(Received for publication, November 20, 1996, and in revised form, January 22, 1997)
From the Department of Pathology, Thomas Jefferson University School of Medicine, Philadelphia, Pennsylvania 19107
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.
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 Gq (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.
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).
AntibodiesThe 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). IB
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).
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 -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
-mercaptoethanol.
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 IP3WB 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 ExtractsAfter 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).
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.
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.
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).
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.
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).
|
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.
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.
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.
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.
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). IB
is an inhibitor of the transcription factor NF
B.
Regulated degradation of I
B
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 I
B
(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.
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.
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.
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.