From the Department of Pharmacology, University of
California, San Diego, La Jolla, California 92093-0636 and the
§ Departments of Neurosciences and Pharmacology, University
of Pennsylvania, Philadelphia, Pennsylvania 19104-6074
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ABSTRACT |
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To identify factors involved in the expression of
ligand-gated ion channels, we expressed nicotinic acetylcholine
receptors in HEK cells to characterize roles for oligosaccharide
trimming, calnexin association, and targeting to the proteasome. The
homologous subunits of the acetylcholine receptor traverse the membrane
four times, contain at least one oligosaccharide, and are retained in
the endoplasmic reticulum until completely assembled into the circular
arrangement of subunits of -
-
-
-
to enclose the ion channel. We previously demonstrated that calnexin is associated with
unassembled subunits of the receptor, but appears to dissociate when
subunits are assembled in various combinations. We used the glucosidase
inhibitor castanospermine to block oligosaccharide processing, and
thereby inhibit calnexin's interaction with the oligosaccharides in
the receptor subunits. Castanospermine treatment reduces the
association of calnexin with the
-subunit of the receptor, and
diminishes the intracellular accumulation of unassembled receptor
subunit protein. However, treatment with castanospermine does not
appear to alter subunit folding or assembly. In contrast, co-treatment
with proteasome inhibitors and castanospermine enhances the
accumulation of polyubiquitin-conjugated
-subunits, and generally reverses the castanospermine induced loss of
-subunit protein. Co-transfection of cDNAs encoding the
- and
-subunits, which leads to the expression of assembled
- and
- subunits, also inhibits the loss of
-subunits expressed in the presence of
castanospermine. Taken together, these observations indicate that
calnexin association reduces the degradation of unassembled receptor
subunits in the ubiquitin-proteasome pathway.
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INTRODUCTION |
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The nicotinic acetylcholine receptor is a prototype molecule of a
family of ligand-gated ion channels, which include GABAA, glycine, and 5HT3 receptors (1, 2). Following binding by agonists to these receptors, a conformational change increases cation
permeability through the central pore of the receptor, eliciting
depolarization of the cell membrane (1, 2). Peptide backbones of each
of the four subunits of the acetylcholine receptor transverse the
membrane four times, and possess at least one Asn-X-Ser/Thr glycosylation signal (2, 3). The subunits are thought to undergo a
maturation pathway which includes oligosaccharide attachment (4),
formation of disulfide bonds (5-7), proline isomerization (8), and
intersubunit contacts at specific interfaces (9-11). Members of this
family of receptors are composed of a multisubunit complex of
glycoproteins which are retained and assembled in the endoplasmic
reticulum prior to transport to the cell surface (9, 12). Acetylcholine
receptor subunits at the neuromuscular junction assemble into a
circular orientation of subunits of -
-
-
-
, to enclose the
central ion channel (2, 13; but see Ref. 14).
The endoplasmic reticulum localized protein, calnexin, is associated with unassembled subunits of the acetylcholine receptor (15-17), but calnexin appears to be absent with combinations of assembled subunits (16). Connolly et al. (18) demonstrated that calnexin is associated with subunits of the GABAA receptor, indicating that calnexin association might be involved in the biogenesis of other multisubunit ion channels. Numerous investigations have established that calnexin associates primarily with monoglucosylated oligosaccharides, which are intermediates in the processing of nascent oligosaccharides or products of reglucosylation by the enzyme UDP-glucose:glycoprotein glucosyltransferase, and that treatment with the glucosidase inhibitor castanospermine can disrupt the interaction (reviewed in Refs. 19 and 20).
Using transient expression of acetylcholine receptor subunits in HEK
cells, we find a role for calnexin association in reducing degradation
of unassembled subunits by the proteasome, since treatment with
castanospermine reduces the subunit-calnexin association and increases
the polyubiquitination of the -subunit. Additionally, co-treatment
with proteasome inhibitors blocks the degradation. Although unassembled
-subunits are degraded at a rapid rate (5), treatment with
glucosidase inhibitors substantially promotes degradation (4). Our data
also indicate that castanospermine treatment does not cause the
-subunit to misfold, as detected by a conformationally sensitive
antibody. Therefore, increased degradation by the proteasome appears to
be independent of the nascent peptide undergoing misfolding in this
system. In contrast, a recent study indicated that treatment of chick
myotubes with castanospermine disrupts
-subunit folding and assembly
(17). Our data also indicate that calreticulin and ERp57, the two other
proteins known to have glycoprotein-associating properties similar to
calnexin (19, 21), do not appear to be bound to the
-subunit.
