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Originally published In Press as doi:10.1074/jbc.M509966200 on November 29, 2005

J. Biol. Chem., Vol. 281, Issue 6, 3722-3730, February 10, 2006
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Mechanism of Proteasomal Degradation of Inositol Trisphosphate Receptors in CHO-K1 Cells*

Cunnigaiper D. Bhanumathy, Steven K. Nakao, and Suresh K. Joseph1

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

Received for publication, September 12, 2005 , and in revised form, November 14, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
myo-Inositol 1,4,5-trisphosphate receptor (IP3R) degradation occurs in response to carbachol (Cch) stimulation of CHO-K1 cells. The response was mediated by endogenous muscarinic receptors and was blocked by atropine or proteasomal inhibitors. We have used these cells to identify the sites of ubiquitination on IP3Rs and study the role of Ca2+ and substrate recognition properties of the degradation system using exogenously expressed IP3R constructs. Employing caspase-3 for IP3R cleavage, we show that Cch promotes polyubiquitination in the N-terminal domain and monoubiquitination in the C-terminal domain. The addition of extracellular Ca2+ to Ca2+-depleted Chinese hamster ovary (CHO) cells initiates IP3R degradation provided Cch is present. This effect is inhibited by thapsigargin. The data suggest that both a sustained elevation of IP3 and a minimal content of Ca2+ in the endoplasmic reticulum lumen is required to initiate IP3R degradation. Transient transfection of IP3R constructs into CHO cells indicated the selective degradation of only the SI(+) splice variant of the type I IP3R. This was also the splice form present endogenously in these cells. A pore-defective, nonfunctional SI(+) IP3R mutant (D2550A) was also degraded in Cch-stimulated cells. The Cch-mediated response in CHO cells provides a convenient model system to further analyze the Ca2+ dependence and structural requirements of the IP3R proteasomal degradation pathway.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The activation of inositol 1,4,5-trisphosphate receptors (IP3Rs)2 by IP3 initiates Ca2+ mobilization from the ER and triggers the Ca2+ signal that underlies alterations in cell function elicited by a diverse array of cell surface stimuli (1, 2). A commonly observed characteristic of cells is that they adapt their responses when chronically stimulated. In the case of cell surface receptors, this is usually the result of phosphorylation and/or internalization. Wojcikiewicz et al. (3, 4) were the first to note that chronic stimulation of cultured SHSY-5Y neuroblastoma cells with carbachol for 6 h causes 90% loss of type I IP3R protein by a mechanism involving the marked acceleration of IP3R degradation. Subsequently, similar effects on IP3R degradation have been described in many different experimental systems with many different agonists (5-16). Down-regulation of IP3R protein is associated with a decrease in the ability of IP3 to mobilize Ca2+ (4, 8). More has shown been recently it that the frequency of elementary Ca2+ puffs that trigger propagating Ca2+ waves in cells is decreased by chronic agonist stimulation (14). The physiological significance of IP3R down-regulation is unknown. However, down-regulation of receptors would be expected to inhibit the global Ca2+ signal elicited by all Ca2+ mobilizing agonists (heterologous desensitization). If this is the only component of the signal transduction system that is down-regulated, then an imbalance in signaling pathways would exist and may have profound consequences for cellular responses. A physiologically relevant example is the response of pituitary cells to GnRH receptor agonists (17). Mammalian GnRH receptors lack a C-terminal tail and therefore do not bind arrestin. Consequently these receptors desensitize and internalize only very slowly. However, sustained activation of GnRH receptors does lead to desensitization of gonadotrophin hormone secretion, which suggests the presence of adaptive mechanisms distal to the cell surface receptor. Treatment with GnRH receptor agonists produce a rapid and pronounced degradation of IP3Rs, and this is associated with a marked reduction in the IP3-mediated Ca2+ signal (15, 18).

The mechanism of IP3R degradation has not been established. Previous studies have shown that chronic elevation of IP3 and IP3 binding to the receptor are required to facilitate IP3R degradation (19, 20). It has been proposed that the sustained elevation of IP3 causes the IP3R to adopt a conformation that exposes sites that become ubiquitinated. The proteasome pathway then degrades the ubiquitinated IP3 receptor. The regions of the IP3R involved in ubiquitination have not been determined. Pretreatment of cells with the SERCA pump inhibitor thapsigargin has been found to inhibit agonist-mediated IP3R degradation, suggesting that Ca2+ also plays a role in IP3R degradation (4). In the present study we have examined IP3R degradation in CHO-K1 cells stimulated with carbachol. This system has been used to examine the domains of IP3Rs that are ubiquitinated and to further explore the role of Ca2+ in IP3R degradation. The ability to readily transfect CHO-K1 cells with various IP3R constructs has allowed an initial characterization of the substrate recognition properties of the IP3R degradation system.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Carbachol, acetylcholine, atropine, ALLN, and ALLM were purchased from Sigma. Lactacystin was purchased from Dr. E. J. Corey (Harvard University). ZL3VS was a kind gift of Dr. Mathew Bogyo (Stanford University). Stabilized acrylamide solution (Protogel) for the preparation of SDS gels was obtained from National Diagnostics (Atlanta, GA). Recombinant caspase-3 was purified from BL-21 bacterial lysates expressing the enzyme as a His6-tagged fusion protein using previously described methods (21). The plasmid encoding caspase-3 was kindly given by Dr. E. S. Alnemri.

