From the Department of Pharmacology, State University of New York Upstate Medical University, Syracuse, New York 13210-2339
Received for publication, July 3, 2002, and in revised form, October 18, 2002
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ABSTRACT |
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In Hormone-induced secretion from anterior pituitary cells is
modulated at many different levels, and among these is regulation of
the activity and abundance of receptors involved in signal transduction
(1, 2). Indeed, recent studies on the InsP3 receptor down-regulation in response to activation of
certain GPCRs has also been seen in other cell types (11-15). This adaptive response is mediated by an increase in the rate of receptor degradation (11, 13); is specific, since other ER and signaling proteins are not simultaneously affected (11, 14); and appears to exist
to allow chronically stimulated cells to reduce the sensitivity of
their Ca2+ stores to InsP3 (4, 5, 10, 14-16).
The event that initiates receptor proteolysis appears to be
InsP3 binding, since only those GPCRs (e.g.
GnRH, cholecystokinin, and muscarinic receptors) that persistently
elevate InsP3 levels cause InsP3 receptor
down-regulation (5, 12, 13), a binding-deficient mutant
InsP3 receptor is resistant to down-regulation (17), and
down-regulation in oocytes is elicited by microinjection of an
InsP3 analogue (18). Whether additional events
(e.g. receptor phosphorylation) are required to trigger
down-regulation remains to be resolved. Furthermore, it is not yet
clear how InsP3 receptors are degraded, and indeed, receptor proteolysis by calpain (12), caspase (19), and the ubiquitin/proteasome pathway (14, 15, 20, 21) have all been described.
Thus, we examined the mechanism of InsP3 receptor
down-regulation in GnRH-stimulated Materials--
Electrophoresis and Immunoblotting--
Samples were subjected
to 5% PAGE and were immunoblotted as described (20). Immunoreactivity
was detected with chemiluminescence using reagents from Pierce and was
digitally imaged and quantitated with a Genegnome (Syngene), working
within the nonsaturating range.
Measurement of InsP3 Receptor Down-regulation in
Measurement of InsP3 Receptor Ubiquitination by
Immunoblotting--
Control or stimulated cells in suspension were
collected by centrifugation (750 × g for 3 min at
4 °C) and were solubilized by incubation for 30 min at 4 °C with
1 ml of lysis buffer. Lysates were then centrifuged (16,000 × g for 10 min at 4 °C), supernatants were collected, and
InsP3 receptors were immunoprecipitated by incubation at
4 °C with CT1h for 1h and then for a further 12-24 h with Protein
A. Immune complexes were then isolated by centrifugation (500 × g for 2 min), were washed three times with ice-cold lysis buffer, and in most experiments were resuspended in 2× gel loading buffer and then immunoblotted with either CT1h or FK2. In additional experiments aimed at further characterizing the ubiquitinated species,
washed immune complexes were released from Protein A and denatured by
incubation at 100 °C for 5 min in 100 µl of 50 mM
Tris, 2% SDS, 2 mM dithiothreitol, pH 7.4, were
centrifuged (16,000 × g for 1 min at 25 °C), were
diluted to 4 ml with lysis buffer, were pretreated with Protein A to
remove residual CT1h, and finally were reimmunoprecipitated with FK2
and Protein A for 12-24 h and resuspended in 2× gel loading buffer.
Measurement of InsP3 Receptor Ubiquitination by
Radiolabeling--
Cell monolayers in 75-cm2 Falcon flasks
were incubated for 48 h with 100 µCi of
[35S]cysteine (NEG022T; PerkinElmer Life Sciences) in
Transfection of Measurement of InsP3
Concentration--
InsP3 concentration in suspensions of
Miscellaneous--
Data shown are representative of at least two
independent experiments. Combined data are mean ± S.E.
(n InsP3 Receptor Down-regulation in
To confirm that persistent GnRH receptor activation and
InsP3 formation were needed for type I InsP3
receptor down-regulation, we utilized the GnRH receptor antagonist
antide, which blocks GnRH-induced InsP3 formation when
added simultaneously with or after GnRH (Fig. 3B). As
expected, antide blocked down-regulation when added simultaneously with
GnRH (Fig. 1B, lane 3). However, we
also observed that the down-regulation seen after a 60-min exposure to
GnRH (lane 2) was not mimicked by exposure to
GnRH alone for 5 min, followed by a further 55-min incubation in
antide-supplemented medium (lane 5). This shows
that acute GnRH receptor activation is not sufficient to program the
cells to subsequently down-regulate InsP3 receptors and is
consistent with the view (13, 20) that persistent elevation of
InsP3 concentration is a prerequisite for
down-regulation.
Proteasome Inhibitors Block Down-regulation and Cause the
Accumulation of Ubiquitinated Receptors--
In order to determine
whether or not GnRH-induced InsP3 receptor down-regulation
is via the ubiquitin/proteasome pathway, we exposed
Consistent with this conclusion and the rapidity of down-regulation
(Fig. 1A), analysis of the time dependence of
polyubiquitination (Fig. 2C) revealed that in the absence of
ALLN, polyubiquitinated receptors accumulated very rapidly (peaking at
5 min) and were detectable only transiently, presumably because they
are degraded rapidly by the proteasome; this also explains why
polyubiquitinated receptors were not detected after incubation with
GnRH alone for 1 h (Fig. 2A, lane
2). In contrast, when ALLN was present, polyubiquitinated receptor accumulation peaked at ~20 min and thereafter did not decline (Fig. 2C). Surprisingly, ALLN also suppressed the
initial rate of receptor polyubiquitination (Fig. 2C). This
was not due to a reduction in the potency of GnRH, which was
half-maximally effective at ~5 nM in the absence or
presence of ALLN,2 and indicates that as well as inhibiting
the degradation of polyubiquitinated species, proteasome inhibitors may
also reduce the rate of polyubiquitination.
Given the mechanistic differences between the proteasome inhibitors, we
also analyzed their kinetics. Fig. 2D shows that the effects
of ALLN are very rapid in onset; ALLN was maximally effective with a
preincubation time of 1 h or more (lanes
5-8) and was close to being maximally effective when added
simultaneously with GnRH (lane 4). Fig.
2E shows that when used at maximally effective concentrations (5-10 times higher than half-maximal values defined in
the legend to Fig. 2A), MG-132 (lanes
4 and 5), like ALLN (lanes 2 and 3), acted rapidly, being similarly
effective with 0- or 2-h preincubation. In contrast, epoxomicin
(lanes 8 and 9) and particularly
lactacystin (lanes 6 and 7) were
slower acting, being much less effective when added simultaneously with
GnRH as compared with 2-h preincubation.
