observations have been made during the identification of other polyubiquitinated proteins including p185c-erbB-2 (36) and the EGF receptor (37). Our experiments show that Ub Ab can immunoprecipitate IP3Rs and recognize a ladder of high molecular weight bands in IP3R Ab immunoprecipitates. We conclude that IP3Rs can be ubiquitinated in WB cells and that Ang II treatment enhances ubiquitination.


Fig. 11. Polyubiquitination of IP3R. WB cells were serum-deprived for 72 h and pretreated with 50 µM ALLN for 90 min followed by incubation in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of 100 nM Ang II for 2 h. The cell extracts were partially purified on heparin-agarose columns and immunoprecipitated with antibodies specific to ubiquitin (top panel) or type I IP3R (bottom panel) as described under "Experimental Procedures." Ubiquitin and IP3R immunoprecipitates (ip, top and bottom panels) were successively immunoblotted with Ub Ab (lanes 1 and 2) and type I IP3R Ab (lanes 3 and 4).
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DISCUSSION

The data presented show that both type I and type III IP3Rs are down-regulated by chronic stimulation of WB cells with Ang II. This effect was specific to Ang II, reversible, and not mimicked by other agonists. Down-regulation of IP3Rs was accompanied by a decreased sensitivity of Ca2+ mobilization in response to agonists and IP3. All of these findings, and the insensitivity of the response to cycloheximide, suggest that Ang II stimulation of IP3R degradation in WB cells have several features in common to IP3R down-regulation described for other agonists in other cell types (10, 11).

The 2-fold elevation of IP3Rs in the presence of NH4Cl or chloroquine suggests that the lysosomal pathway plays a significant role in the basal turnover of IP3Rs. On the other hand, Ang II-induced degradation of IP3Rs was not blocked by NH4Cl or chloroquine, excluding a primary role for lysosomes in the degradation of IP3Rs triggered by chronic Ang II stimulation. An important conclusion of the present study is that Ang II-stimulated degradation of IP3Rs occurs via the ubiquitin proteasome pathway. This conclusion is supported by experiments using pharmacological compounds that are known to inhibit the ubiquitin proteasome pathway. Peptide aldehyde inhibitors such as ALLN and ALLM have been shown to inhibit cysteine proteases. However, at low concentrations (<50 µM), only ALLN inhibits the chymotryptic activity of the 26 S proteasome complex (31). Down-regulation of IP3Rs by Ang II was blocked by ALLN but not by an equal concentration of its structural analogue ALLM. A large difference in potency of ALLN and ALLM has also been observed for the effects of these inhibitors on the degradation of CFTR protein (38, 39), connexin 43 (33), and peptide presentation by major histocompatibility complex class I molecules (31). The conclusion that proteasomes are involved in the regulated degradation of IP3Rs is further supported by the finding that 10 µM lactacystin can block the effect of Ang II. Lactacystin (100 µM) has been shown to have no effect on the activity of cysteine or serine proteases and is the most specific proteasomal inhibitor currently available (32). Although most proteins degraded by the proteasomal pathway are ubiquitinated, there are also examples of substrates that are not (e.g. ornithine decarboxylase (22)). Our experiments using Ub Ab indicates that high molecular weight polyubiquitinated species of IP3R accumulate in WB cells treated with ALLN and Ang II. This suggests that Ang II enhancement of Ub conjugation to the IP3R may act as a signal to mark this protein for proteasomal degradation. We have been unable to detect the appearance of any additional proteolytic fragments after Ang II addition to cells. The absence of intermediate proteolytic products has also been noted during the sterol-regulated degradation of hydroxymethylglutaryl-CoA reductase, an integral ER membrane enzyme, the regulated degradation of which is also blocked by lactacystin (40).

A protein degradation system which removes incompletely assembled or misassembled complexes, is known to be present in the ER as part of the quality control function of this organelle (reviewed in Ref. 41). The components of this degradation pathway have not been identified. Recent studies, using an in vitro yeast microsomal system, suggest the involvement of cytosolic components, ATP hydrolysis, and calnexin (42). There have also been several reports to suggest that the regulated degradation of integral membrane proteins in the ER may involve the proteasome pathway. Examples include a mutant form of the CFTR protein (38, 39), hydroxymethylglutaryl-CoA reductase (40), and cytochrome P4502E1 (43). In yeast, genetic evidence has been presented to suggest that disassembled components of the Sec61p complex (responsible for the translocation of nascent polypeptides into the ER) are also degraded by the proteasome pathway (44). With the exception of hydroxymethylglutaryl-CoA reductase, all these proteins have been demonstrated to be ubiquitinated. The exact mechanism by which a polytopic transmembrane protein is degraded in the ER is not well understood, but presumably the concerted action of proteolytic systems on both sides of the membrane is involved. A recent study has suggested that even luminal proteins of the ER can undergo retrograde transport across the ER membrane to be exposed to cytosolic proteasomal complexes (45).