Antibodies—The type I polyclonal antibody was raised to unique amino acids 2731-2749 present at the C terminus of the rat type I IP3R (CT-1) and has been previously characterized (22, 23). An N-terminal polyclonal antibody was raised to amino acids 326-341 of the type I IP3R (NT-1). This Ab was used after affinity purification using the immobilized antigenic peptide as a column matrix. The type III IP3R monoclonal antibody was purchased from Transduction Laboratories (Lexington, KY). Monoclonal Ab to ubiquitin was purchased from StressGen (Victoria, Canada). Myc monoclonal Ab (9E11) was obtained from the Cell Center of the University of Pennsylvania.

Expression Constructs—The cDNA encoding rat type I IP3R SI(-)/SII(+)/SIII(+) splice variant in pCMV3 was the kind gift of Dr. Thomas Sudhof (University of Texas Southwestern Medical Center) (24). The cDNA encoding the rat type I IP3R SI(+) variant was the kind gift of Dr. Gregory Mignery (Loyola University Chicago) (25). The D2550A pore-defective mutant in SI(-) (26) was transferred to the SI(+) IP3R using BstBI/XbaI restriction sites. The Myc-tagged constructs were made using PCR as described previously (27).

Cell Culture and Pretreatment—CHO-K1 cells were obtained from ATCC (catalog number CCL-61) and from Dr. John Pastorino. The cells were grown in Dulbecco's minimal essential medium containing 5% fetal bovine serum and 1% penicillin/streptomycin. The confluent cells were deprived of serum (1-2 h) and treated with carbachol for the indicated times. 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 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 protease inhibitor mixture (Roche Applied Science) (solubilization buffer). Insoluble material was removed by centrifugation for 10 min at 25,000 x g. 20 µg of protein was loaded 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—Changes in cytosolic [Ca2+]in individual CHO cells plated on coverslips were measured by digital imaging fluorescence microscopy as previously described (28). Briefly, the cells were loaded with Fura-2 by incubating with 5 µM Fura-2AM in 0.03% pluronic F-127 in the presence of 100 µM sulfinpyrazone for 30 min at 37 °C. The coverslips were washed with HEPES buffer (10 mM HEPES, 120 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 5 mM NaHCO3, 10 mM glucose, pH 7.4) containing 2% bovine serum albumin and then in HEPES buffer containing 0.25% bovine serum albumin. Coverslips with Fura-2-loaded cells were transferred into a chamber with 1 ml of HEPES buffer with or without Ca2+ and mounted onto the stage of an inverted microscope thermostatically maintained at 37 °C. Fluorescence images with a 4-s delay were collected alternately at excitation wavelengths of 340 and 380 nm with an emission wavelength of 460-600 nm. [Ca2+]i for each individual cell was calculated from the fluorescence values at each time point using the ratio method as described previously (28).

Measurement of IP3R Ubiquitination—To measure ubiquitination CHO-K1 cells were pretreated for 1 h with or without proteasomal inhibitors and then treated for 4-6 h with 1 mM carbachol. The cells were then treated for 10 min at room temperature with phosphate-buffered saline containing 1 mM NEM and lysed in solubilization buffer supplemented with 1 mM NEM. The NEM was added to block deubiquitinating enzymes. The lysate was then immunoprecipitated overnight with CT-1 Ab. The immunoprecipitates were electrophoresed on 5% SDS-PAGE, transferred to nitrocellulose, and probed with a 1:5000 dilution of anti-ubiquitin Ab (StressGen). To resolve IP3R domains that contained ubiquitin, we employed a Myc-tagged Ub plasmid (kindly given by Dr. Ron Kopito) that was transfected together with the type I IP3R into 4 x 150-mm plates (20 µg of DNA each/plate) using LT-1 as a transfection reagent (Mirus Corp., Madison, WI). After 48 h the plates were treated for 1 h with a mixture of proteasomal inhibitors consisting of 10 µM lactacystin, 50 µM ZL3VS, and 50 µM ALLN. The plates were then treated with 1 mM carbachol, lysed as described above in NEM-containing medium, and immunoprecipitated overnight with CT-1 Ab. After washing twice in solubilization buffer, an aliquot of the immunoprecipitate (80%) was digested with caspase-3 in 200 µl of a buffer containing 50 mM HEPES (pH 7.2), 1 mM dithiothreitol, 1 mM EDTA, 0.25 mM phenylmethylsulfonyl fluoride, 0.1% Triton X-100, and 1 µg recombinant caspase-3. 20% of this sample was quenched with SDS-PAGE buffer and was used as the uncut control sample. The digested fragments were released from the PrA-Sepharose beads by 5 min of treatment at 95 °C with 0.35 M glycine (pH 2.8) and 0.1% Triton X-100. The sample was diluted in lysis buffer and reimmunoprecipitated with Myc-Ab to recover the ubiquitinated fragments. The uncut control sample and the Myc reimmunoprecipitated samples were processed on 10% SDS-PAGE and immunoblotted with IP3R Abs to locate the ubiquitinated fragments.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Basic Characterization of IP3R Down-regulation in CHO-K1 Cells— Our previous studies on IP3R degradation were carried out in WB rat liver epithelial cells stimulated with angiotensin II (8, 30). These cells have proved very difficult to transiently transfect with IP3R constructs, and stable WB cell lines lose their IP3R degradation response to angiotensin II (data not shown). We therefore examined other cells types that were more amenable to transfection for an agonist-mediated IP3R degradation response. Fig. 1A shows that carbachol (Cch) addition to CHO-K1 cells stimulated IP3R down-regulation. Similar Cch effects were seen when the blots were reprobed for the type III IP3R isoform (data not shown). The effect was specific to Cch because ATP, which also elevates Ca2+ in these cells (cf. Fig. 5), did not induce significant IP3R degradation. To further characterize these effects we examined time course and dose-response relationships (Fig. 1, B and C). Densitometric analysis of immunoblots showed that IP3R down-regulation was detected within 2 h and peaked between 4 and 6 h of Cch stimulation (Fig. 1B). This pattern of response is comparable with what has been observed for agonist-mediated IP3R down-regulation in other systems (8). Fig. 1C shows the dose response for IP3R down-regulation using carbachol, methacholine, and acetylcholine as agonists. The dose response for the carbachol effect was complex and showed two distinct components with half-maximal inhibitory effects at ~20 and 80-100 µM. Methacholine was somewhat more potent than carbachol (IC50 = ~10 µM). Acetylcholine was the least potent agonist with half-maximal effects at 50 µM and only a 40% down-regulation even at 1 mM. These data, together with the finding that the carbachol effects on down-regulation are completely blocked by atropine (Fig. 2), suggest that IP3R down-regulation is mediated by endogenous muscarinic receptors in CHO-K1 cells.