InsP3 Receptor Deubiquitination--
Since
proteasome inhibitors completely block GnRH-induced InsP3
receptor down-regulation, it would be expected that a large proportion
of cellular InsP3 receptors would accumulate as
polyubiquitinated species when the proteasome was inhibited. However,
this was not the case, since only 9 ± 1% of receptors were
polyubiquitinated in the presence of ALLN plus GnRH, and maximal
accumulation of polyubiquitinated receptors in the presence of ALLN was
only approximately twice that seen in its absence (Fig. 2C).
Thus, we examined whether deubiquitination might be counteracting the
accumulation of polyubiquitinated receptors. Antide was used for these
studies, since it blocks InsP3 formation and
InsP3 receptor polyubiquitination when added simultaneously
with GnRH (Fig. 3B,
left panel, and Fig. 3A,
lane 6) and rapidly (within 10 min) returns
InsP3 concentration to basal levels when added to
GnRH-stimulated cells (Fig. 3B, right). Fig.
3A (lanes 1-5) shows that the
addition of antide to ALLN-preincubated, GnRH-stimulated cells results
in a rapid decline in the level of polyubiquitinated InsP3
receptors, indicating that they are being deubiquitinated. Thus,
deubiquitinating enzymes (27, 28) are active in Thapsigargin, TPEN, and Glycerol Inhibit Polyubiquitination and
Down-regulation--
The effects of potential inhibitors that might
provide insight into the mechanism of polyubiquitination and
down-regulation were also tested (Fig.
4). Thapsigargin inhibits
Ca2+-ATPases that pump Ca2+ into the ER,
reduces intraluminal Ca2+ concentration, and disrupts ER
function (29). Fig. 4 (A, lane 4, and
B, lane 2) shows that thapsigargin
inhibits GnRH-induced InsP3 receptor down-regulation and
polyubiquitination without affecting InsP3 formation (Fig.
4C), suggesting that Ca2+ binding to
intraluminal regions of the type I InsP3 receptor (6-8) or
to other ER proteins that interact with the type I InsP3 receptor (29-31) is required for this process. TPEN chelates
Zn2+ with high affinity and has been shown to inhibit the
activity of purified RING domain-containing E3 ubiquitin-protein
ligases, presumably by removing the Zn2+ that is normally
complexed with the RING domain (28, 32-34). Fig. 4 (A,
lane 6, and B, lane
3) shows that TPEN inhibits InsP3 receptor
down-regulation and polyubiquitination, and Fig. 4C shows that this is not due to inhibition of InsP3
formation.3 These data
suggest that a RING domain-containing E3 mediates InsP3
receptor ubiquitination. Finally, glycerol has been proposed to act as
a "chemical chaperone," acting to enhance the proper folding and
suppress the degradation of either misfolded ER-associated proteins or
proteins destined for ER-associated degradation (35, 36). Glycerol did
inhibit down-regulation and polyubiquitination of InsP3
receptors (Fig. 4, A, lane 8, and
B, lane 4). However, it also
completely inhibited InsP3 formation (Fig. 4C),
making it impossible to draw conclusions related to its action as a
chaperone.
GnRH-induced Ubiquitination of Exogenous
InsP3 Receptors in Transfected Cells--
Having
characterized the polyubiquitination of endogenous receptors (Figs.
1-4), we next examined whether exogenous receptors, introduced by
transient transfection, could be polyubiquitinated in a
GnRH-dependent manner, since this would provide a system for the analysis of mutant receptors. Pilot studies utilizing cDNA
encoding green fluorescent protein and a variety of transfection reagents revealed that 5-10% of
In order to assess the ubiquitination of just the exogenous receptors,
we immunoprecipitated with HA11 (Fig. 5B), which purifies only HA-tagged receptors (see Fig. 5C). Initial analysis of
InsP3RHAwt revealed, somewhat surprisingly, the
existence of a Myc-immunoreactive band very similar in size to
unmodified InsP3RHAwt (Fig. 5B,
upper panel, lane 4), even
in the absence of GnRH. This is likely to be
InsP3RHAwt modified by one or a very small
number of Myc-ubiquitin residues, which hereafter is referred to as
"monoubiquitinated" receptor. Importantly, however, exposure of
these cells to GnRH led to a large increase in Myc-polyubiquitination
(upper panel, lanes 5 and
6), confirming that exogenous
InsP3RHAwt is polyubiquitinated. In contrast,
in cells expressing InsP3RHA
However, it must be noted that the processing of exogenous and
endogenous receptors was not identical, since exogenous receptors were
"monoubiquitinated." This was not dependent on GnRH stimulation or
InsP3 binding, since both
InsP3RHAwt and
InsP3RHA The Role of Phosphorylation in Ubiquitination--
Phosphorylation
has been shown to trigger the polyubiquitination of many proteins (27,
28) and could contribute to triggering InsP3 receptor
polyubiquitination, since the type I InsP3 receptor is
phosphorylated by protein kinase A (PKA) (6-8) under conditions that
lead to down-regulation (38). PKA-mediated phosphorylation of the mouse
type I receptor occurs at serine residues 1588 and 1755 (39); thus, we
created and analyzed a phosphorylation-resistant mutant receptor
(InsP3RHAA/A) in which both sites are converted
to alanine. Fig. 5B (lanes 10-12)
shows that exogenous InsP3RHAA/A was
Myc-polyubiquitinated in an identical manner to
InsP3RHAwt (lanes 4-6),
indicating that PKA-dependent phosphorylation does not
contribute to the triggering of InsP3 receptor polyubiquitination.
In summary, the data presented show that GnRH-induced
InsP3 receptor down-regulation in Importantly, to the best of our knowledge, this study represents the
first analysis of the ubiquitin/proteasome pathway in anterior
pituitary cells and the first demonstration that a hypothalamic releasing factor, such as GnRH, can utilize this pathway to regulate protein levels. Indeed, ubiquitin/proteasome pathway-mediated InsP3 receptor down-regulation is likely to contribute to
the mechanism by which long term administration of GnRH and its
analogues to patients suppresses luteinizing
hormone/follicle-stimulating hormone secretion and produces a
hypogonadal state (2, 3). Further, these data raise the possibility
that other proteins, perhaps those involved signal transduction (26,
40, 41), might also be targeted by the ubiquitin/proteasome pathway in anterior pituitary cells upon GPCR activation.
