From the Department of Pharmacology, University of
California, San Diego, La Jolla, Califronia 92093, ¶ Departments of Neurosciences and Pharmacology, University of
Pennsylvania, Philadelphia, Pennsylvania 19104-6074, and
Department of Neurosciences, University of California, San
Diego, La Jolla, California 92093
Received for publication, January 24, 2001, and in revised form, February 14, 2001
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ABSTRACT |
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The nicotinic acetylcholine receptor in muscle is
a ligand-gated ion channel with an ordered subunit arrangement of
Multimeric transmembrane proteins, including complex ligand-gated
ion channels represented by nicotinic acetylcholine receptors (nAchR),1 generally require
subunit assembly to be transported beyond the endoplasmic reticulum
(ER) into the secretory pathway leading to the cell surface (1-3). As
integral membrane components of lipid trafficking vesicles, unassembled
subunits are re-localized to the ER, stabilized by chaperones and
either assembled with neighboring subunits or targeted for degradation
by ubiquitination and cleavage in the proteasome (4, 5). Cellular
mechanisms that distinguish whether a protein is folded and/or
assembled and directed to the cell surface or misfolded and/or
unassembled and shuttled into a degradative pathway are poorly
understood, especially for the polytypic membrane proteins represented
by ligand-gated ion channels. The importance for shedding light on this
topic is borne out by several debilitating disorders associated with
mutations that are believed to cause misfolding and inhibit the
trafficking of physiologically important ion channels. Examples include
inherited mutations in potassium channel subunits that increase the
propensity to develop cardiac arrhythmias (6, 7) and amino acid
substitutions in cystic fibrosis transmembrane conductance regulator
that result in cystic fibrosis (8-10). In this study, we employ the
nAChR The nAChR in muscle is a ligand-gated ion channel composed of two
We therefore hypothesized that trafficking signals positioned in the
Antibodies--
Western blotting and immunofluorescence
protocols aimed at detecting the Plasmids, Transfections, and Cells--
The cDNAs encoding
the mouse nAChR subunits are inserted in the EcoRI site in
the expression vector pBR4 (Invitrogen, San Diego, CA). Mutations were
introduced in the wild-type cDNA template by employing the
"Quickchange" method (Stratagene, San Diego, CA). Subsequently,
double-stranded DNA subjected to mutagenesis was subcloned into
plasmids not exposed to the polymerase chain reaction-based mutagenesis
procedures, and the introduced fragment was checked by automated
sequencing. HEK-293 and ts20 (16) cells were employed in these studies,
which do not express nAChR at detectable levels. The receptor subunits
were transiently expressed in these cells following gene transfection.
Calcium phosphate precipitation was used for transfection in HEK cells.
For immunofluorescence, cells were grown in 35-mm glass bottom dishes
to ~80% confluency and transfected with 3 µg of plasmid DNA
encoding the
In ts20 cells, 1 µg of plasmid DNA was added to a 35-mm plate, and
transfection employed a liposome incorporation method (LipofectAMINE, Life Technologies, Inc.). ts20 cells express a mutant
temperature-labile ubiquitin-activating enzyme (16). At 30 °C, the
ts20 cells maintain their ability to ubuiquitinate proteins, but at
40 °C the mutant ubiquitin-activating enzyme is inactive, and cells
lose this capacity. Following transfection, ts20 cells were grown for
24 h at 30 °C and then shifted to 40 °C and grown for
another 15 h. ts20 cells were then fixed in paraformaldehyde and
processed for immunofluorescence in the same manner as the HEK cells
detailed below.