The role of different proteolytic pathways in IP3R degradation was investigated using various protease inhibitors (Fig. 2). The most pronounced inhibition of Cch-mediated IP3R degradation was observed with the proteasomal inhibitor lactacystin. Rather surprisingly, 50 µM of the peptide aldehyde inhibitor ALLN, which is effective at preventing proteasomal degradation of IP3Rs in WB cells (8), exerted only a small inhibitory effect on Cch-mediated IP3R degradation. A small inhibitory effect of 100 µM chloroquine was also observed. This is probably an indirect effect and unlikely to reflect the involvement of the lysosomal pathway because 5 mM NH4Cl was without effect. Similarly the lack of effects of ALLM, DEVD-CHO, and calpain inhibitor would rule out the involvement of general cysteine proteases, caspases, and calpains, respectively. Thus the data would suggest that the major proteolytic system involved in Cch-mediated IP3R degradation is the proteasomal pathway.


Figure 1
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  		FIGURE 1.
Time course and dose response of IP3R down-regulation by carbachol in CHO cells. A, cells were serum-deprived overnight and treated with ATP (1 mM) or Cch (1 mM). The cell lysates were electrophoresed on a 5% gel, transferred to nitrocellulose, and probed with type I IP3R Ab. The upper panel shows a representative blot. The lower panel shows quantitation of the blots by densitometric analysis of three to five independent experiments. The data are expressed as percentages of IP3R levels in untreated control samples. B, CHO cells were incubated with 1 mM carbachol for the indicated time periods and were analyzed for type I IP3R by immunoblotting. C, cells were incubated with different concentrations of carbachol (bullet), acetylcholine ({circ}), or methacholine ({blacksquare}) for 4 h and were analyzed for type I IP3R immunoreactivity. The data in both panels are expressed as percentages of the control untreated sample and are the means ± S.E. of three experiments.

 


Figure 2
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  		FIGURE 2.
The effect of protease inhibitors on carbachol-mediated IP3R degradation. CHO cells were incubated with 1 mM carbachol for 6 h in the absence or presence of protease inhibitors and were analyzed for type I IP3R by immunoblotting. The concentration of drugs used were as follows: atropine (100 µM), ALLN (50 µM), ALLM (50 µM), lactacystin (10 µM), caspase inhibitor DEVD-CHO (100 µm), NH4Cl (5 mM), chloroquine (1 mM), and calpain inhibitor MDL-28170 (20 µM). None of the agents changed IP3R levels when added alone with the exception of NH4Cl and chloroquine, which increased levels by 44 + 14 and 35 + 7.9%, respectively. *, p > 0.05 significant from carbachol alone. The data are the means ± S.E. for three to five experiments.

 
Ubiquitination Domains in IP3R—We first carried out experiments to confirm that the proteasomal degradation of IP3Rs was accompanied by ubiquitination. CHO cell lysates were treated with Cch in the presence or absence of lactacystin, and the immunoprecipitated IP3R was probed for ubiquitin with anti-Ub Ab (Fig. 3A). The data show that ubiquitinated IP3R accumulated only when the cells were treated with a combination of Cch and lactacystin (Fig. 3A, lane 3). There are 159 cytosol-exposed lysines in the primary sequence of the type I IP3R that could potentially serve as sites for ubiquitination. Our limited objective in the present study was to identify domains in the IP3R that may serve as targets of ubiquitination. The strategy we chose to employ was to digest the ubiquitinated IP3R with a protease that would cleave the IP3R but not ubiquitin. A protease that satisfies this criteria is caspase-3, which is known to cleave IP3Rs (31, 32). Initial experiments to detect endogenous Ub attached to caspase-3-cleaved IP3R fragments were limited by the poor sensitivity of the available anti-Ub Abs. For this reason we chose to transfect the CHO cells with Myc-tagged Ub. However, transfection with Myc-tagged Ub alone did not significantly label endogenous IP3Rs immunoprecipitated from CHO cells treated with Cch and lactacystin (Fig. 3B, lane 1). At high exposures of the blot it was possible to detect a single band at the expected migration of IP3R, indicating that endogenous receptors may become monoubiquitinated under these conditions (Fig. 3B, lane 5). A weak labeling of endogenous receptors by Myc-tagged Ub has also been observed in {alpha}T3-1 anterior pituitary cells (18). In part, this may be related to the low transfection efficiency of our cells, which was estimated at 10-20% by indirect immunofluorescence measurements (data not shown). To maximize the signal we cotransfected CHO cells with both Myc-tagged Ub and IP3R cDNA. Under these conditions a significant increase in Myc ubiquitinated IP3R was observed when IP3R was immunoprecipitated from CHO cells treated with Cch and lactacystin (Fig. 3B, lane 2).