These studies also show that it is the ubiquitin/proteasome pathway,
and not other candidate proteolytic systems (12, 19), that accounts for
GnRH-mediated InsP3 receptor down-regulation. Thus, in
response to GnRH receptor activation, InsP3 receptors are
targeted by members of the E2/E3 enzyme family that conjugate ubiquitin
to proteins (27, 28). In analyzing this response in We have also been able to show that exogenous transiently expressed
receptors are polyubiquitinated in response to GPCR activation. This is
significant, because in order to study the triggering and site
specificity of polyubiquitination, it will be necessary to assess a
large number of mutant receptors, which can be most easily accomplished
by expressing them transiently. In summary, we found that exogenous
InsP3RHAwt was processed in response to GnRH
receptor activation similarly to endogenous InsP3 receptor
but with the exception that it was also constitutively
"monoubiquitinated" (modified with one or a very small number of
Myc-ubiquitin residues). Significantly, whereas
InsP3RHAwt was both poly- and
monoubiquitinated, InsP3RHA With regard to events that trigger type I InsP3 receptor
polyubiquitination, the analysis of InsP3RHA In conclusion, our studies show that the ubiquitin/proteasome pathway
is active in T3-1 mouse anterior pituitary gonadotropes,
chronic activation of gonadotropin-releasing hormone (GnRH) receptors
causes inositol 1,4,5-trisphosphate (InsP3) receptor
down-regulation (Willars, G. B., Royall, J. E., Nahorski,
S. R., El-Gehani, F., Everest, H. and McArdle, C. A. (2001)
J. Biol. Chem. 276, 3123-3129). In the current study,
we sought to define the mechanism behind this adaptive response. We
show that GnRH induces a rapid and dramatic increase in
InsP3 receptor polyubiquitination and that proteasome
inhibitors block InsP3 receptor down-regulation and cause
the accumulation of polyubiquitinated receptors. Thus, the ubiquitin/proteasome pathway is active in
T3-1 cells, and GnRH regulates the levels of InsP3 receptors via this mechanism.
Given these findings and further characterization of this system, we also examined the possibility that
T3-1 cells could be used to examine the ubiquitination of exogenous InsP3 receptors
introduced by cDNA transfection. This was found to be the case,
since exogenous wild-type InsP3 receptors, but not
binding-defective mutant receptors, were polyubiquitinated in a
GnRH-dependent manner, and agents that inhibited the
polyubiquitination of endogenous receptors also inhibited the
polyubiquitination of exogenous receptors. Further, we used this system
to determine whether phosphorylation was involved in triggering
InsP3 receptor polyubiquitination. This was not the case,
since mutation of serine residues 1588 and 1755 (the predominant
phosphorylation sites in the type I receptor) did not inhibit
polyubiquitination. In total, these data show that the
ubiquitin/proteasome pathway is active in anterior pituitary cells,
that this pathway targets both endogenous and exogenous
InsP3 receptors in GnRH-stimulated
T3-1 cells, and that,
in contrast to the situation for many other substrates, phosphorylation
does not trigger InsP3 receptor polyubiquitination.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T3-1 mouse gonadotrope cell
line have indicated that the suppression of secretion from gonadotropes
in patients treated chronically with gonadotropin-releasing hormone
(GnRH)1 receptor agonists (2,
3) may result from a reduction in the expression of inositol
1,4,5-trisphosphate (InsP3) receptors (2, 4, 5).
InsP3 receptors are a family of three proteins (termed type
I, II, and III receptors) that form tetrameric ion channels in
endoplasmic reticulum (ER) membranes, and upon binding of
InsP3, the channels open, and Ca2+ stored
within the ER flows into the cytoplasm (6-8). Thus, InsP3 receptors play a pivotal role in linking G-protein-coupled receptor (GPCR)-mediated InsP3 formation to increases in cytoplasmic
free Ca2+ concentration (9). A reduction in their
expression (i.e. their down-regulation) would, therefore, be
expected to suppress Ca2+ mobilization (4, 5, 10) and
secretion (2).
T3-1 cells and show that it
occurs via the ubiquitin/proteasome pathway. In characterizing this
adaptive response, we identified major differences in the properties of commonly used proteasome inhibitors and found that deubiquitination of
InsP3 receptors occurs rapidly and is likely to limit the
accumulation of ubiquitinated receptors and that InsP3
receptor ubiquitination is Zn2+-dependent.
Importantly, we also used
T3-1 cells to develop conditions for the
analysis of exogenous InsP3 receptor ubiquitination, and by
expressing mutant receptors, we show that InsP3 binding is important in triggering this event but that phosphorylation is not.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T3-1 cells were kindly provided by Dr. P. Mellon (University of California, San Diego, CA) and were cultured as
monolayers in Falcon Integrid tissue culture dishes in Dulbecco's
modified Eagle's medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum; cells were subcultured every 3-7 days using 0.25% trypsin, 1 mM EDTA. Rabbit
polyclonal antisera CT1h and CT1w were raised against the C terminus of
the rat type I receptor and were affinity-purified and shown to
specifically recognize endogenous type I InsP3 receptors
(13). CT1h immunoprecipitated both endogenous receptors and exogenous
epitope-tagged type I receptors and was used in all
immunoprecipitations. Surprisingly, however, this antiserum did not
recognize epitope-tagged receptors in immunoblots. Thus, CT1w was used
to probe for type I InsP3 receptor expression in
transfected cells, since this antiserum recognized both endogenous
receptors and exogenous epitope-tagged receptors. Mouse monoclonal
anti-ubiquitin (FK2), which recognizes both mono- and polyubiquitinated
proteins, was purchased from Affiniti Research Products Limited,
anti-hemagglutinin (HA) epitope (HA11) was from Babco, and anti-c-Myc
(9E10) was from Roche Molecular Biochemicals. Peroxidase-conjugated
antibodies, molecular mass markers, SDS, Triton X-100, protease
inhibitors,
N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and receptor agonists were obtained from Sigma; Protein A-Sepharose CL-4B (Protein A) was from Amersham Biosciences;
dithiothreitol was from Bio-Rad; lactacystin and MG-132 were from
Biomol; N-acetyl-Leu-Leu-norleucinal (ALLN) and thapsigargin
were from Alexis; and epoxomicin was a kind gift from Dr. C. Crews
(Yale University, New Haven, CT).
T3-1 Cells--
Cell suspensions were prepared by detaching
adherent cells with HBSE (155 mM NaCl, 10 mM
HEPES, 0.7 mM EDTA, pH 7.4) and vigorous pipetting in
culture medium. Cells were then pipetted into wells of Falcon six-well
plates (2 ml/well), were incubated with stimuli or inhibitors, were
collected by centrifugation (750 × g for 3 min at
4 °C), and were solubilized by incubation for 30 min at 4 °C with
lysis buffer (50 mM Tris, 150 mM NaCl, 1%
Triton X-100, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, 10 µM pepstatin, 0.2 µM soybean trypsin
inhibitor, 1 mM dithiothreitol, pH 8.0). Following
centrifugation (16,000 × g for 10 min at 4 °C),
supernatants containing solubilized receptors were mixed with 2× gel
loading buffer and were electrophoresed and immunoblotted with CT1h as
described (20).