Immunofluorecence and Confocal
Microscopy--
Immunofluorescence methods were performed in
Triton-permeabilized cells to detect intracellular protein or in
non-permeabilized cells to detect Biotinylation of Proteins Located at the Cell Surface--
Cells
were washed in PBS adjusted to pH 8.0 and incubated in 0.5 mg/ml
sulfo-NHS-biotin (Pierce) in PBS for 30 min at room temperature. The
reaction was quenched in 50 mM glycine in PBS; cells were
washed further and then lysed in 1% Triton, 150 mM NaCl, 5 mM EDTA, 20 mM Tris-HCl, pH 8.0, on ice with
protease inhibitors (protease inhibitor mixture, Roche Molecular
Biochemicals). Streptavidin-Sepharose beads (Pierce) were added to the
mixture to isolate biotinylated protein. The beads were washed and
sedimented, and Laemmli sample buffer was added to elute the bound
protein. Other cells, transfected with the same calcium phosphate
mixture, were lysed in the above buffer, and acetylcholine receptor
subunits were co-immunoprecipitated with anti- Immunoprecipitation and Western Blots--
Similar numbers of
harvested cells were rinsed in PBS, sedimented, and solubilized in 0.15 M NaCl, 2 mM EDTA, 20 mM Tris-HCl, pH 7.5, 0.5% Triton with protease inhibitors (protease inhibitor mixture; Roche Molecular Biochemicals) on ice. The insoluble materials were removed by centrifugation, and a stoichiometric excess of antibody
to 125I- Altering the Adjacent Basic Signal
Arg313-Lys314 into
Arg313-Gln314 Promotes Trafficking of the
For control and comparison, transfected wild-type
In contrast to the wild-type
To examine whether cytoplasmically positioned adjacent basic amino acid
sequences modulate the trafficking of nAChR
To verify that the observed co-localization is due to spatial overlap
in the three-dimensional field of the confocal image as opposed to
artificial channel crossover caused by overly bright emission,
wavelengths corresponding to FITC and rhodamine were individually
blocked to examine whether images became apparent in the non-emitting
channel. Since images were not observed in the non-emitting channel,
crossover was excluded as the cause for the yellowish coloration
observed in the merged images. Furthermore, since the steady state
expression levels of the wild-type and K314Q Interactions of the Adjacent Basic Residue Signal with the Cellular
Trafficking Machinery--
Proteins that have been retrieved back to
the ER are incorporated into COP I vesicles that migrate in an
anterograde direction from the intermediate compartment to the ER (1).
Exposed adjacent basic amino acid sequences are known to interact with
the COP I components that incorporate the retrieved proteins into the COP I vesicles (21-23). Therefore, in contrast to the isolated wild-type
The experimental observations indicate that wild-type
In contrast to the interaction of the wild-type The K314Q Ubiquitination Modulates Placement of the K314Q
Two pathways for the post-Golgi sorting of the unassembled K314Q
To determine whether ubiquitination modulates post-Golgi trafficking in
the context of expressing unassembled protein, The findings of this study reveal that at least two distinct
and sequential mechanisms target nAChR -
-
-
-
. The subunits are sequestered in the endoplasmic
reticulum (ER) and assembled into the pentameric arrangement prior to
their exit to the cell surface. Mutating the
Arg313-Lys314 sequence in the
large cytoplasmic loop of the
-subunit to K314Q promotes the
trafficking of the mutant unassembled
-subunit from the ER to the
Golgi in transfected HEK cells, identifying an important determinant
that modulates the ER to Golgi trafficking of the subunit. The
association of the K314Q
-subunit with
-COP, a component of COP I
coats implicated in Golgi to ER anterograde transport, is diminished to
a level comparable to that observed for wild-type
-subunits when
co-expressed with the
-,
-, and
-subunits. This suggests that
the Arg313-Lys314 sequence is masked when the
subunits assemble, thereby enabling ER to Golgi trafficking of the
-subunit. Although unassembled K314Q
-subunits accumulate in the
Golgi, they are not detected at the cell surface, suggesting that a
second post-Golgi level of capture exists. Expressing the K314Q
-subunit in the absence of the other subunits in ubiquitinating
deficient cells (ts20) results in detecting this subunit at the cell
surface, indicating that ubiquitination functions as a post-Golgi
modulator of trafficking. Taken together, our findings support the
hypothesis that subunit assembly sterically occludes the trafficking
signals and ubiquitination at specific sites. Following the masking of
these signals, the assembled ion channel expresses at the cell surface.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits as a model to identify factors that modulate the
trafficking of ion channel proteins into the secretory pathway. We
approached this question by identifying mechanisms that restrict the
placement of unassembled
-subunits at the cell surface and asked how
subunit assembly abrogates these processes.