Assembly with the -subunit reduces the degradation of the
-subunit when
- and
-subunits are co-expressed in the presence of castanospermine, indicating that glucose trimming, calnexin association, assembly and entrance into the proteasome are linked in
the expression of the receptor. Connections among these phenomena are
also likely to be critical to the fidelity of expression of other
multisubunit glycoproteins.
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MATERIALS AND METHODS |
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Castanospermine Treatment and Transfections-- Castanospermine (Calbiochem, San Diego, CA) was solubilized in Dulbecco's modified Eagle's medium at a concentration of 100 µg/ml, and immediately added to 10-cm plates of cells. Cells were treated with castanospermine 2 h prior to transfection, transfected with receptor subunit cDNAs, allowed to grow for 16 h, or replenished with fresh castanospermine in Dulbecco's modified Eagle's medium and raised for another 24 h.
Transfections employed the calcium phosphate precipitation method, as described in Keller et al. (16). Generally, 15 µg of plasmid DNA encoding each receptor subunit were added to plates of cells, unless noted otherwise. In transfections whereDetergent Solubilization, Immunoprecipitation, Electrophoresis,
and Western Blots--
In experiments involving immunoprecipitations
with antibodies to calnexin, calreticulin, ERp57, and the receptor
subunits, the solubilization buffer consisted of 0.5%
CHAPS,1 150 mM
NaCl, 1 mM CaCl2, 20 mM HEPES, pH
8.0, and the protease inhibitors: benzamidine, aprotinin, leupeptin,
and pepstatin A. The characteristics of the antibodies used for these
immunoprecipitations, mAb 35 or mAb 61 to precipitate the -subunit,
mAb 111 to precipitate the
-subunit and mAb 137 to precipitate the
-subunit, were described previously (22). Anticalnexin,
antipolyubiquitin, and anti-calreticulin were purchased from Stressgen
(British Columbia, Canada). Solubilization of cells and
immunoprecipitations using anti-polyubiquitin were in 50 mM
Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.4%
deoxycholate, 1% Nonidet P-40, 0.1% SDS, phenylmethanesulfonyl
fluoride, N-ethylmaleimide, and the other protease
inhibitors listed above. The ratio of solubilization buffer volume
relative to the number of cell plates were the same for each sample
within an experiment. Following solubilization, samples were
centrifuged at 10,000 × g for 5 min. Dilution ratios for antibody in all immunoprecipitations were approximately 1:100.
Proteasome Inhibitors-- Proteasome inhibitors were solubilized in Me2SO and added 3 h after transfection. Equivalent concentrations of Me2SO were added to untreated cells. The final concentration of Me2SO in a plate of cells was at most 0.3%. Cells were grown for an additional 16 h and then solubilized. The proteasome inhibitor benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO) was obtained from Dr. F. Mercurio (Signal Pharmaceuticals, San Diego, CA) and used at a final concentration of 20 µM. MG-132 (carbobenzoxyl-leucinyl-leucinyl-leucinal) and lactacystin were purchased from Calbiochem (San Diego, CA) and used at 50 and 10 µM, respectively. Calpain inhibitor I (N-Ac-Leu-Leu-norleucinal; Calbiochem, San Diego, CA) was used at final concentration of 100 µM.
125I--Bungarotoxin Binding and Density Scans of
Western Blots--
The snake toxin
-bungarotoxin (
-Bgt) binds to
both unassembled and assembled
-subunits, whereas carbamoylcholine
recognizes only
-subunits assembled with
-,
-, or
-subunits
(10). Transfected and untransfected cells were permeabilized in a 0.1%
saponin buffer (11), exposed to an excess of
125I-
-bungarotoxin (125I-
-Bgt) for 3 h, washed, and
-radiation was counted. The binding of
125I-
-Bgt to untransfected cells was subtracted from its
binding to transfected cells. Data were standardized to the average of 125I-
-Bgt bound to cells untreated with castanospermine.
