Mechanism of Proteasomal Degradation of Inositol Trisphosphate Receptors in CHO-K1 Cells*

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.

The activation of inositol 1,4,5-trisphosphate receptors (IP 3 Rs) 2 by IP 3 initiates Ca 2ϩ mobilization from the ER and triggers the Ca 2ϩ 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 IP 3 R protein by a mechanism involving the marked acceleration of IP 3 R degradation. Subsequently, similar effects on IP 3 R degradation have been described in many different experimental systems with many different agonists (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). Downregulation of IP 3 R protein is associated with a decrease in the ability of IP 3 to mobilize Ca 2ϩ (4,8). More recently it has been shown that the frequency of elementary Ca 2ϩ puffs that trigger propagating Ca 2ϩ waves in cells is decreased by chronic agonist stimulation (14). The physiological significance of IP 3 R down-regulation is unknown. However, down-regulation of receptors would be expected to inhibit the global Ca 2ϩ signal elicited by all Ca 2ϩ 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 IP 3 Rs, and this is associated with a marked reduction in the IP 3mediated Ca 2ϩ signal (15,18).
The mechanism of IP 3 R degradation has not been established. Previous studies have shown that chronic elevation of IP 3 and IP 3 binding to the receptor are required to facilitate IP 3 R degradation (19,20). It has been proposed that the sustained elevation of IP 3 causes the IP 3 R to adopt a conformation that exposes sites that become ubiquitinated. The proteasome pathway then degrades the ubiquitinated IP 3 receptor. The regions of the IP 3 R involved in ubiquitination have not been determined. Pretreatment of cells with the SERCA pump inhibitor thapsigargin has been found to inhibit agonist-mediated IP 3 R degradation, suggesting that Ca 2ϩ also plays a role in IP 3 R degradation (4). In the present study we have examined IP 3 R degradation in CHO-K1 cells stimulated with carbachol. This system has been used to examine the domains of IP 3 Rs that are ubiquitinated and to further explore the role of Ca 2ϩ in IP 3 R degradation. The ability to readily transfect CHO-K1 cells with various IP 3 R constructs has allowed an initial characterization of the substrate recognition properties of the IP 3 R degradation system.

EXPERIMENTAL PROCEDURES
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 His 6 -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 IP 3 R (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 IP 3 R (NT-1). This Ab was used after affinity purification using the immobilized antigenic peptide as a column matrix. The type III IP 3 R 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 IP 3 R SI(Ϫ)/ SII(ϩ)/SIII(ϩ) 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 IP 3 R SI(ϩ) variant was the kind gift of Dr. Gregory Mignery (Loyola University Chicago) (25). The D2550A poredefective mutant in SI(Ϫ) (26) was transferred to the SI(ϩ) IP 3 R 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 ϫ g. 20 g 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 ␤-mercaptoethanol.
Measurement of Ca 2ϩ Mobilization-Changes in cytosolic [Ca 2ϩ ] 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 M Fura-2AM in 0.03% pluronic F-127 in the presence of 100 M 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 KH 2 PO 4 , 1.2 mM MgSO 4 , 5 mM NaHCO 3 , 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 Ca 2ϩ 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. [Ca 2ϩ ] 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 IP 3 R 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 phosphatebuffered 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 IP 3 R 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 IP 3 R into 4 ϫ 150-mm plates (20 g 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 M lactacystin, 50 M ZL3VS, and 50 M ALLN. The plates were then treated with 1 mM carbachol, lysed as described above in NEMcontaining 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 l 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 g 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 IP 3 R Abs to locate the ubiquitinated fragments.

Basic Characterization of IP 3 R Down-regulation in CHO-K1 Cells-
Our previous studies on IP 3 R 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 IP 3 R constructs, and stable WB cell lines lose their IP 3 R degradation response to angiotensin II (data not shown). We therefore examined other cells types that were more amenable to transfection for an agonist-mediated IP 3 R degradation response. Fig. 1A shows that carbachol (Cch) addition to CHO-K1 cells stimulated IP 3 R down-regulation. Similar Cch effects were seen when the blots were reprobed for the type III IP 3 R isoform (data not shown). The effect was specific to Cch because ATP, which also elevates Ca 2ϩ in these cells (cf. Fig. 5), did not induce significant IP 3 R 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 IP 3 R 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 IP 3 R down-regulation in other systems (8). Fig. 1C shows the dose response for IP 3 R 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 ϳ20 and 80 -100 M. Methacholine was somewhat more potent than carbachol (IC 50 ϭ ϳ10 M). Acetylcholine was the least potent agonist with half-maximal effects at 50 M 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 IP 3 R down-regulation is mediated by endogenous muscarinic receptors in CHO-K1 cells.