T3-1 cell culture medium supplemented with sufficient nonradioactive
cysteine (200 µM) to allow for normal cell growth. Cells
were then preincubated for 1 h with ALLN (20 µg/ml), stimulated
for 1 h with GnRH (2 µM), and harvested in lysis
buffer, and type I InsP3 receptors were immunoprecipitated
with CT1h and electrophoresed as described. Gels were stained with
Coomassie Blue, and regions corresponding to unmodified and
polyubiquitinated InsP3 receptors were excised, homogenized, and assessed for radioactivity in 4 ml of scintillation fluid. Because ubiquitin does not contain cysteine, it does not become
radiolabeled; thus, the percentage of receptors polyubiquitinated can
be calculated from the amount of radioactivity migrating in the region
corresponding to polyubiquitinated receptors relative to total receptor radioactivity.
T3-1 Cells--
Cells were harvested using
0.25% trypsin/1 mM EDTA, were seeded into six-well Falcon
plates at a density of 2 × 106/well, and were
transfected 24 h later by adding 1 ml of fresh culture medium
containing a complex of cDNA and 9 µl of Superfect (Qiagen),
prepared according to the manufacturer's instructions. The cDNAs
used were as follows: pCW7, which encodes His6, c-Myc epitope-tagged yeast ubiquitin (Myc-ubiquitin) and was a kind gift from
Dr. R. R. Kopito (Stanford University) (22); pcDNA3 (empty
vector); pcWIHA (17), which encodes wild-type mouse type I
InsP3 receptor tagged at the C terminus with an HA epitope
(InsP3RHAwt); pcWIHA
(17), which encodes a
binding-defective, HA-tagged mutant mouse type I receptor
(InsP3RHA
) that lacks residues 316-352; and
pcWIHAA/A, which encodes an HA-tagged receptor
(InsP3RHAA/A) with serine
alanine mutations
at positions 1588 and 1755. This mutant was created using the
QuikChangeTM kit (Stratagene). In brief, pcWIHA was
first mutated to introduce alanine at position 1755 using primer pair
5'-GGAAGAAGAGAGGCGCTTACCAGCTTTGG-3' and
5'-CCAAAGCTGGTAAGCGCCTCTCTTCTTCC-3'. This mutant was then further
mutated to introduce alanine at position 1588 using primer pair
5'-CGCAGAGACGCTGTACTGGCAGCTAGCAGAGACTAC-3' and
5'-GTAGTCTCTGCTAGCTGCCAGTACAGCGTCTCTGCG-3'. The first and second
primer pairs also introduced HaeII and NheI sites, respectively, to facilitate screening. The correct introduction of the desired mutations into the polymerase-generated region was
confirmed by sequencing. 48 h after transfection, the cells were
exposed to stimuli or inhibitors and were harvested and solubilized by
incubation for 30 min at 4 °C in 1 ml of lysis buffer. After centrifugation (16,000 × g for 10 min at 4 °C),
type I InsP3 receptors were immunoprecipitated with CT1h
(to purify endogenous and exogenous receptors) or HA11 (to purify
exogenous HA-tagged receptors only), and immunoprecipitates were
immunoblotted with HA11, 9E10, CT1h, or CT1w.
T3-1 cells incubated at 37 °C was measured with a radioreceptor
assay exactly as described (20).
3) or mean range (n = 2).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T3-1
Cells--
Initial measurements of type I InsP3 receptor
levels in lysates from GnRH-stimulated cells showed that receptor
down-regulation in response to GnRH (0.1 µM) was
half-maximal at 15 ± 2 min (Fig. 1A). This is considerably more
rapid than that seen in other cell types (11-15), most likely because
GnRH receptors are refractory to desensitization and thus elevate
InsP3 concentration profoundly and persistently (2, 4).
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Fig. 1.
Down-regulation of type I
InsP3 receptors. T3-1 cells in
suspension were incubated for the times indicated with GnRH (0.1 µM) and in some cases with added antide (3 µM). Cells were then harvested, lysates were prepared,
and type I receptor content was determined in immunoblots with CT1h.
The 220-320-kDa regions of gels are shown, and the arrows
mark the position of type I receptor (~260 kDa). A,
kinetics of GnRH-induced InsP3 receptor down-regulation. In
four quantitated independent experiments, the half-maximal decline in
type I receptor immunoreactivity occurred at 15 ± 2 min, and the
maximal reduction in immunoreactivity was 66 ± 6%. B,
acute stimulation with GnRH does not lead to InsP3 receptor
down-regulation. Cells were incubated with GnRH alone for 60 or 5 min
(lanes 2 and 4) or for 60 min either
with GnRH and antide, added simultaneously (lane
3), or with antide added 5 min after GnRH (lane
5).
T3-1 cells to
GnRH in the absence or presence of a range of proteasome inhibitors and
monitored type I InsP3 receptor levels and associated
ubiquitin immunoreactivity. Three of these inhibitors, ALLN, MG-132,
and lactacystin, are widely employed, the first two being peptides that
are reversibly acting transition state analogues and the latter being a
structurally different pseudosubstrate that covalently modifies the
active site (23). The remaining inhibitor used, epoxomicin, is a novel,
highly potent, irreversible inhibitor (24). In the absence of
inhibitor, incubation with GnRH for 1 h caused InsP3
receptor down-regulation (Fig. 2A, lane
2, lower panel) but did not cause the
accumulation of ubiquitinated species (Fig. 2A,
lane 2, upper panel). In
contrast, when the cells were preincubated with inhibitors for 2 h, GnRH-induced InsP3 receptor down-regulation was blocked,
and a parallel increase in the level of ubiquitin immunoreactivity
associated with InsP3 receptors was observed (Fig.
2A, lanes 3-10). Whereas they
exhibited different potencies (see legend to Fig. 2A), the
four inhibitors were equally efficacious in causing the accumulation of
ubiquitinated species and completely blocked down-regulation at maximal
concentration (Fig. 2, A and E, lanes
2, 4, 6, and 8). Control
experiments (e.g. Fig. 2D, lane
3) showed that the inhibitors alone did not increase basal
InsP3 receptor levels or cause the accumulation of
ubiquitinated species. Additional controls showed that the
ubiquitinated species were indeed modified type I receptors, since when
purified, they were clearly immunoreactive with type I receptor
antiserum (Fig. 2B, lower panel,
lane 2). The inability of the same antiserum to
detect ubiquitinated receptors in crude type I receptor
immunoprecipitates (Fig. 2A, lower
panel) is most likely explained by the low abundance of
ubiquitinated receptors relative to unmodified receptors. To address
this issue, the proportion of type I InsP3 receptors
ubiquitinated was defined in experiments in which cells were
radiolabeled with [35S]cysteine, and in maximally
stimulated cells it was found to be 9 ± 1% of total
(n = 3). The ubiquitinated receptors
migrated as a "smear" (~275-380 kDa) (Fig. 2A,
upper panel, and Fig. 2B) slightly
less rapidly than unmodified type I receptor (~260 kDa) (Fig.