Fig. 4A shows the location of eight potential DXXD caspase-3 cleavage sites in the type I IP3R and the expected size of N- and C-terminal fragments for cleavage at each of these sites. Previous studies have documented a single cleavage site distal to the DEVD sequence at aspartate 1892 (31, 32). This would be expected to generate a 98-kDa C-terminal fragment and a 215-kDa N-terminal fragment. IP3R immunoprecipitates were prepared from CHO cells cotransfected with Myc-Ub and type I IP3R plasmids that were then treated with Cch in the presence of proteasomal inhibitors. IP3R was immunoprecipitated from these lysates with CT-1 Ab and then cleaved with caspase-3. The fragments formed were reimmunoprecipitated with Myc-Ab to bring down the Myc-tagged cleavage fragments (Fig. 4B). Analysis of the fragments by immunoblotting with Myc Ab showed a prominent single band at 95 kDa and a smeared signal that extended from 119 to 230 kDa (Fig. 4B, lane 2). The 95-kDa band was cross-reactive with the CT-1 Ab and corresponds to the expected C-terminal caspase-3 cleavage product (Fig. 4B, lane 4). Because it appears as a single band, we propose that the C-terminal segment of IP3Rs can be monoubiquitinated. The smeared signal seen in the Myc Ab blots was cross-reactive with the N-terminal Ab (lane 6) but not the C-terminal Ab (lane 4). We conclude that polyubiquitination is confined to the N-terminal segments of the IP3R. When caspase-3-cleaved IP3R was examined with N-terminal Ab, a fragment of ~95 kDa was observed rather than the expected 215-kDa fragment (Fig. 4B, lanes 7 and 8). This suggests that in vitro caspase may cleave at other sites in addition to aspartate 1892. The size of the 95-kDa N-terminal fragment is compatible with cleavage at aspartate 820, which would locate the polyubiquitination sites proximal to this amino acid. The spread of the Myc-Ub signal would suggest heterogeneity in the size of the polyubiquitin chains and/or the use of multiple attachment sites in the N-terminal domain.


Figure 3
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  		FIGURE 3.
Ubiquitination of IP3Rs in CHO cells. A, CHO cells were treated with Cch for 4 h with and without a preincubation for 30 min with 10 µM lactacystin (LC). The cells were lysed in buffer containing 1 mM NEM, and endogenous type I IP3Rs were immunoprecipitated (IP) with CT-1 Ab. The immunoprecipitates were processed on 5% SDS-PAGE and immunoblotted (IB) with Anti-Ub Ab. B, CHO cells were transfected with Myc-Ub plasmid alone (lane 1) or together with type I IP3R DNA (lanes 2-4). The cells were treated as in A except that proteasomal inhibitors (50 µM) of ZL3VS and ALLN were included together with the LC. Where indicated Cch (1 mM) was added for 5 h. The lysates were immunoprecipitated with CT-1 Ab and immunoblotted with anti-Myc monoclonal antibody to visualize ubiquitinated IP3R. A high exposure of lane 1 is shown in lane 5. The single Myc-reactive band was shown to be at the position expected of IP3R by reprobing the blot with CT-1 Ab (data not shown).

 


Figure 4
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  		FIGURE 4.
Localization of ubiquitination sites in the type I IP3R using Myc-tagged Ub sites of ubiquitination in type I IP3R. A, the schematic diagram shows the location of eight putative caspase-3 cleavage sites in the type I IP3R based on the presence of a DXXD consensus sequence. The calculated size of the C-terminal fragment (indicated in bold type) and the N-terminal fragment (italics) for cleavage at each of the sites is shown. The open arrows indicate the locations of the epitopes of the CT-1 and NT-1 Abs used in this study. The locations of the splice sites are also shown. B, lysates were prepared from CHO cells transfected with Myc-tagged Ub and type I IP3R and then treated with 1 mM Cch for 6 h. The lysates were immunoprecipitated overnight with CT-1 Ab, and 20% of this sample was quenched with SDS-PAGE buffer and used as the uncut control sample (lanes 1, 3, and 5). The remaining 80% was digested with caspase-3 and reimmunoprecipitated with Myc Ab as described under "Experimental Procedures." Both uncut and Myc reimmunoprecipitated samples were processed on 10% SDS-PAGE and consecutively immunoblotted with the indicated Abs. The brackets and arrows indicate the putative polyubiquitinated and monoubiquitinated species, respectively. In lanes 7 and 8 nonubiquitinated lysates were immunoprecipitated with CT-1 Ab, and control and caspase-3-digested samples were probed by immunoblotting with NT-1 Ab to locate the N-terminal cleavage fragment (arrowhead).