The role of different proteolytic pathways in IP 3 R degradation was investigated using various protease inhibitors (Fig. 2). The most pronounced inhibition of Cch-mediated IP 3 R degradation was observed with the proteasomal inhibitor lactacystin. Rather surprisingly, 50 M of the peptide aldehyde inhibitor ALLN, which is effective at preventing proteasomal degradation of IP 3 Rs in WB cells (8), exerted only a small inhibitory effect on Cch-mediated IP 3 R degradation. A small inhibitory effect of 100 M chloroquine was also observed. This is probably an indirect effect and unlikely to reflect the involvement of the lysosomal pathway because 5 mM NH 4 Cl 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 IP 3 R degradation is the proteasomal pathway.
Ubiquitination Domains in IP 3 R-We first carried out experiments to confirm that the proteasomal degradation of IP 3 Rs was accompanied by ubiquitination. CHO cell lysates were treated with Cch in the presence or absence of lactacystin, and the immunoprecipitated IP 3 R was probed for ubiquitin with anti-Ub Ab (Fig. 3A). The data show that ubiquitinated IP 3 R 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 IP 3 R that could potentially serve as sites for ubiquitination. Our limited objective in the present study was to identify domains in the IP 3 R that may serve as targets of ubiquitination. The strategy we chose to employ was to digest the ubiquitinated IP 3 R with a protease that would cleave the IP 3 R but not ubiquitin. A protease that satisfies this criteria is caspase-3, which is known to cleave IP 3 Rs (31,32). Initial experiments to detect endogenous Ub attached to caspase-3-cleaved IP 3 R 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 IP 3 Rs 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 IP 3 R, 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 ␣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 IP 3 R cDNA. Under these conditions a significant increase in Myc ubiquitinated IP 3 R was observed when IP 3 R 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 IP 3 R 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. IP 3 R immunoprecipitates were prepared from CHO cells cotransfected with Myc-Ub and type I IP 3 R plasmids that were then treated with Cch in the presence of proteasomal inhibitors. IP 3 R 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 IP 3 Rs 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 IP 3 R. When caspase-3-cleaved IP 3 R was examined with N-terminal Ab, a fragment of ϳ95 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.
The Role of Ca 2ϩ in Carbachol-mediated IP 3 R Degradation- Fig. 5A shows the Ca 2ϩ transients recorded in Fura-2-loaded CHO-K1 cells in response to a maximal dose of Cch that triggers IP 3 R degradation. Cch addition elicited a Ca 2ϩ signal that remained elevated for a prolonged period (Ͼ30 min). By contrast a Ca 2ϩ -mobilizing stimulus such as ATP, which did not cause IP 3 R degradation, produced a smaller and more transient Ca 2ϩ signal. The ATP responses observed in cells that were pretreated with Cch for 6 h was substantially blunted as would be anticipated if IP 3 Rs were degraded by Cch pretreatment (Fig. 5B). To further examine the role of Ca 2ϩ , we pretreated the cells for 30 min with thapsigargin (to empty intracellular stores) or EGTA (to remove extracellu-   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). lar Ca 2ϩ ). 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 Ca 2ϩ transient by loading the cell permeant BAPTA-AM chelator is shown in Fig. 6B. BAPTA-AM completely blocked Cch-mediated IP 3 R 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 IP 3 R observed at 6 h. This indicates that a continuously elevated cytosolic Ca 2ϩ is not required for IP 3 R degradation and that the signals that irreversibly commit the IP 3 R 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.