2A, lower panel), indicative of the
formation of a spectrum of polyubiquitinated species and typical of the
migration of other polyubiquitinated proteins (25, 26). In total, the
finding that the four structurally and mechanistically different
proteasome inhibitors all have the same effect shows that the
ubiquitin/proteasome pathway mediates InsP3 receptor
down-regulation in
T3-1 cells. This conclusion is supported by
findings that specific inhibitors of other candidate proteolytic
pathways (20 µM
benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl ketone, a caspase
inhibitor, and 20 µM PD150606, a calpain inhibitor) did
not block GnRH-induced InsP3 receptor
down-regulation.2
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Fig. 2.
Proteasome inhibitors block InsP3
receptor down-regulation and cause the accumulation of
polyubiquitinated receptors. T3-1 cells in suspension were
incubated with or without 0.1 µM GnRH and proteasome
inhibitors and were harvested, lysates were prepared, InsP3
receptors were immunoprecipitated with CT1h, and ubiquitin and type I
receptor immunoreactivity was assessed in immunoblots with FK2
(upper panels) and CT1h (lower
panels), respectively. The 220-420-kDa regions of gels are
shown, and the arrows and brackets mark the
respective positions of unmodified type I receptor (~260 kDa) and
polyubiquitinated type I receptor (~275-380 kDa). A, dose
dependence of proteasome inhibitor effects. Cells were preincubated for
2 h with ALLN (lanes 3-6) or MG-132
(lanes 7-10) at the concentrations indicated and
were then exposed to GnRH for 1 h (lanes
2-10). Half-maximal accumulation of polyubiquitinated
species and inhibition of down-regulation occurred at 4 µg/ml ALLN,
0.3 µg/ml MG-132, 0.5 µM lactacystin, and 0.04 µM epoxomicin (mean, n
2 independent
experiments). B, immunoreactivity of purified
polyubiquitinated species. Extracts of CT1h-derived immunoprecipitates
from control cells (lane 1) or 20 µg/ml
ALLN-pretreated, GnRH-stimulated cells (lane 2)
were reimmunoprecipitated with FK2 to purify ubiquitinated species and
were then probed with FK2 or CT1h. C, time course of
GnRH-induced type I InsP3 receptor polyubiquitination.
Cells were preincubated without or with 20 µg/ml ALLN for 2 h
and were then incubated with GnRH for 0-60 min. The ubiquitin
immunoreactivity of immunoprecipitated receptors was then assessed and
quantitated (mean ± S.E., n
3). D,
time dependence of ALLN effects. Cells were preincubated for 0-4 h
with 20 µg/ml ALLN (lanes 3-8) and were then
exposed to GnRH for 1 h (lanes 2 and
4-8). E, time dependence of proteasome inhibitor
effects on ubiquitination. Cells were preincubated for 2 or 0 h
with 20 µg/ml ALLN (lanes 2 and 3),
2 µg/ml MG132 (lanes 4 and 5), 3 µM lactacystin (lanes 6 and
7), or 0.3 µM epoxomicin (lanes
8 and 9) and were then exposed to GnRH for 1 h.
T3-1 cells and
appear to participate in suppressing the build-up of polyubiquitinated
InsP3 receptors.
View larger version (44K):
[in a new window]
Fig. 3.
InsP3 receptors are
deubiquitinated upon blockade of GnRH receptors. A,
InsP3 receptor deubiquitination. Cell suspensions,
preincubated with 20 µg/ml ALLN for 2 h, were incubated with 0.1 µM GnRH plus 3 µM antide for 20 min
(lane 6), or with 0.1 µM GnRH for
20 min, followed by further incubations for the times indicated with
added 3 µM antide (lanes 1-5).
Ubiquitin immunoreactivity associated with immunoprecipitated type I
InsP3 receptors was then assessed as in Fig. 2.
B, effects of antide on InsP3 formation. Cells
in suspension were incubated for 20 min with 0.1 µM GnRH
alone or with 3 µM antide plus 0.1 µM GnRH
(left panel), for 30 min with 0.1 µM GnRH alone, or for 20 min with 0.1 µM
GnRH followed by a further 10-min incubation after 3 µM
antide addition (right panel). Data shown are
mean ± S.E. or range of replicate samples.
View larger version (47K):
[in a new window]
Fig. 4.
Effects of inhibitors on InsP3
receptor down-regulation and polyubiquitination. Cells in
suspension were incubated without or with 0.1 µM GnRH in
the absence or presence of 1 µM thapsigargin, 100 µM TPEN, or 10% glycerol as indicated. A,
inhibition of down-regulation. Cells were incubated for 60 min and were
probed with CT1h as in Fig. 1. B, inhibition of
polyubiquitination. Cells preincubated with 20 µg/ml ALLN for 2 h were incubated for 20 min, and ubiquitin immunoreactivity was
assessed as in Fig 2. C, effects on InsP3
formation. Cells were incubated for 20 min, and InsP3
concentration was assessed as in Fig. 3. Data shown are mean ± S.E. of triplicate samples (*, p < 0.02).
T3-1 cells could be transfected and that expression of exogenous wild-type type I receptor was insufficient to reproducibly increase the InsP3 receptor or
polyubiquitin content of CT1h-derived immunoprecipitates probed as in
Figs. 2-4.2 Thus, we sought to selectively measure
exogenous InsP3 receptor ubiquitination in the limited
population of transfectable cells, by co-expressing Myc-ubiquitin and
HA-tagged InsP3 receptors. Fig.
5A illustrates the feasibility
of this approach, since when Myc-ubiquitin was expressed alone,
GnRH-induced Myc-polyubiquitination of endogenous receptors
(upper panel, lanes 1-3)
paralleled the profile of endogenous ubiquitin incorporation into type
I receptors (Fig. 2C), indicating that Myc-ubiquitin is
incorporated into polymeric chains capable of mediating proteasomal
degradation. Further, co-expression of
InsP3RHAwt (lanes 4-6)
at levels insufficient to increase the total InsP3 receptor
content of CT1h immunoprecipitates (lower panel,
compare lanes 1-3 with lanes
4-6) considerably enhanced Myc-polyubiquitination (upper panel, lanes 4-6),
indicating that exogenous receptors are efficiently polyubiquitinated.
It is noteworthy that whereas endogenous receptors were down-regulated
by GnRH (lower panel), the exogenous HA-tagged
receptors were not (middle panel,
lanes 4-6). This is consistent with other
studies showing that the HA tag inhibits InsP3
receptor down-regulation (17).
View larger version (43K):
[in a new window]
Fig. 5.