-subunits and single
-,
-, and
-subunits which surround a
central cation channel pore. As determined by subcellular
fractionation, the unassembled
-subunits are confined primarily to
the ER compartment (11). Insignificant amounts of unassembled
-subunit are detected on the cell surface following transfection,
transient expression in mammalian cells, and
125I-
-bungarotoxin exposure (12). In contrast,
appreciable binding of 125I-
-bungarotoxin is detected on
the cell surface when
-subunits are co-expressed with the
-,
-, and
-subunits, demonstrating that subunit assembly is a
requirement for the cell surface expression (12, 13). As the nAchR
assembles into a pentamer, assembled
-
and
-
dimers and
other intermediates are detected in intracellular pools of the cell
(12-14). However, the
-
and
-
dimers are also sequestered
intracellularly, as evidenced by the lack of 125I-
-bungarotoxin binding to the surface of intact
cells. (12, 13). Co-expression of the
-subunit with the
-,
-,
and
-subunits is required to transport the assembled subunits to the
cell surface.
-subunit at the interface that assembles with the
-subunit are
enclosed when the subunits assemble to form a circular pentamer. The
trafficking signals then become sterically occluded from the cellular
machinery that otherwise would retrieve proteins back to the ER. The
major aim of this study was to identify the ER retrieval sequences in
the
-subunit that inhibit the trafficking of the unassembled subunit
beyond the ER. Our experimental findings demonstrate that the adjacent
basic amino acid signal Arg313-Lys314 in the
large cytoplasmic loop of the
-subunit regulates trafficking of the
-subunit from the ER to the Golgi. This is revealed in the
trafficking characteristics of the
-subunit with the K314Q mutation
that proceeds from the ER and accumulates in the Golgi when expressed
in the absence of the other receptor subunits. Although the altered
-subunit proceeds to the Golgi, it does not express at the cell
surface. Inhibition of ubiquitination results in detecting the
-subunit with the K314Q alteration at the cell surface. We therefore
conclude that ubiquitination is a modulator for the Golgi to cell
surface trafficking of the
-subunit. Thus, we disclose two
mechanisms that regulate the trafficking of nAChR. One of these
involves masking of ER retrieval sequences by assembly of the
heterologous subunits that regulate the ER to Golgi trafficking of the
-subunit. The second mechanism regulates Golgi to cell surface
trafficking and is modulated by ubiquitination. These two mechanisms
may operate together to provide the "quality control" of the
completed receptor that progresses to the cell surface.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunit employed the rat
monoclonal antibody (mAb) 210, which recognizes an epitope in the
extracellular domain (15). The antibody to
-mannosidase II used to
detect the medial-trans-Golgi was a gift of Dr. M. Farquhar, University
of California, San Diego. Antibody to
-COP was provided by Dr. C. Harter, University of Heidelberg, Heidelberg, Germany, antibody to
-COP was purchased from Sigma, and antibody to calnexin was acquired
from Stressgen (Victoria, British Columbia, Canada). The secondary
antibodies employed for immunofluorescence were purchased from Jackson
Laboratories (West Grove, PA), as were the peroxidase-labeled secondary
antibodies used in Western blot detection.
-subunit. For immunoprecipitation and Western blotting,
cells were grown in 10-cm dishes, and 15 µg of plasmid DNA encoding
the
-subunit was added to each transfected plate of cells. Plasmids
encoding the other subunits were transfected at one-half the mass
employed for the
-subunit. Following transfection, cells were grown
at 37 °C for ~48 h and then prepared for immunofluorescence or immunoprecipitation.