The interpretation of the finding that thapsigargin, EGTA, and BAPTA-AM inhibit IP 3 R degradation is complicated by the known Ca 2ϩ sensitivity of phospholipase C (PLC) in CHO cells (33). Because it has been established that chronic elevation of IP 3 and IP 3 binding to the IP 3 R are prerequisite for obtaining IP 3 R degradation (19,20), the perturbing effect of Ca 2ϩ chelating and mobilizing agents could reflect an inadequate PLC-dependent generation of IP 3 . To distinguish between Ca 2ϩ -dependent and IP 3 -dependent effects, we carried out the experiments shown in Fig. 7. Extracellular Ca 2ϩ was chelated with EGTA, and the cells were stimulated with Cch. This resulted in a Ca 2ϩ elevation that was transient and returned to base line within 5 min (Fig. 7A). This transient elevation of Ca 2ϩ is clearly insufficient to sustain IP 3 R degradation (Fig. 6A). Depletion of intracellular stores is known to activate Ca 2ϩ entry channels in the plasma membrane. This can be experimentally monitored as a marked elevation of cytosolic Ca 2ϩ when extracellular Ca 2ϩ is added back to store-depleted cells (Fig. 7A). Using this protocol we noted that the addition of Ca 2ϩ also caused marked IP 3 R degradation (Fig. 7B) and occurred even when Ca 2ϩ was added back 1 h after carbachol. The effect of Ca 2ϩ was not mimicked by the addition of Mn 2ϩ , Ba 2ϩ , or Sr 2ϩ (Fig. 7C). There was an absolute requirement for receptor stimulation because Ca 2ϩ addition in the absence of Cch did not cause IP 3 R degradation (Fig. 7C, lane 8) or promote Ca 2ϩ entry (Fig.  7A). The effect of Ca 2ϩ addition was completely blocked by pretreatment with proteasomal inhibitors (Fig. 7D).
The addition of thapsigargin prior to Ca 2ϩ should prevent Ca 2ϩ entry into the ER lumen without preventing the elevation of Ca 2ϩ in the cytosol or the consequent activation of PLC. Thapsigargin addition was found to inhibit the IP 3 R degradation caused by Ca 2ϩ addition, indicating that Ca 2ϩ entry into the lumen of the ER is necessary for initiating IP 3 R degradation (Fig. 7E). The importance of luminal ER Ca 2ϩ is further re-enforced by the experiment shown in Fig. 7F where CHO cells were incubated for 30 min with 10 mM Ca 2ϩ to load the intracellular stores before removal of extracellular Ca 2ϩ with EGTA. Under these conditions EGTA treatment failed to block Cch-induced IP 3 R degradation.
Degradation of Exogenously Transfected IP 3 R Constructs-The ability of Cch to induce the degradation of epitope-tagged IP 3 Rs was examined in transiently transfected CHO cells (Fig. 8). Previous studies have shown that stably transfected C-terminally hemagglutinin-tagged type I  In all cases the cells were lysed after 6 h of treatment with agonist and analyzed for type I IP 3 R by immunoblotting. The Cch-mediated Ca 2ϩ 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 IP 3 R were compared with cells continuously exposed to Cch for 4 h. IP 3 Rs were degraded more slowly than wild-type IP 3 Rs in SH-SY5Y cells (19) or were not degraded at all in transiently transfected ␣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 Myctagged constructs were not degraded in transiently transfected CHO cells (Fig. 8A). However, untagged IP 3 R 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 IP 3 R contains 3 sites that are alternatively spliced (Fig. 4A). All of the IP 3 R constructs used so far in these studies are derived from the rat cDNA and correspond to the SI(Ϫ),SII(ϩ),SIII(Ϫ) splice variant (24). Fig. 8 (A and B) shows that the untagged SI(ϩ),SII(ϩ),SIII(Ϫ) 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(ϩ) splice variant, whereas the SII(Ϫ) form is present in all peripheral tissues (35). We utilized an Ab to a sequence within the SII(ϩ) splice domain (36) and confirmed that it preferentially recognized the IP 3 R 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(ϩ) splice variant was degraded (Fig. 8D).