Ubiquitination of exogenous InsP3
receptors. T3-1 cell monolayers were transfected with 0.5 µg
of pCW7 (encoding Myc-ubiquitin) and 0.05 µg of either pcDNA3
(empty vector), pcWIHA (encoding InsP3RHAwt),
pcWIHA
(encoding InsP3RHA
),
or pcWIHAA/A (encoding InsP3RHAA/A)
and were incubated with 0.1 µM GnRH under the conditions
indicated. Cells were then harvested, lysates were prepared,
InsP3 receptors were immunoprecipitated (IP)
with CT1h or HA11, and immunoblots (IB) were probed for
Myc-ubiquitin immunoreactivity with 9E10 or for InsP3
receptor immunoreactivity with HA11, CT1w, or CT1h, as indicated. The
220-420-kDa regions of gels are shown, and the migration
positions of endogenous receptor (~260 kDa; arrows),
unmodified or monoubiquitinated HA-tagged exogenous receptors (~265
or ~270 kDa; arrowheads), and Myc-polyubiquitinated
receptor (~275-380 kDa; brackets) are indicated.
A and B, Myc-ubiquitin and InsP3
receptor immunoreactivity of immunoprecipitates from GnRH-stimulated
transfected cells. C, InsP3 receptor
immunoreactivity of immunoprecipitates from unstimulated transfected
cells (lanes 1-4) and of a sample of endogenous
T3-1 cell type I receptor (lane 5).
D, inhibitory effects of 1 µM thapsigargin and
3 µM antide on Myc-polyubiquitination in cells
co-transfected with pCW7 and pcWIHA and stimulated with GnRH for 60 min.
, which does not
bind InsP3 (17), the level of Myc-ubiquitination was
unaffected by GnRH (upper panel, lanes
7-9), indicating that InsP3RHA
is not polyubiquitinated. Parallel analysis of HA-tagged receptor content (lower panel) showed that both
InsP3RHAwt and
InsP3RHA
were expressed and that
InsP3RHA
(~265 kDa) migrated slightly more
rapidly than InsP3RHAwt (~270 kDa). To
demonstrate that only the HA-tagged receptors were purified and that
endogenous receptors did not co-immunoprecipitate, we probed the
HA11-derived immunoprecipitates with CT1w, which recognizes both
HA-tagged and endogenous receptors in immunoblots, and with CT1h, which
recognizes only endogenous receptors in immunoblots. Fig. 5C
(upper panel, lane 2) shows
that InsP3RHAwt migrates more slowly than
endogenous receptor (lane 5), as previously described (17), and that no endogenous receptor was
co-immunoprecipitated. This was confirmed by the observation that CT1h
(lower panel) did not immunoreact with
lane 2. Likewise, CT1h did not immunoreact with
immunoprecipitated InsP3RHA
(lane
3), showing again that endogenous receptors do not
co-immunoprecipitate with the exogenous
receptors.4 Importantly,
these data rule out the possibility that endogenous receptors might
contribute to the ubiquitination seen in Fig. 5B. Finally,
Myc-ubiquitin immunoreactivity in lysates, which was predominantly in
high molecular mass conjugates, was the same in cells expressing
InsP3RHAwt and
InsP3RHA
,2 indicating that the
lack of Myc-polyubiquitination in the latter was indeed due to the
deletion in the InsP3 receptor. Thus, polyubiquitination of
transiently expressed exogenous receptors is mediated by
InsP3 binding, indicating that they interact appropriately
with InsP3 and are subject to the same regulatory processes
as stably expressed receptors (37). Furthermore, their processing
paralleled that of endogenous receptors, since GnRH-induced
Myc-polyubiquitination of InsP3RHAwt was
inhibited by thapsigargin and antide (Fig. 5D).
were modified (Fig. 5B),
was not blocked by thapsigargin or antide, and, significantly, became
more prominent at InsP3 receptor cDNA levels >0.1
µg,2 indicating that it may result from the
overexpression of exogenous protein. Nevertheless, by expressing
relatively low amounts of exogenous InsP3 receptor, the
contribution of monoubiquitination to the overall ubiquitination signal
could be minimized, and it was possible to use transient receptor
expression to probe the events that trigger polyubiquitination.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
T3-1 cells is mediated
by the ubiquitin/proteasome pathway, that transiently expressed
exogenous InsP3 receptors are polyubiquitinated similarly
to endogenous receptors, and that InsP3 binding, but not
PKA-mediated InsP3 receptor phosphorylation, is a key event
in the process that leads to polyubiquitination. In addition, we
demonstrate that deubiquitination limits the accumulation of
polyubiquitinated InsP3 receptors and that both
thapsigargin and TPEN inhibit polyubiquitination.
T3-1 cells, we
have extended our understanding of InsP3 receptor polyubiquitination and the ubiquitin/proteasome pathway in general in
several ways. First, our data show that the accumulation of polyubiquitinated InsP3 receptors in the presence of
proteasome inhibitors is countered by deubiquitination. Currently,
virtually all work on deubiquitination has been done on purified
proteins or disrupted cells (27, 42), and very little is known about the role of this activity in intact cells, apart from a recent study
showing that a novel enzyme specifically deubiquitinates and stabilizes
p53 (43). Whereas the nature of the activity that deubiquitinates
InsP3 receptors in intact cells was not examined, its
existence explains why the accumulation of polyubiquitinated species in
proteasome inhibitor-treated cells is relatively minor, amounting to
only 9 ± 1% of the total receptor complement. It also suggests
that in cells not exposed to proteasome inhibitors, polyubiquitinated
InsP3 receptors will be subject to the competing effects of
deubiquitinating enzymes (causing stabilization) and the proteasome
(causing degradation). These and other findings (43) raise the
possibility that this situation is the norm for all polyubiquitinated
proteins. Second, with regard to the mechanism of ubiquitination, the
ability of TPEN to inhibit InsP3 receptor polyubiquitination implicates a RING domain-containing E3 in this process, perhaps one of the recently identified E3s involved in the
degradation of ER proteins (36, 44). Mechanistic insight was also
obtained using thapsigargin, which depletes ER Ca2+ (29,
30) and completely inhibited InsP3 receptor
polyubiquitination. This indicates that intraluminal Ca2+
plays a role in InsP3 receptor polyubiquitination.
Intriguingly, ubiquitin/proteasome pathway-mediated processing of other
ER proteins is also inhibited by depletion of ER Ca2+ (45,
46). Thus, InsP3 receptor polyubiquitination appears to be
via a pathway common to all ER proteins targeted via the ubiquitin/proteasome pathway that is dependent on the normal storage of
Ca2+ in the ER and perhaps on Ca2+ binding to
one or more of the many Ca2+-binding ER proteins (29-31).