Bands on Western blots were scanned with Deskscan (Hewlett Packard) and integrated with the program Collage (Fotodyne, New Berlin, WI).
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RESULTS |
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Castanospermine Diminishes Accumulation of -,
-, and
-Subunits in the Cell, without Generally Influencing the
Accumulation of Other Cellular Proteins--
Cells were treated with
the glucosidase inhibitor castanospermine, to identify roles for
glucose trimming in the biogenesis of acetylcholine receptors expressed
in HEK cells. Treatment with castanospermine, prior to and during the
transfection period, inhibits the glucosidase I and glucosidase II
enzymes, leaving oligosaccharides capped with three glucose residues.
Castanospermine treatment, therefore, maintains larger sized
oligosaccharides which are attached to glycoproteins confined to the
endoplasmic reticulum. Since unassembled receptor subunits and
-
dimers are retained intracellularly (23, 24), treatment with
castanospermine should maintain larger oligosaccharides attached to the
receptor subunits. A side reaction, which may result from a residual
fraction of glucosidase enzymes remaining active, is that, nascent
oligosaccharide chains whose glucosyl residues have been trimmed, in
turn, become reglucosylated by
UDP-glucose:glycoprotein-dependent glucosyltransferase. Reglucosylated receptor subunits may accumulate transiently because most glucosidase enzymes are inhibited (20).
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Polyubiquitinated -Subunits Accumulate when Cells Are Treated
with Proteasome Inhibitors--
To identify mechanisms which regulate
receptor subunit degradation, experiments were designed to ascertain
whether treatment with castanospermine increases conjugation of
polyubiquitin to
-subunits. Cells were treated or untreated with
castanospermine, transfected with cDNA encoding the
-subunit,
and maintained in the presence of proteasome inhibitors: calpain
inhibitor I and benzyloxycarbonyl-Leu-Leu-phenylalaninal (Z-LLF-CHO;
26). Calpain inhibitor I and Z-LLF-CHO were used in this experiment,
because other investigations have demonstrated their efficacy for
detecting the conjugation of polyubiquitin chains to proteins (27, 28). Detergent extracts were immunoprecipitated with an antibody to polyubiquitin, and Western blots were developed with mAb 210 to detect
-subunits (Fig. 2). The appearance of
a characteristic ladder pattern near the top of the gel indicates that
the proteins are polyubiquitinated (27-30). A high molecular weight
ladder pattern is observed when
-subunits are expressed in the
presence of castanospermine (Fig. 2A, lane 2), indicating
that the
-subunits become polyubiquitinated. In comparison,
-subunits expressed in the absence of castanospermine are also
polyubiquitinated, but the high molecular weight ladder pattern is
fainter (Fig. 2A, compare lanes 1 and
2). The high molecular weight pattern is absent when
Z-LLF-CHO is omitted in the experiment (Fig. 2B); this
finding indicates that treatment with Z-LLF-CHO inhibits the
degradation of polyubiquitinated
-subunits in the proteasome,
thereby allowing the conjugated intermediates to accumulate. Treatment
with calpain inhibitor I alone does not lead to the detection of
polyubiquitinated
-subunits.
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Treatment with the Proteasome Inhibitors MG-132 or Lactacystin
Inhibits the Degradation of -Subunits--
To further characterize
the mechanisms of degradation, cells were treated with the proteasome
inhibitors lactacystin or MG-132, to examine whether degradation of
-subunits is reduced. Lactacystin is a irreversible inhibitor which
binds specifically to the proteasome, as demonstrated by affinity
labeling and peptide sequencing (31). MG-132 is a congener of
Z-LLF-CHO, also a peptide-aldehyde that blocks the proteolytic
activities of the proteasome (32). Cells were treated or untreated with
castanospermine, transfected with the same plasmid DNA transfection
mixture in all plates, and then treated or untreated with a proteasome
inhibitor. Following an incubation period, cells were solubilized, and
-subunits were immunoprecipitated and detected on Western blots. As
displayed in Fig. 3, lanes 3 and 4, and in previous experiments, treatment with
castanospermine results in a loss of
-subunit protein; however, inclusion of lactacystin diminishes the loss of
-subunit protein (Fig. 3, lanes 1 and 2). Similar results were
obtained with MG-132 (Fig. 4C,
lanes 3 and 4). These data indicate that
-subunits, which may have been polyubiquitinated and subjected to isopeptidase activity (33), are degraded in the proteasome in cells treated with
castanospermine.