 
The Role of Ca2+ in Carbachol-mediated IP3R Degradation—Fig. 5A shows the Ca2+ transients recorded in Fura-2-loaded CHO-K1 cells in response to a maximal dose of Cch that triggers IP3R degradation. Cch addition elicited a Ca2+ signal that remained elevated for a prolonged period (>30 min). By contrast a Ca2+-mobilizing stimulus such as ATP, which did not cause IP3R degradation, produced a smaller and more transient Ca2+ signal. The ATP responses observed in cells that were pretreated with Cch for 6 h was substantially blunted as would be anticipated if IP3Rs were degraded by Cch pretreatment (Fig. 5B). To further examine the role of Ca2+, we pretreated the cells for 30 min with thapsigargin (to empty intracellular stores) or EGTA (to remove extracellular Ca2+). Fig. 6A show that both treatments blocked Cch-mediated degradation, in agreement with previous studies on other cell types (4). The effect of buffering the cytosolic Ca2+ transient by loading the cell permeant BAPTA-AM chelator is shown in Fig. 6B. BAPTA-AM completely blocked Cch-mediated IP3R degradation when added 30 min prior to Cch (data not shown), but its effectiveness declined progressively when the chelator was added at different times after Cch. Thus BAPTA-AM loaded into the cells 30 min after Cch addition was unable to prevent the degradation of IP3R observed at 6 h. This indicates that a continuously elevated cytosolic Ca2+ is not required for IP3R degradation and that the signals that irreversibly commit the IP3R to degradation are formed within a 30-min interval of Cch addition. A similar conclusion was reached from experiments in which the Cch stimulus was terminated at various times by the addition of atropine (Fig. 6C). These data show that carbachol exposure for 1 h has the same effect as the continuous exposure to carbachol for 4 h.


Figure 5
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  		FIGURE 5.
Calcium transients in CHO-K1 cells in response to agonists. A, cytosolic Ca2+ changes were monitored in Fura-2-loaded cells incubated in normal extracellular Ca2+ and stimulated with 1 mM Cch or ATP. B shows cytosolic Ca2+ changes in cells challenged with two concentrations of ATP after being exposed to Cch pretreatment (1 mM; 6 h). For both experiments each trace represents the average of three coverslips (30 cells/coverslip).

 


Figure 6
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  		FIGURE 6.
The effect of inhibitors of Ca2+ mobilization on Cch-mediated IP3R degradation. A, CHO cells were incubated with 1 mM Cch for 4 h with or without pretreatment with 2 µM thapsigargin or 1 mM EGTA for 30 min. The lysates were analyzed for type I IP3R by immunoblotting. B shows the effect of BAPTA-AM (100 µM) addition at the indicated times after the addition of 1 mM carbachol. In all cases the cells were lysed after 6 h of treatment with agonist and analyzed for type I IP3R by immunoblotting. The Cch-mediated Ca2+ transient was completely inhibited within 5 min of addition of BAPTA-AM to the cells (data not shown). C, CHO cells were exposed to 1 mM Cch for 15, 30, 60, and 90 min prior to the addition of 0.1 mM atropine followed by an additional 4-h incubation. The levels of type I IP3R were compared with cells continuously exposed to Cch for 4 h.

 
The interpretation of the finding that thapsigargin, EGTA, and BAPTA-AM inhibit IP3R degradation is complicated by the known Ca2+ sensitivity of phospholipase C (PLC) in CHO cells (33). Because it has been established that chronic elevation of IP3 and IP3 binding to the IP3R are prerequisite for obtaining IP3R degradation (19, 20), the perturbing effect of Ca2+ chelating and mobilizing agents could reflect an inadequate PLC-dependent generation of IP3. To distinguish between Ca2+-dependent and IP3-dependent effects, we carried out the experiments shown in Fig. 7. Extracellular Ca2+ was chelated with EGTA, and the cells were stimulated with Cch. This resulted in a Ca2+ elevation that was transient and returned to base line within 5 min (Fig. 7A). This transient elevation of Ca2+ is clearly insufficient to sustain IP3R degradation (Fig. 6A). Depletion of intracellular stores is known to activate Ca2+ entry channels in the plasma membrane. This can be experimentally monitored as a marked elevation of cytosolic Ca2+ when extracellular Ca2+ is added back to store-depleted cells (Fig. 7A). Using this protocol we noted that the addition of Ca2+ also caused marked IP3R degradation (Fig. 7B) and occurred even when Ca2+ was added back 1 h after of by the addition of carbachol. The effect Ca2+ was not mimicked Mn2+, Ba2+, or Sr2+ (Fig. 7C). There was an absolute requirement for receptor stimulation because Ca2+ addition in the absence of Cch did not cause IP3R degradation (Fig. 7C, lane 8) or promote Ca2+ entry (Fig. 7A). The effect of Ca2+ addition was completely blocked by pretreatment with proteasomal inhibitors (Fig. 7D).

The addition of thapsigargin prior to Ca2+ should prevent Ca2+ entry into the ER lumen without preventing the elevation of Ca2+ in the cytosol or the consequent activation of PLC. Thapsigargin addition was found to inhibit the IP3R degradation caused by Ca2+ addition, indicating that Ca2+ entry into the lumen of the ER is necessary for initiating IP3R degradation (Fig. 7E). The importance of luminal ER Ca2+ is further re-enforced by the experiment shown in Fig. 7F where CHO cells were incubated for 30 min with 10 mM Ca2+ to load the intracellular stores before removal of extracellular Ca2+ with EGTA. Under these conditions EGTA treatment failed to block Cch-induced IP3R degradation.