Finally, comparison of the effects of different proteasome inhibitors
showed that they varied considerably in their rate of action, with ALLN
and MG-132 acting much more rapidly than lactacystin and epoxomicin.
This kinetic variation most likely reflects the mechanistic differences
between the inhibitors (23, 24) and clearly should be taken into
account when these inhibitors are used.
, which does not
bind InsP3 (17), was only monoubiquitinated, indicating
that only polyubiquitination occurs in response to InsP3
binding. Furthermore, InsP3RHAwt
polyubiquitination, but not monoubiquitination, was blocked by thapsigargin and antide. Thus, monoubiquitination appears to be a
process mechanistically discrete from that which mediates
polyubiquitination and may be a response to the overexpression of
exogenous receptors, a view supported by the observation that the
prominence of monoubiquitination increased as exogenous receptor
expression increased. Thus, it is possible that the capacity of
T3-1
cells to accommodate and correctly process transiently expressed
exogenous InsP3 receptors is relatively limited, and above
that capacity, the receptors are monoubiquitinated. The fact that
exogenous receptors did not associate to a detectable extent with
endogenous receptors lends credence to this view. An alternative
explanation is that InsP3 receptor monoubiquitination is a
normal cellular event that has been revealed because of the high
sensitivity of the Myc epitope antibody. In this regard, it has
recently been shown that other receptors and their associated proteins
can be monoubiquitinated (40, 48) as a prelude to their trafficking to
lysosomes. It is an intriguing possibility that InsP3
receptors could be similarly processed.
shows that InsP3 binding, and presumably the conformational
changes that result from this binding (49), cause the receptor to
become polyubiquitinated. The possibility that phosphorylation might be
involved in triggering polyubiquitination was also examined, since the
type I InsP3 receptor is phosphorylated stoichiometrically in response to PKA activation in intact cells (6-8) and, indeed, in
response to activation of the Gq-linked GPCRs that lead to InsP3 receptor down-regulation (38). However,
PKA-dependent phosphorylation of the receptor was clearly
not required for polyubiquitination, since
InsP3RHAA/A was polyubiquitinated equivalently
to InsP3RHAwt.
T3-1 anterior pituitary cells and mediates InsP3 receptor degradation in response to activation of
GnRH receptors. Since transiently expressed exogenous receptors are
polyubiquitinated similarly to endogenous receptors, use of this cell
type will allow for the analysis of a range of mutant receptors and the dissection of the molecular events that lead to InsP3
receptor polyubiquitination.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. T. Furuichi and K. Mikoshiba for providing the mouse type I InsP3 receptor cDNA that was used to create the constructs described.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant 5RO1DK49194, American Heart Association Grant 0256225T, and the Pharmaceutical Research and Manufacturers of America Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Pharmacology,
SUNY Upstate Medical University, 750 E. Adams St., Syracuse, NY
13210-2339. Tel.: 315-464-7956; Fax: 315-464-8014; E-mail: wojcikir@mail.upstate.edu.
Published, JBC Papers in Press, November 5, 2002, DOI 10.1074/jbc.M206607200
2 R. J. H. Wojcikiewicz and Q. Xu, unpublished results.
3 Surprisingly, TPEN significantly raised InsP3 concentration, most likely due to relief of inhibitory effects of Zn2+ on phosphoinositidase C activity.
4 This lack of co-immunoprecipitation indicates that the exogenous and endogenous receptors do not associate in transiently transfected cells. This contrasts with the situation in stably transfected cells, where some heterotetramer formation and co-immunoprecipitation was observed (17). The basis for this discrepancy was not examined, but it may reflect differences in the rate of receptor synthesis resulting from the two modes of receptor expression.
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ABBREVIATIONS |
---|
The abbreviations used are:
GnRH, gonadotropin-releasing hormone;
InsP3, inositol
1,4,5-trisphosphate;
InsP3RHAwt, wild-type type
I InsP3 receptor;
InsP3RHA, type
I InsP3 receptor lacking residues 316-352;
InsP3RHAA/A, type I InsP3 receptor
with serine
alanine mutations at positions 1588 and 1755;
ER, endoplasmic reticulum;
GPCR, G protein-coupled receptor;
ALLN, N-acetyl-Leu-Leu-norleucinal;
HA, hemagglutinin;
Protein A, Protein A-Sepharose CL-4B;
TPEN, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine);
PKA, protein kinase A;
E2, ubiquitin-conjugating enzyme;
E3, ubiquitin-protein isopeptide ligase.
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REFERENCES |
---|
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---|
1. | Shacham, S., Harris, D., Ben-Shlomo, H., Cohen, I., Bonfil, D., Przedecki, F., Lewy, H., Ashkenazi, I. E., Seger, R., and Naor, Z. (2001) Vitam. Horm. 63, 63-90[CrossRef][Medline] [Order article via Infotrieve] |
2. |
McArdle, C. A.,
Franklin, J.,
Green, L.,
and Hislop, J. N.
(2002)
J. Endocrinol.
173,
1-11 |
3. | Barbieri, R. L. (1992) Trends Endocrinol. Metab. 3, 30-34 |
4. |
McArdle, C. A.,
Willars, G. B.,
Fowkes, R. C.,
Nahorski, S. R.,
Davidson, J. S.,
and Forrest-Owen, W.
(1996)
J. Biol. Chem.
271,
23711-23717 |
5. |
Willars, G. B.,
Royall, J. E.,
Nahorski, S. R., El-,
Gehani, F.,
Everest, H.,
and McArdle, C. A.
(2001)
J. Biol. Chem.
276,
3123-3129 |
6. | Patel, S., Joseph, S. K., and Thomas, A. P. (1999) Cell Calcium 25, 247-264[CrossRef][Medline] [Order article via Infotrieve] |
7. | Taylor, C. W., Genazzani, A. A., and Morris, S. A. (1999) Cell Calcium 26, 237-251[CrossRef][Medline] [Order article via Infotrieve] |
8. | Thrower, E. C., Hagar, R. E., and Ehrlich, B. E. (2001) Trends Pharmacol. Sci. 22, 580-586[CrossRef][Medline] [Order article via Infotrieve] |
9. | Berridge, M. J., Lipp, P., and Bootman, M. D. (2000) Nature Rev. Mol. Cell. Biol. 1, 11-21[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Tovey, S. C.,
de Smet, P.,
Lipp, P.,
Thomas, D.,
Young, K. W.,
Missiaen, L., De,
Smedt, H.,
Parys, J. B.,
Berridge, M. J.,
Thuring, J.,
Holmes, A.,
and Bootman, M. D.
(2001)
J. Cell Sci.
114,
3979-3989 |
11. |
Wojcikiewicz, R. J. H.,
Furuichi, T.,
Nakade, S.,
Mikoshiba, K.,
and Nahorski, S. R.