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Treatment with Castanospermine Disrupts an Interaction between
Calnexin and the -Subunit--
To ascertain whether glucose
trimming influences interactions between calnexin and
-subunits,
cells were treated with castanospermine, and then transfected with
various amounts of plasmid DNA encoding the
-subunit (Fig. 4). In
some experiments, cells were also treated with proteasome inhibitors to
reduce degradation and to facilitate the distinction between altered
recognition by calnexin from increased degradation. Sequential
immunoprecipitations were conducted with an antibody to calnexin
(c), followed by immunoprecipitation of the unbound material
with an anti-
- subunit antibody (mAb 61), to assess the extent of
calnexin-
-subunit recognition. In Fig. 4, sequentially precipitated
-subunits are displayed, with lane 1 in sections
A-C displaying
-subunits bound to calnexin, and lane 3 exhibiting the
-subunits not cleared with
calnexin; likewise, lane 2 in sections A-C
displays
-subunits bound to calnexin and lane 4 exhibits
-subunits not cleared with calnexin. Densities of
-subunit bands
were quantified, and density ratios for
-subunits expressed in the
presence relative to the absence of castanospermine were calculated.
Each histogram bar was calculated separately for
-subunits
precipitated with anti-calnexin or anti-
-subunit antibodies; for
example, in Fig. 4, A-C, the histogram bars designated TFT:
and IP to: c, were calculated for
-subunits co-immunoprecipitated with calnexin by anti-calnexin
antibody, by dividing the band densities for
-subunits expressed in
the presence (shown in lane 2) and absence of
castanospermine (shown in lane 1). Similarly, the histogram
bar designated as TFT:
and IP to:
, was
calculated for
-subunits immunoprecipitated with anti-
-subunit
antibody, by dividing the band densities for
-subunits expressed in
the presence and absence of castanospermine (displayed in lane
3). A comparison in the density ratios for
-subunits
co-immunoprecipitated with calnexin relative to those
immunoprecipitated with anti-
-antibody provides an estimate of the
extent of calnexin-
-subunit recognition.
Assembly with -Subunits Reduces the Loss of
-Subunits
Expressed in the Presence of Castanospermine--
To examine whether
-subunit assembly with the
-subunit ameliorates the loss of
-subunits expressed in the presence of castanospermine, the mass of
cDNA encoding the
-subunit was kept constant at 15 µg of
plasmid DNA/plate and the plasmid DNA encoding the
-subunit was
transfected at two different ratios to
-subunit. Accumulation of
-subunits was measured using 125I-
-Bgt binding to
permeabilized cells (Fig. 5):
125I-
-Bgt binding was also quantified in a similar
manner with untransfected cells. Standardized values for
125I-
-Bgt binding are displayed in Fig. 5, where
-toxin binding to untransfected cells is subtracted from the
values in transfected cells.
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Accumulation of -
Dimers Expressed in the Presence of
Castanospermine Is Dependent on the Transfected Ratios of
- and
-Subunit cDNAs--
To ascertain whether treatment with
castanospermine and the stoichiometric ratios of cDNAs encoding
- and
-subunits in the transfection influence the accumulation of
-
dimers, cells were treated with castanospermine and
co-transfected with plasmid DNAs encoding the
- and
-subunits at
ratios of 8:1 and 3:1. The mass of plasmid DNA encoding the
-subunit
was kept constant at 15 µg of plasmid DNA/plate and the mass of
plasmid DNA encoding the
-subunit was varied. The
-subunit was
immunoprecipitated with anti-
-antibody (mAb 61), and the Western
blot was developed with antibodies to the
- and
-subunits (mAb
210 and 137, respectively; Fig.