Degradation of Exogenously Transfected IP3R Constructs—The ability of Cch to induce the degradation of epitope-tagged IP3Rs was examined in transiently transfected CHO cells (Fig. 8). Previous studies have shown that stably transfected C-terminally hemagglutinin-tagged type I IP3Rs were degraded more slowly than wild-type IP3Rs in SH-SY5Y cells (19) or were not degraded at all in transiently transfected {alpha}T3-1 cells (18). In the latter study it was shown that the tagged construct was still a substrate for ubiquitination. We confirmed that the C- and N-terminal Myc-tagged constructs were not degraded in transiently transfected CHO cells (Fig. 8A). However, untagged IP3R was also not degraded (Fig. 8B, closed circles). This was observed over a wide range of expression levels as determined by blotting with CT-1 Ab, which would detect both endogenous and overexpressed receptors. The inability to detect even the degradation expected for endogenous receptors may suggest that the untagged receptor exerts a dominant negative effect in these experiments. The type I IP3R contains 3 sites that are alternatively spliced (Fig. 4A). All of the IP3R constructs used so far in these studies are derived from the rat cDNA and correspond to the SI(-),SII(+),SIII(-) splice variant (24). Fig. 8 (A and B) shows that the untagged SI(+),SII(+),SIII(-) variant could be degraded when transiently transfected into CHO cells. To be able to specifically recognize the transfected construct without using epitope tags, we made use of the observation that only neuronal cells contain the SII(+) splice variant, whereas the SII(-) form is present in all peripheral tissues (35). We utilized an Ab to a sequence within the SII(+) splice domain (36) and confirmed that it preferentially recognized the IP3R from cerebellum but not the endogenous receptor in CHO cells (Fig. 8, C and D). The SII Ab was used to detect the transiently transfected SI splice variants in CHO cells and confirmed our observation that only the SI(+) splice variant was degraded (Fig. 8D).


Figure 7
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  		FIGURE 7.
The effect of extracellular Ca2+ addition and luminal Ca2+ on Cch-mediated IP3R degradation. A, cytosolic Ca2+ was measured in Fura-2-loaded CHO cells in response to Cch (1 mM) in the absence of external Ca2+ (medium containing 2.5 mM EGTA). At 30 min the cells were challenged with 4 mM extracellular CaCl2. As a control 4 mM CaCl2 was added back to unstimulated cells incubated in calcium-free medium (dotted line). The traces represent the averages of 90 cells from three different coverslips. B, cells were deprived of extracellular calcium by the addition of 2.5 mM EGTA before treatment with 1 mM carbachol. CaCl2 (4 mM) was added at the indicated time points after carbachol addition. The cells were lysed after 6 h of incubation, and the lysates were probed with CT-1 antibody. C shows the specificity of Ca2+ in causing down-regulation of IP3R. The cells were treated with 2.5 mM EGTA and 1 mM carbachol and subsequently treated with 4 mM Mn2+, Ba2+, Sr2+, or Ca2+ for 6 h. The lysates were immunoblotted with CT-1 antibody. D, Cch and extracellular additions of Ca2+ were carried out as described above. Where indicated, the cells were pretreated for 30 min with a mixture of proteasome inhibitors consisting of 20 µM ALLN, 5 µMlactacystin, and 15 µM ZL3VS. E, cells were treated with EGTA and Cch as above, and 30 min later 4 mM extracellular CaCl2 was added to induce IP3R down-regulation. Where present 2 µM thapsigargin was added 5 min after Cch. The cells were lysed at the end of a 6-h incubation and immunoblotted with CT-1 Ab. F, lanes 1-4, the cells were treated with 1 mM Cch for 6 h in the presence or absence of 2.5 mM EGTA. Lanes 5 and 6, the cells were preloaded with 10 mM Ca2+ for 30 min to overfill intraluminal Ca2+ stores. The cells were washed and then incubated in 2.5 mM EGTA before being challenged with 1 mM Ca2+.

 
An explanation for this preference for the SI(+) form may be that it reflects the endogenously occurring form of the IP3R in CHO cells. To test this we cleaved the IP3R in microsomes from CHO cells with trypsin and compared the size of the N-terminal tryptic fragment with the corresponding fragments from cerebellum microsomes and COS-7 cell microsomes prepared from SI(+) and SI(-) transfected cells (Fig. 8E). Cerebellum microsomes generate a doublet of bands at 38 and 43 kDa corresponding to the presence of both SI(+) and SI(-) forms in this tissue (36). CHO cells contained only a single band corresponding to the SI(+) form.