Why is Ang II unique in being the only Ca2+-mobilizing agonist in WB cells that causes down-regulation of IP3Rs? The Ang II receptor on the WB cell is predominantly the type 1 subtype,3 which is known to be coupled to phospholipase C. It has been proposed that the common feature of cell surface receptors that down-regulate IP3Rs is their ability to maintain a prolonged activation of phospholipase C. The sustained elevation of IP3 is postulated to induce a conformation of the IP3R that makes the receptor particularly susceptible to proteolytic cleavage (10). Our IP3 measurements clearly indicate that phospholipase C does remain activated 6 h after continuous exposure of WB cells to Ang II. However, it is difficult to distinguish from these data whether the elevated IP3 levels are a primary regulatory factor or simply accompany the activation of other signaling pathways that cause down-regulation. We observed that exposure to Ang II for short periods was sufficient to initiate down-regulation and that a continuous exposure to Ang II was not required (Fig. 5). This implies that a maintained elevation of IP3 may not be required. If the changes in IP3 levels do play a role, they must exert their effects at a relatively early stage in the sequence of events that lead to IP3R degradation.

Agonist-mediated Ca2+ mobilization in WB cells has been shown to stimulate multiple tyrosine kinases and to result in the tyrosine phosphorylation of a large number of proteins (24, 46). The kinases affected include members of the mitogen-activated protein kinase family, with a 200-fold activation of c-Jun N-terminal kinase occurring within 30-60 min of Ang II stimulation (47). However, these changes are also induced by EGF, vasopressin, and thapsigargin with a similar magnitude and time course as Ang II (47, 48). Because only Ang II down-regulates IP3Rs, it is unlikely that these particular kinases are involved. However, we cannot exclude the possibility that unique tyrosine kinases are activated in WB cells by Ang II stimulation. Ubiquitin-conjugating enzymes and proteasomal complexes have also been shown to be regulated by phosphorylation (49). Additional studies are needed to unravel the signaling pathway utilized by Ang II in stimulating IP3R degradation.

While this manuscript was in preparation, Wojcikiewicz and Oberdorf (50) published the results of experiments on the mechanism of IP3R down-regulation induced by carbachol in SH-SY5Y human neuroblastoma cells. These authors showed that 50 mM NH4Cl did not block carbachol-mediated IP3R down-regulation. In contrast to our observations in WB cells, this treatment did not affect basal IP3R levels. As in the present study, the authors found that ALLN blocked agonist-mediated IP3R down-regulation. The effects of ALLM or lactacystin were not reported. From these results they concluded that a cysteine protease was involved. A model was proposed in which carbachol stimulation results in very high local concentrations of Ca2+ in the vicinity of IP3Rs, which in turn triggers receptor degradation as a result of the activation of calpain, a Ca2+-sensitive cysteine protease. Whether different cell surface receptors in different cell types utilize different mechanisms to down-regulate IP3Rs remains to be experimentally evaluated.


FOOTNOTES

*   This work was supported by Postdoctoral Training Grant T32-AA07463 (to S. B.) and Grants RO1-DK34804 and RO1-AA10971 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Rm. 230A, JAH, 1020 Locust St., Philadelphia, PA 19107. Tel.: 215-503-1222; Fax: 215-923-6813; E-mail: josephs{at}jeflin.tju.edu.
1   The abbreviations used are: IP3, myo-inositol 1,4,5-trisphosphate; IP3R, myo-inositol 1,4,5-trisphosphate receptor; Ang II, angiotensin II; EGF, epidermal growth factor; TPA, 12-O-tetradecanoylphorbol-13-acetate; Ab, antibody; PAGE, polyacrylamide gel electrophoresis; ALLM, N-acetyl-L-leucinyl-L-leucinyl-L-methioninal; ALLN, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; Ub, ubiquitin; SDS, sodium dodecyl sulfate; ER, endoplasmic reticulum; CFTR, cystic fibrosis transmembrane conductance regulator.
2   This band was not immunoprecipitated by type I IP3R Ab. It was also not recognized on immunoblots by nonimmune serum, type III IP3R Ab, or Ab to type I N-terminal amino acids 401-414 (data not shown). We therefore conclude that the band represents a C-terminal fragment of the IP3R.
3   S. Fluharty, personal communication.

ACKNOWLEDGEMENTS

We thank Drs. Fluharty, Helenius, Haas, Lau, and Whitson for generously supplying us with reagents used in this study. The helpful suggestions of Drs. Mark Hochstrasser, Edward Mimnaugh, and Alan Schwartz are also acknowledged.


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