-subunits at the cell surface.
Cells were fixed in 4% paraformaldehyde in PBS, rinsed, and quenched
in PBS/glycine. Cells were then permeabilized and blocked in 0.1%
Triton X-100, 1% fish gelatin (Sigma), and 1% bovine serum albumin in
PBS. Exposure to primary antibody was for 1 h in the
permeabilization solution diluted with an equivalent volume of PBS.
Exposure to secondary antibody conjugated to fluorophores was also for
1 h in the same buffer. Non-permeabilized cells were processed in
the same manner except Triton X-100 was omitted. Following exposure to
antibodies, cells were preserved in gelvatol and stored in the dark
under refrigeration. Confocal images were taken with a Bio-Rad MRC 1024 laser-scanning system attached to a Zeiss Axiovert microscope using a
40× oil NA 1.3 objective, and processed with Adobe Photoshop (San
Jose, CA). The brightly fluorescent cells show evidence of gene
transfection; other fainter appearing cells in the microscope field
apparently do not express the transfected gene.
-subunit antibody (mAb 111).
-COP or
-COP (clone M3A5, Sigma) was added to the soluble
fraction for 30 min, followed by addition of IgG-Sepharose beads for
another 45 min. Equivalent volumes of sample were resolved in 10%
SDS-polyacrylamide gels (NOVEX, San Diego, CA) and transferred to
nitrocellulose. Western blots were developed with chemiluminescent techniques.
-Bungarotoxin Expression Assay--
Cells
were transfected with the indicated subunit combinations, grown for
48 h, and blocked first with 10 mM carbamylcholine followed by exposure to 10 nM
125I-
-bungarotoxin for 1 h; the resultant
radioactive counts correspond to the residual nonspecific binding.
Other cells, transfected with the same calcium phosphate mixture, were
exposed directly to 10 nM
125I-
-bungarotoxin.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-Subunit to the Golgi--
Acetylcholine receptor
-subunits are
representative of the family of ligand-gated ion channels that display
the general topology shown in Fig.
1A, consisting of one major
extracellular domain, four transmembrane spans, and a large and small
cytoplasmic loop (15, 17). The largest span of sequence is
extracellular and is predicted to consist of the first 210 residues
starting at the N terminus (17). The major cytoplasmic loop is thought
to be located between residues 299 and 408 in the
-subunit and
includes the trafficking signals examined in this study. Conservation
in the adjacent basic sequences corresponding to positions 313-314 in
the
-subunit is observed among most receptor subunits when alignments of the major cytoplasmic loops are examined (Fig.
1B), suggesting that this sequence may encode an important
conservation of function. Therefore, an adjacent basic sequence at
position 313-314 in the
-subunit (18) was selected as a candidate
to be altered by site-directed mutagenesis from
Arg313-Lys314 to
Arg313-Gln314 (designated as the K314Q
-subunit) to examine whether subsequent changes occur in the
trafficking characteristics. The potential trafficking signal RK was
altered to RQ on the basis of its presence at an identical position in
the
-subunit (Fig. 1B), the subunit that facilitates
transport of the assembled receptor to the cell surface (12,
13).
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Fig. 1.
A, proposed topology of the nicotinic
acetylcholine receptor subunits (15, 17). The N-terminal domain, which
extends from residues 1 to 210 in the -subunit, is extracellular in
the cell surface receptor. A major cytoplasmic loop is situated between
residues 299 and 408 and includes the
Arg313-Lys314 signal. An Endo-H-sensitive
N-linked glycosylation site is located at Asn141
in the
-subunit. B, alignments of sequences in the major
cytoplasmic loop among the acetylcholine receptor subunits employed in
this study; note the conservation in alignment of the adjacent basic
amino acid sequences among the subunits corresponding to positions
313-314 in the
-subunit.