Figure 8
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  		FIGURE 8.
Degradation of exogenously transfected IP3 receptors. A, CHO-K1 cells were transfected with 10 µg of each of the indicated tagged or untagged constructs. After 48 h the cells were treated with and without Cch (1 mM) for 6 h, and 20 µg of cell lysates were analyzed by SDS-PAGE. The Western blotting Ab is indicated below each blot. B, CHO cells were transfected with either SI(-) or SI(+) splice variants. Immunoblots probed with CT-1 Ab were quantitated by densitometry, and the fold elevation above basal levels obtained by transfection in independent experiments has been plotted against the percentage of down-regulation obtained with Cch. C, 5 µg of cerebellum lysate (CB) and 20 µg of CHO cell lysate was run out on 5% SDS-PAGE and immunoblotted with either Ab specific for the C terminus of the type I IP3R or an Ab raised to a peptide sequence within the SII(+) splice site. D, the degradation of IP3Rs in mock-transfected cells or cells transfected with SI(-) or SI(+) variants was analyzed by immunoblotting with the SII-specific Ab shown in the previous experiment. E, crude microsomal membranes were prepared from cerebellum, CHO cells, or COS-7 cells transfected with the indicated SI splice constructs. The membranes (25 µg) were digested with trypsin at a ratio of protein/trypsin of 10:1 for 5 min. The N-terminal tryptic cleavage fragment was detected on immunoblots with NT-1-specific Ab. The mobility of the two SI splice variants are indicated by arrows. F, Cch-mediated IP3R degradation was evaluated in CHO cells that were mock-transfected or transfected with a D2550A mutant SI(+) IP3R, in which the channel pore is nonfunctional. C-term, C-terminal; N-term, N-terminal.

 
It has been suggested that Ca2+ translocation through a functional IP3R channel and the local accumulation of Ca2+ may play roles in IP3R degradation (5, 18). We have previously shown that the point mutation D2550A in the pore domain of the IP3R is inactive as a Ca2+ channel (26, 27). The data in Fig. 8F show that this mutant construct expressed in the SI(+) background was degraded in response to Cch stimulation. We conclude that there is no requirement for a functional channel in degradation, presumably because the Ca2+ requirement for degradation is supplied by intraluminal Ca2+ and by active endogenous receptors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study we have investigated various aspects of the mechanism of IP3R degradation including sites of receptor ubiquitination, Ca2+ dependence of IP3R degradation, and splice variant specificity of the degradation process. All of these studies were performed using CHO-K1 cells and Cch as an agonist. Previous studies have shown that 1 mM Cch for 18 h can effectively degrade type I IP3Rs in CHO cells stably overexpressing M1 or M3 muscarinic subtypes that are coupled to phospholipase C but not in CHO cells overexpressing M2 muscarinic receptors, which are coupled to adenylate cyclase inhibition (4). In the present study we used the parental (untransfected) CHO-K1 cells, which are normally considered to lack endogenous muscarinic receptors as measured by radioligand binding studies (37). However, in our experiments these cells displayed a robust mobilization of Ca2+ in response to Cch stimulation (Figs. 5 and 7) as well as an IP3R degradation response. Others have reported that Cch addition to parental CHO-K1 results in the generation of nitric oxide, even though muscarinic receptors cannot be detected by radioligand binding assays (37). This suggests that low levels of endogenous muscarinic receptors in the parental CHO-K1 cells are still capable of eliciting functional responses when stimulated by the relatively high concentrations of Cch employed in the present study. Although Cch-mediated Ca2+ signals and IP3R degradation were blocked by atropine, we have not attempted to identify the exact muscarinic subtype(s) involved. CHO-K1 cells stably expressing the M1 receptor subtype also showed Cch-mediated IP3R degradation, but surprisingly the dose response for Cch was not significantly different from the parental CHO-K1 cells (data not shown).

A common feature of agonist-mediated IP3R degradation is that it is accompanied by ubiquitination (8, 16, 18, 19). Recent studies have shown that IP3R ubiquitination involves Ubc7 (38), an ER-associated E2-ligase, that also plays a role in the ER degradation of other substrates (39, 40). In the present study we localized the IP3R domains that become ubiquitinated and found that both monoubiquitin and polyubiquitin become attached at different regions of the receptor. The monoubiquitination site is in the C-terminal region of the protein. This site becomes monoubiquitinated even when ligand-binding defective mutants are transfected in COS cells together with Myc-tagged Ub (data not shown). Because polyubiquitination and agonist-mediated IP3R degradation are prevented in ligand-binding defective mutants (19), this suggests that monoubiquitination is unrelated to agonist-mediated IP3R degradation. However, the attachment of monoubiquitin plays a role in the endocytic targeting/degradation of a number of cell surface receptors (41) and could potentially play a similar role in the basal turnover of IP3Rs (42). Polyubiquitination appeared to be confined to an N-terminal segment of the receptor containing the ligand-binding domain. It is possible that the attachment of long Ub chains at lysines adjacent to the ligand-binding site may disrupt the structure of the site or sterically interfere with access of IP3, thereby inactivating the receptor as a prelude to degradation.