6A). The appearance of
co-immunoprecipitated
- and
-subunit bands appear to display
correspondingly similar changes in density when coexpressed in the
absence or presence of castanospermine, indicating that subunit
assembly is not disrupted (Fig. 6A). The density of the
-subunit band on the blot provides an indication of the accumulation
of
-
dimers. When cells are co-transfected with a large
stoichiometric imbalance of subunits, using plasmid DNAs encoding
-
and
-subunits at a 8:1 ratio, there are significantly diminished
- and
-subunit bands in the sample expressed in the presence of
castanospermine, indicating
-
dimers are less prevalent (Fig.
6A, compare lanes 1 to 2). However,
treatment with castanospermine has a smaller impact on altering the
density of
- and
-subunit bands when plasmid DNAs are
co-transfected at a 3:1 ratio, a closer stoichiometric balance of
subunits (Fig. 6A, compare lanes 3 to 4). Similar results with respect to changes in the
accumulation of assembled
-subunits are displayed in Fig.
6B, in an experiment where plasmid DNAs encoding
and
are co-transfected at ratios of 8:1 and 1:1. These data indicate that
when
-subunits have a higher probability of contacting
-subunits,
glucose trimming has a smaller impact on the accumulation of
-
dimers. These data also indicate that the ratios of nascent unassembled
subunits and glucose trimming have a combined influence on the
accumulation of assembled receptor subunits, which should ultimately
dictate the expression levels of fully assembled receptors at the cell surface.
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Calreticulin and ERp57 Association with -Subunits Is not
Detectable under Conditions where Calnexin Association Is
Evident--
Similar to the binding properties of calnexin,
calreticulin and ERp57 are thought to preferentially associate with
newly synthesized glycoproteins which possess oligosaccharides capped
with a single glucose residue (19, 21). Immunoprecipitation and Western blotting experiments were performed to ascertain whether calreticulin and ERp57 are associated with
-subunits. Calreticulin is expressed in HEK cells, as detected in a Western blot of a detergent extract (Fig. 7A, lane 1) and upon
immunoprecipitation with an antibody to calreticulin (Fig. 7A,
lane 4). However, co-immunoprecipitation of
-subunits with
calreticulin was not appreciably detected (Fig. 7B, lane 2),
although calreticulin was immunoprecipitated in this sample (Fig.
7A, lane 4),
-subunits were present in the extraction mixture (Fig. 7B, lane 4), and these conditions reveal
co-immunoprecipitation of
-subunits with calnexin (Fig. 7B,
lane 1). Association of
-subunits with calreticulin was also
not detected when the detergent extract was immunoprecipitated with
anti-
-antibody (mAb 61) and the Western blot developed with an
antibody to calreticulin (data not shown). Experiments with the
antibodies available to us indicate calnexin is more prevalently
associated with
-subunits than is calreticulin.
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Castanospermine Treatment Does Not Appear to Alter the Folding of
-Subunits, as Detected with a Conformationally Sensitive Antibody,
whereas Assembly with
-Subunits Increases the Fraction of Folded
-Subunits in the Cell--
We used mAb 35 reactivity to identify
whether treatment with castanospermine influences the folding of
unassembled
-subunits expressed in HEK cells. Cells were treated or
untreated with castanospermine, and transfected with 15 µg of plasmid
DNA/plate encoding the
-subunit. Sequential immunoprecipitations
using mAb 35, which binds primarily to folded
-subunits (5),
followed by an immunoprecipitation of the unbound material with mAb 61, which binds independently of conformation (5), were used to assess
folding (Fig. 8A). Only a
fraction of the
-subunit pool is recognized by mAb 35, indicating
the
-subunit pool is predominantly unfolded when expressed in the
absence of other subunits in HEK cells (Fig. 8A, lanes 1-4); this observation is consistent with previous observations in other cells (10, 34). An estimate of the fraction of folded
-subunits was obtained by measuring the density of
-subunit bands
in Fig. 8 and dividing the respective mAb 35 density by the mAb 61 density (Fig. 8C). The fractions of
-subunits
immunoprecipitated with mAb 35 relative to mAb 61 are similar when
expressed in the absence or presence of castanospermine (22 and 29%,
respectively; Fig. 8C). Within resolution limits of our
assay, and the capacity of mAb 35 to detect folded subunits, glucose
trimming does not appear to influence
-subunit folding.