(1994)
J. Biol. Chem.
269,
7963-7969 |
12. |
Wojcikiewicz, R. J. H.,
and Oberdorf, J. A.
(1996)
J. Biol. Chem.
271,
16652-16655 |
13. |
Wojcikiewicz, R. J. H.
(1995)
J. Biol. Chem.
270,
11678-11683 |
14. |
Bokkala, S.,
and Joseph, S. K.
(1997)
J. Biol. Chem.
272,
12454-12461 |
15. | Sipma, H., Deelman, L., De, Smedt, H., Missiaen, L., Parys, J. B., Vanlingen, S., Henning, R. H., and Casteels, R. (1998) Cell Calcium 23, 11-21[Medline] [Order article via Infotrieve] |
16. |
Wojcikiewicz, R. J. H.,
and Nahorski, S. R.
(1991)
J. Biol. Chem.
266,
22234-22241 |
17. |
Zhu, C. C.,
Furuichi, T.,
Mikoshiba, K.,
and Wojcikiewicz, R. J. H.
(1999)
J. Biol. Chem.
274,
3476-3484 |
18. | Brind, S., Swann, K., and Carroll, J. (2000) Dev. Biol. 223, 251-265[CrossRef][Medline] [Order article via Infotrieve] |
19. |
Hirota, J.,
Furuichi, T.,
and Mikoshiba, K.
(1999)
J. Biol. Chem.
274,
34433-34437 |
20. | Oberdorf, J., Webster, J. M., Zhu, C. C., Luo, S. G., and Wojcikiewicz, R. J. H. (1999) Biochem. J. 339, 453-461[CrossRef][Medline] [Order article via Infotrieve] |
21. | Wojcikiewicz, R. J. H., Ernst, S. A., and Yule, D. I. (1999) Gastroenterology 116, 1194-1201[Medline] [Order article via Infotrieve] |
22. | Ward, C. L., Omura, S., and Kopito, R. R. (1995) Cell 83, 121-127[Medline] [Order article via Infotrieve] |
23. | Lee, D. H., and Goldberg, A. L. (1998) Trends Cell Biol. 8, 397-403[CrossRef][Medline] [Order article via Infotrieve] |
24. |
Meng, L.,
Mohan, R.,
Kwok, B. H. B.,
Elofsson, M.,
Sin, N.,
and Crews, C. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
10403-10408 |
25. |
Ravid, T.,
Doolman, R.,
Avner, R.,
Harats, D.,
and Roitelman, J.
(2000)
J. Biol. Chem.
275,
35840-35847 |
26. |
Shenoy, S. K.,
McDonald, P. H.,
Kohout, T. A.,
and Lefkowitz, R. H.
(2001)
Science
294,
1307-1313 |
27. | Pickart, C. M. (2001) Annu. Rev. Biochem. 70, 503-533[CrossRef][Medline] [Order article via Infotrieve] |
28. | Weissman, A. M. (2001) Nat. Rev. Mol. Cell. Biol. 2, 169-178[CrossRef][Medline] [Order article via Infotrieve] |
29. | Paschen, W. (2001) Cell Calcium 29, 1-11[CrossRef][Medline] [Order article via Infotrieve] |
30. | Meldolesi, J., and Pozzan, T. (1998) Trends Biochem. Sci. 23, 10-14[CrossRef][Medline] [Order article via Infotrieve] |
31. |
Yoo, S. H.,
and Lewis, M. S.
(2000)
J. Biol. Chem.
275,
30293-30300 |
32. |
Lorick, K. L.,
Jensen, J. P.,
Fang, S.,
Ong, A. M.,
Hatakeyama, S.,
and Weissman, A. M.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11364-11369 |
33. |
Fang, S.,
Jensen, J. P.,
Ludwig, R. L.,
Vousden, K. H.,
and Weissman, A. M.
(2000)
J. Biol. Chem.
275,
8945-8951 |
34. | Bays, N. W., Gardner, R. G., Seelig, L. P., Joazeiro, C. A., and Hampton, R. (2001) Nat. Cell Biol. 3, 24-29[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Sato, S.,
Ward, C. L.,
Krouse, M. E.,
Wine, J. J.,
and Kopito, R. R.
(1996)
J. Biol. Chem.
271,
635-638 |
36. |
Gardner, R. G.,
Shearer, A. G.,
and Hampton, R.
(2001)
Mol. Cell. Biol.
21,
4276-4291 |
37. | Zhu, C. C., and Wojcikiewicz, R. J. H. (2000) Biochem. J. 348, 551-556[CrossRef][Medline] [Order article via Infotrieve] |
38. |
Wojcikiewicz, R. J. H.,
and Luo, S. G.
(1998)
J. Biol. Chem.
273,
5670-5677 |
39. |
Haug, L. S.,
Jensen, V.,
Hvalby, O.,
Walaas, S. I.,
and Ostvold, A. C.
(1999)
J. Biol. Chem.
274,
7467-7473 |
40. |
Marchese, A.,
and Benovic, J. L.
(2001)
J. Biol. Chem.
276,
45509-45512 |
41. |
Penela, P,
Ruiz-Gomez, A.,
Castano, J. G.,
and Mayor, F.
(1998)
J. Biol. Chem.
273,
35238-35244 |
42. | Rajapurohitam, V., Bedard, N., and Wing, S. S. (2002) Am. J. Physiol. 282, E739-E745[Medline] [Order article via Infotrieve] |
43. | Li, M., Chen, D., Shiloh, A., Luo, J., Nikolaev, A. Y., Qin, J., and Gu, W. (2002) Nature 416, 648-653[CrossRef][Medline] [Order article via Infotrieve] |
44. |
Fang, S.,
Ferrone, M.,
Yang, C.,
Jensen, J. P.,
Tiwari, S.,
and Weissman, A. M.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
14422-14427 |
45. |
Inoue, S.,
and Simoni, R. D.
(1992)
J. Biol. Chem.
267,
9080-9086 |
46. |
Durr, G.,
Strayle, J.,
Plemper, R.,
Elbs, S.,
Klee, S. K.,
Catty, P.,
Wolf, D. H.,
and Rudolph, H. K.
(1998)
Mol. Biol. Cell
9,
1149-1162 |
47. |
Joseph, S. K.,
Bokkala, S.,
Boehning, D.,
and Zeigler, S.
(2000)
J. Biol. Chem.
275,
16084-16090 |
48. | Polo, S., Sigismund, S., Faretta, M., Guidi, M., Capura, M. R., Bossi, G., Chen, H., De, Camilli, P., and Di Fiore, P. P. (2002) Nature 416, 451-455[CrossRef][Medline] [Order article via Infotrieve] |
49. | Mignery, G. A., and Sudhof, T. C. (1990) EMBO J. 9, 3893-3898[Abstract] |