We have investigated the role of Ca2+ in IP3R degradation. Studies with pleckstrin homology domain probes suggest that PLC generation of IP3 is Ca2+-dependent in CHO cells (33). The experimental evidence suggests that sustained elevation of IP3 is a prerequisite for initiating IP3R degradation (19, 20). Thus the absence of agonist-mediated IP3R degradation when Ca2+ is removed may be the consequence of a limited generation of IP3 rather than an effect of Ca2+ on the degradation process. We show in the present study that the readdition of Ca2+ CHO to cells, incubated in a Ca2+-free medium, initiates IP3R degradation provided the cells have been exposed to Cch. Although this Ca2+ readdition is expected to stimulate IP3 generation, this cannot be the sole factor responsible for IP3R degradation because the effect of Ca2+ addition was blocked by thapsigargin. Under these conditions thapsigargin does not interfere with the activation of PLC (4, 33) or the elevation of cytosolic Ca2+ but would prevent accumulation of Ca2+ into the lumen of the ER. The inhibition of IP3R degradation seen when Ca2+ is removed can be prevented by first overloading the intracellular stores with Ca2+ (Fig. 7F). This suggests that there may be a minimal level of ER intraluminal Ca2+ that is necessary to facilitate degradation. From our data we conclude that both a sustained IP3 elevation and intraluminal Ca2+ are necessary for agonist-mediated IP3R degradation. Pretreatment with thapsigargin has been shown to block agonist-mediated IP3R ubiquitination (18). This supports the idea that Ca2+ is required for initiating the early steps of IP3R degradation, although this observation does not distinguish between a requirement for cytosolic or luminal Ca2+. Intraluminal Ca2+ may promote interactions of IP3Rs with ER-resident chaperones or act as a permissive factor allowing IP3Rs to adopt a degradation-sensitive conformational state upon prolonged IP3 binding. Elevated cytosolic Ca2+ promotes large conformational changes in the receptor (43), which may expose ubiquitination sites. Both cytosolic and luminal Ca2+ could therefore play regulatory roles at different stages of IP3R degradation. A recently published study has examined the role of Ca2+ in the ubiquitination and degradation of IP3Rs in gonadotrophin (GnRH)-responsive {alpha}T3-1 cells (44). The authors found that the combination of 25 µM BAPTA-AM and 1 µM nifedipine did not prevent ubiquitination of wild-type IP3Rs. These conditions did not entirely eliminate the Ca2+ signal mediated by GnRH, and it was suggested that local increases in cytosolic Ca2+ are sufficiently large, even with BAPTA-AM and nifedipine, to facilitate ubiquitination. In our hands a 30-min preincubation of CHO cells with 100 µM BAPTA-AM was sufficient to entirely eliminate the carbachol-mediated Ca2+ signal and the accompanying IP3R degradation (data not shown). The D2550A pore-inactive mutant stably expressed in {alpha}T3-1 cells was ubiquitinated and down-regulated in response to GnRH, albeit with a slightly reduced efficiency. Overall, the study in Ref. 44 and the present report are in general agreement that both Ca2+ and IP3 binding to the IP3R are required for IP3R degradation.

Transient transfection experiments in CHO cells with exogenous IP3R constructs have provided insights into the specificity of the degradation process. We noted that only the SI(+) splice variant of the type I IP3R was degraded and that this was also the only form detectable endogenously in CHO cells. The 15 amino acids of the SI splice site are located in the ligand-binding domain of the receptor. Functional studies comparing the SI(-) and SI(+) forms of the type IIP3R have not revealed any marked differences in ligand binding or channel function (25, 45), although a recent study noted differences in the Ca2+ sensitivity of the two variants (29). We have also observed that the exposure of highly reactive surface thiol groups in the ligand-binding domain are different for the SI(+) and SI(-) variants (34). By contrast the SII splice site does not appear to be critical because both exogenous SI(+)/SII(+) and endogenous SI(+)/SII(-) forms were degraded. Interestingly, the only previous studies demonstrating the degradation of exogenous receptors used the mouse SI(+)/SII(+) clone transfected into SH-SY5Y neuroblastoma cells (19) or {alpha}T3-1 anterior pituitary cells (18). However, the SI splice status of the endogenous receptor in these cells has not been determined. The preference for the SI(+) splice variant is unlikely to be related to ubiquitination because the SI(-) variant was readily ubiquitinated (Fig. 4). Nevertheless, only a crude analysis of IP3R ubiquitination domains was carried out in the present study, and detailed differences in ubiquitination sites between splice constructs cannot be excluded. Previous studies by others have noted that insertion of a C-terminal tag has a strong inhibitory effect on IP3R degradation, although ubiquitination of the tagged receptor was not noticeably affected (18, 19). After ubiquitination, the large tetrameric IP3R complex in the ER membrane is presumably subject to complex unfolding and disassembly steps. These processes may also have strict structural requirements that are disrupted by the insertion of epitope tags or inappropriate SI splice variants. The presence of a Cch-sensitive IP3R degradation system in CHO cells and the ability to transfect these cells with mutant IP3Rs should allow us to further probe the structural specificity of the IP3R proteasomal degradation pathway.


   FOOTNOTES
 
* This study was supported by National Institutes of Health Grant DK34804. 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

1 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: suresh.joseph{at}mail.tju.edu.

2 The abbreviations used are: IP3, myo-inositol 1,4,5-trisphosphate; IP3R, IP3 receptor; Cch, carbachol; Ab, antibody; NEM, N-ethylmaleimide; ER, endoplasmic reticulum; GnRH, gonadotrophic hormone; ALLM, N-acetyl-L-leucinyl-L-leucinyl-L-methioninal; ALLN, N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal; ZL3VS, peptide vinyl sulfone carboxybenzyl-leucyl-leucylleucine vinyl sulfone; PLC, phospholipase C; CHO, Chinese hamster ovary; Ub, ubiquitin; BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester. Back


   ACKNOWLEDGMENTS
 
We thank Professor Mikoshiba for the initial gift of SII(+) Ab and Dr. Madesh Muniswamy for assistance with Ca2+ imaging.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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