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DISCUSSION |
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Of the three proteins thought to be involved in the biogenesis of
glycoproteins, and to associate primarily with glycoproteins possessing
oligosaccharides capped with one glucose residue (19, 21), only
calnexin was found to associate with the receptor -subunits.
Calreticulin and ERp57 could be associated with
-subunits in
vivo, but the interaction was undetectable in vitro, by
using antibodies available to us and techniques which readily detect calnexin in association with
-subunits. Based on data presented in
this study, the processing events in receptor biosynthesis which are
altered by castanospermine treatment appear to be related to calnexin
association.
Recognition of -subunits by calnexin appears to be rather complex,
with calnexin displaying association to both oligosaccharide structures
and the polypeptide backbone. Calnexin binding to the
-subunit
oligosaccharide is dependent on oligosaccharide trimming, because
castanospermine treatment reduces the interaction. The importance for
oligosaccharide trimming in this interaction becomes apparent when
expression levels are low, and degradation is inhibited with a
proteasome inhibitor (Fig. 4, B and C). These
observations may indicate that the calnexin-oligosaccharide interaction
displays a higher affinity with unassembled subunits, than the
calnexin-polypeptide interaction. In fact, several investigations have
suggested that calnexin displays a sufficient affinity to bind directly
to oligosaccharide structures irrespective of the polypeptide backbone
(19, 35).
The additional interaction between calnexin and the -subunit
polypeptide backbone appears to be dependent on subunit assembly, because assembly with the
-subunit reduces calnexin
association, and this interaction is not significantly altered by
castanospermine treatment (Fig. 4A, lanes 5-8). Subunit
assembly may inhibit the calnexin-polypeptide interaction, because the
newly formed contact interface between subunits occludes the lower
affinity association with calnexin. Alternatively,
-subunits which
fold upon assembly may bury an interface which is required for calnexin
attachment.
In agreement with our observations that castanospermine increases
receptor degradation, an earlier study with BC3H-1 cells showed that,
treatment with the glucosidase inhibitor 1-deoxynojirimycin decreased
the stability of unassembled -subunits expressed from the endogenous
gene (4). Investigations on other transmembrane spanning glycoproteins
have also revealed a decreased stability of glycoproteins expressed
under conditions which impair glucose trimming (36-38). In contrast,
studies of secretory protein processing indicate that calnexin
association correlates with the instability of these proteins (39,
40).
The ubiquitin-proteasome pathway appears to be involved in the
degradation of -subunits expressed in the presence of
castanospermine, because polyubiquitinated
-subunits accumulate in
cells treated with proteasome inhibitors. Moreover, the degradation of
-subunits is inhibited when cells are treated with the specific
proteasome inhibitor lactacystin (31), localizing the proteasome as the site for the degradation. Several investigations have established that
proteins embedded in the ER membrane are susceptible to degradation in
the proteasome, with examples including the cystic fibrosis transmembrane conductance regulator (29, 41), major histocompatibility complex subunits (42-44), and T-cell receptor subunits (45). Treatment
with proteasome inhibitors can result in the detection of conjugated
polyubiquitin chains (29), and an enhanced accumulation of proteins
which would otherwise be targeted for degradation (29, 45).
Investigations on major histocompatibility complex subunits (42-44)
and T-cell receptor
-subunits (45) have also revealed that
degradation of misfolded or unassembled proteins follows a pathway
which involves retrograde transport through the Sec61 protein
translocator channel (43, 44), cytoplasmic deglycosylation, and
degradation in the proteasome (43-45). The
-subunit of the
acetylcholine receptor has numerous lysine residues positioned in its
two cytoplasmic loops (i1-2 and i3-4); these
and other lysines positioned at the cytoplasmic and luminal boundaries
of the transmembrane domains, and in the extracellular domains, may
serve as polyubiquitin attachment sites, which target receptor subunits
to the proteasome.