An explanation for this preference for the SI(ϩ) form may be that it reflects the endogenously occurring form of the IP 3 R in CHO cells. To test this we cleaved the IP 3 R in microsomes from CHO cells with trypsin FIGURE 7. The effect of extracellular Ca 2؉ addition and luminal Ca 2؉ on Cch-mediated IP 3 R degradation. A, cytosolic Ca 2ϩ was measured in Fura-2-loaded CHO cells in response to Cch (1 mM) in the absence of external Ca 2ϩ (medium containing 2.5 mM EGTA). At 30 min the cells were challenged with 4 mM extracellular CaCl 2 . As a control 4 mM CaCl 2 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. CaCl 2 (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 Ca 2ϩ in causing down-regulation of IP 3 R. The cells were treated with 2.5 mM EGTA and 1 mM carbachol and subsequently treated with 4 mM Mn 2ϩ , Ba 2ϩ , Sr 2ϩ , or Ca 2ϩ for 6 h. The lysates were immunoblotted with CT-1 antibody. D, Cch and extracellular additions of Ca 2ϩ were carried out as described above. Where indicated, the cells were pretreated for 30 min with a mixture of proteasome inhibitors consisting of 20 M ALLN, 5 M lactacystin, and 15 M ZL3VS. E, cells were treated with EGTA and Cch as above, and 30 min later 4 mM extracellular CaCl 2 was added to induce IP 3 R down-regulation. Where present 2 M 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 Ca 2ϩ for 30 min to overfill intraluminal Ca 2ϩ stores. The cells were washed and then incubated in 2.5 mM EGTA before being challenged with 1 mM Ca 2ϩ . 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(ϩ) and SI(Ϫ) transfected cells (Fig. 8E). Cerebellum microsomes generate a doublet of bands at 38 and 43 kDa corresponding to the presence of both SI(ϩ) and SI(Ϫ) forms in this tissue (36). CHO cells contained only a single band corresponding to the SI(ϩ) form.
It has been suggested that Ca 2ϩ translocation through a functional IP 3 R channel and the local accumulation of Ca 2ϩ may play roles in IP 3 R degradation (5,18). We have previously shown that the point mutation D2550A in the pore domain of the IP 3 R is inactive as a Ca 2ϩ channel (26,27). The data in Fig. 8F show that this mutant construct expressed in the SI(ϩ) background was degraded in response to Cch stimulation. We conclude that there is no requirement for a functional channel in deg-radation, presumably because the Ca 2ϩ requirement for degradation is supplied by intraluminal Ca 2ϩ and by active endogenous receptors.

DISCUSSION
In the present study we have investigated various aspects of the mechanism of IP 3 R degradation including sites of receptor ubiquitination, Ca 2ϩ dependence of IP 3 R 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 IP 3 Rs 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, FIGURE 8. Degradation of exogenously transfected IP 3 receptors. A, CHO-K1 cells were transfected with 10 g 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 g 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(Ϫ) or SI(ϩ) 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 g of cerebellum lysate (CB) and 20 g 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 IP 3 R or an Ab raised to a peptide sequence within the SII(ϩ) splice site. D, the degradation of IP 3 Rs in mock-transfected cells or cells transfected with SI(Ϫ) or SI(ϩ) 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 g) 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 IP 3 R degradation was evaluated in CHO cells that were mock-transfected or transfected with a D2550A mutant SI(ϩ) IP 3 R, in which the channel pore is nonfunctional. C-term, C-terminal; N-term, N-terminal. 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 Ca 2ϩ in response to Cch stimulation (Figs. 5 and 7) as well as an IP 3 R 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 Ca 2ϩ signals and IP 3 R 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 IP 3 R 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 IP 3 R degradation is that it is accompanied by ubiquitination (8,16,18,19). Recent studies have shown that IP 3 R 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 IP 3 R 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 IP 3 R degradation are prevented in ligand-binding defective mutants (19), this suggests that monoubiquitination is unrelated to agonist-mediated IP 3 R 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 IP 3 Rs (42). Polyubiquitination appeared to be confined to an N-terminal segment of the receptor containing the ligandbinding 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 IP 3 , thereby inactivating the receptor as a prelude to degradation.