Treatment with castanospermine may perturb the assembly pathway, where
calnexin association is a critical step which stabilizes unassembled
receptor subunits. Disruption of this interaction, and bi-directional
motions of the -subunit, may lead to increased exposure of ubiquitin
attachment sites at the cytoplasmic boundary, and promote targeting of
the subunit to the proteasome. Furthermore, the association of calnexin
with the
-subunit may occlude the ability of the
-subunit-calnexin complex to move through a relatively narrow Sec61
channel (46). A low affinity interaction between calnexin and
unassembled
-subunits, in the absence of artificially impaired
glucose trimming with castanospermine, might release proteins to enter
the degradative pathway. In support of the notion that retrograde
transport is a component of the degradative pathway for acetylcholine
receptor subunits, we have preliminary data indicating that treatment
with castanospermine and proteasome inhibitors results in an enhanced
accumulation of unglycosylated
-subunits.2
Ubiquitin-conjugating enzymes have been identified to reside on the cytoplasmic exposure of the ER membrane in yeast (47, 48), and attachment of ubiquitin chains is a requirement for efficient degradation via retrograde transport and proteasome hydrolysis (48). The presence of a homologous enzyme in HEK cells might conjugate ubiquitin molecules to sites on acetylcholine receptor subunits exposed just outside the cytoplasmic face of the ER membrane. In contrast, an attachment of calnexin to secretory proteins may result in their localization near to the ER membrane, and this may contribute to their destabilization. Secretory proteins which are positioned at the ER membrane may become dissociated from calnexin, translocated through the Sec61 channel, and degraded in the proteasome.
Assembly with -subunits may further stabilize the
-subunits, by
constraining the folded conformation, increase tethering in position in
the membrane, and physically inhibit the ability of the larger
-
dimer complex from moving through the Sec61 channel. The juxtaposition
of
- and
-subunits may also limit the accessibility of the
ubiquitin conjugating machinery to previously exposed lysine residues.
The above hypotheses concerning the degradative pathway for receptor
-subunits are consistent with observations that: (a)
calnexin dissociation diminishes the accumulation of receptor subunits,
and (b) assembly of
-subunits with
-subunits correlates with further folding and stabilization of
-subunits.
Our experiments indicate that castanospermine treatment does not
significantly alter the folding of -subunits, or the assembly of
-subunits with
-subunits. Supportive data in two other
investigations have also indicated that
-subunit folding and
assembly are not significantly impacted by impaired glucose trimming
(4, 49). In contrast, castanospermine treatment was observed to disrupt folding and assembly of
-subunits expressed in chick myotubes (17).
This investigation also indicated that overexpression of calnexin, by
transfection of a cDNA encoding calnexin, led to enhanced assembly
and surface expression of
-subunits in COS and HEK cells (17).
Glucose trimming and calnexin association may have their greatest
impact on the expression of multisubunit glycoprotein complexes when
the translation product of any subunit is a minor component of the
total protein embedded in the endoplasmic reticulum membrane, and there
is a stoichiometric imbalance in the expression of each subunit. This
hypothesis is consistent with the observation that the accumulation of
-
dimers, which are expressed in the presence of castanospermine,
is significantly diminished when
-subunits are in a large
stoichiometric deficiency to
-subunits. Using muscle cells, several
investigations have revealed that endogenous acetylcholine receptor
subunits are expressed in stoichiometric imbalances (24, 50, 51).
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ACKNOWLEDGEMENTS |
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We thank Dr. J. Holtzman for providing an antibody to ERp57 and Drs. J. DiDonato and F. Mercurio for providing Z-LLF-CHO.
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FOOTNOTES |
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* This work was supported by United States Public Health Service Fellowship NS09715 (to S. H. K.), National Institutes of Health Grant NS11323, MDA, and Smokeless Tobacco Research Council, Inc. grants (to J. L.), and United States Public Health Service Grant GM18360 (to P. T.).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.
1 The abbreviations used are: CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; Bgt, bungarotoxin; CST, castanospermine; ER, endoplasmic reticulum; HEK, human embryonic kidney; Me2SO, dimethyl sulfoxide; LAC, lactacystin; Z-LLF-CHO, benzyloxycarbonyl-Leu-Leu-phenylalaninal; mAb, monoclonal antibody.
2 S. Keller, unpublished data.
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REFERENCES |
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