We have investigated the role of Ca 2ϩ in IP 3 R degradation. Studies with pleckstrin homology domain probes suggest that PLC generation of IP 3 is Ca 2ϩ -dependent in CHO cells (33). The experimental evidence suggests that sustained elevation of IP 3 is a prerequisite for initiating IP 3 R degradation (19,20). Thus the absence of agonist-mediated IP 3 R degradation when Ca 2ϩ is removed may be the consequence of a limited generation of IP 3 rather than an effect of Ca 2ϩ on the degradation process. We show in the present study that the readdition of Ca 2ϩ to CHO cells, incubated in a Ca 2ϩ -free medium, initiates IP 3 R degradation provided the cells have been exposed to Cch. Although this Ca 2ϩ readdition is expected to stimulate IP 3 generation, this cannot be the sole factor responsible for IP 3 R degradation because the effect of Ca 2ϩ addition was blocked by thapsigargin. Under these conditions thapsigargin does not interfere with the activation of PLC (4,33) or the elevation of cytosolic Ca 2ϩ but would prevent accumulation of Ca 2ϩ into the lumen of the ER. The inhibition of IP 3 R degradation seen when Ca 2ϩ is removed can be prevented by first overloading the intracellular stores with Ca 2ϩ (Fig.  7F). This suggests that there may be a minimal level of ER intraluminal Ca 2ϩ that is necessary to facilitate degradation. From our data we conclude that both a sustained IP 3 elevation and intraluminal Ca 2ϩ are necessary for agonist-mediated IP 3 R degradation. Pretreatment with thapsigargin has been shown to block agonist-mediated IP 3 R ubiquitination (18). This supports the idea that Ca 2ϩ is required for initiating the early steps of IP 3 R degradation, although this observation does not distinguish between a requirement for cytosolic or luminal Ca 2ϩ . Intraluminal Ca 2ϩ may promote interactions of IP 3 Rs with ER-resident chaperones or act as a permissive factor allowing IP 3 Rs to adopt a degradation-sensitive conformational state upon prolonged IP 3 binding. Elevated cytosolic Ca 2ϩ promotes large conformational changes in the receptor (43), which may expose ubiquitination sites. Both cytosolic and luminal Ca 2ϩ could therefore play regulatory roles at different stages of IP 3 R degradation. A recently published study has examined the role of Ca 2ϩ in the ubiquitination and degradation of IP 3 Rs in gonadotrophin (GnRH)-responsive ␣T3-1 cells (44). The authors found that the combination of 25 M BAPTA-AM and 1 M nifedipine did not prevent ubiquitination of wild-type IP 3 Rs. These conditions did not entirely eliminate the Ca 2ϩ signal mediated by GnRH, and it was suggested that local increases in cytosolic Ca 2ϩ are sufficiently large, even with BAPTA-AM and nifedipine, to facilitate ubiquitination. In our hands a 30-min preincubation of CHO cells with 100 M BAPTA-AM was sufficient to entirely eliminate the carbachol-mediated Ca 2ϩ signal and the accompanying IP 3 R degradation (data not shown). The D2550A poreinactive mutant stably expressed in ␣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 Ca 2ϩ and IP 3 binding to the IP 3 R are required for IP 3 R degradation.
Transient transfection experiments in CHO cells with exogenous IP 3 R constructs have provided insights into the specificity of the degradation process. We noted that only the SI(ϩ) splice variant of the type I IP 3 R 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(Ϫ) and SI(ϩ) forms of the type I IP 3 R have not revealed any marked differences in ligand binding or channel function (25,45), although a recent study noted differences in the Ca 2ϩ 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(ϩ) and SI(Ϫ) variants (34). By contrast the SII splice site does not appear to be critical because both exogenous SI(ϩ)/SII(ϩ) and endogenous SI(ϩ)/SII(Ϫ) forms were degraded. Interestingly, the only previous studies demonstrating the degradation of exogenous receptors used the mouse SI(ϩ)/SII(ϩ) clone transfected into SH-SY5Y neuroblastoma cells (19) or ␣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(ϩ) splice variant is unlikely to be related to ubiquitination because the SI(Ϫ) variant was readily ubiquitinated (Fig. 4). Nevertheless, only a crude analysis of IP 3 R 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 IP 3 R degradation, although ubiquitination of the tagged receptor was not noticeably affected (18,19). After ubiquitination, the large tetrameric IP 3 R 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 IP 3 R degradation system in CHO cells and the ability to transfect these cells with mutant IP 3 Rs should allow us to further probe the structural specificity of the IP 3 R proteasomal degradation pathway.