-subunits
expressed in ts20 cells (at 30 °C) generally display the
reticulate-diffuse pattern reminiscent of proteins deposited in the ER
(Fig. 2A, panels a
and b, red) and appear to overlap in pattern with endogenous calnexin (Fig. 2A, panels a and
c, blue), a diagnostic marker for the ER. Wild-type
-subunits expressed in ts20 cells also display minimal
co-localization (Fig. 2A, panel a)
with a GFP-Golgi protein marker (panel d,
green, GFP linked to the Golgi localization signal of
galactosyltransferase (19)), which was expressed in these cells by
co-transfection with the plasmid DNA encoding the
-subunit. Since
the GFP-Golgi protein marker was expressed from a transfected gene,
only a subset of cells display its expression. Moreover, wild-type
-subunits expressed in HEK cells display minimal overlap with the
endogenous medial-trans-Golgi marker
-mannosidase II (Fig.
2B, panel e), further substantiating
that the unassembled wild-type
-subunits are sequestered primarily in the ER. In support of our experimental observations on the trafficking characteristics of unassembled wild-type
-subunits as
revealed by transfection and confocal microscopy, subcellular separations employing sucrose gradients of
-subunits expressed in
muscle cells from the endogenous gene also demonstrated that unassembled
-subunits are restricted to the ER (11). Under the
numerous transfection and expression experiments employed over the
course of our study, minimal overlap was observed between the
unassembled wild-type
-subunits and endogenous
-mannosidase II.
Thus, high levels of overexpression from the transfected gene did not
appear to contribute to artificially induced trafficking patterns.
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Fig. 2.
Wild-type
-subunits expressed alone in cells are deposited
primarily in the ER, as evidenced by confocal microscopy.
A, transfected wild-type
-subunits (a and
b, red) expressed in ts20 cells (30 °C) overlap
extensively with endogenous calnexin, a diagnostic marker for the ER
(a and c, blue), but minimally with a
co-transfected GFP-Golgi compartment marker protein (a and
d, green, GFP linked to the Golgi localization
signal of galactosyltransferase (19)). ts20 cells were co-transfected
with plasmids encoding
-subunits and the GFP-Golgi marker. Cells
were permeabilized and exposed to antibodies detecting the
-subunit
(mAb 210) and calnexin (Stressgen, Victoria, British Columbia, Canada).
B, transfected wild-type (wt)
-subunits
expressed alone in HEK cells display the reticulate-diffuse pattern
reminiscent of the ER (panels e and f, green) and
display minimal co-localization with endogenous
-mannosidase II
(
-ManII) (panels e and g,
red), a diagnostic marker for the medial-trans-Golgi. Cells were
transfected to express the
-subunit, permeabilized, and exposed to
antibodies to the
-subunit (mAb 210) and
-mannosidase II.
-subunits expressed alone, cells
co-expressing
-subunits with
-,
-, and
-subunits display appreciable co-localization between the
-subunits and
-mannosidase II (Fig. 3a),
coinciding with the finding that co-transfection of plasmid DNAs
encoding the entire complement of subunits in HEK cells results in
detection of the receptor at the cell surface (13). Note that although
significant co-localization between the
-subunits and
-mannosidase II is observed when the receptor subunits are
co-expressed, a major fraction of
-subunits also display the
reticulate-diffuse pattern suggestive of localization in the ER (Fig.
3, a and b): this likely reflects the fraction of
-subunits not assembled with the other subunits or assembled receptor protein that did not exit the ER. As a further note,
-subunits co-expressed with the other receptor subunits in HEK cells
yield glycosylated
-subunits that traffic to the cell surface and
are fully cleavable with Endo-H in whole cell lysates (20). Thus,
Endo-H cleavage was not employed in this study as a tool to
characterize
-subunit trafficking.
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Fig. 3.
Wild-type -subunits
co-expressed with the
-,
-, and
-subunits
co-localize with
-mannosidase II in
permeabilized cells (a). HEK cells were
co-transfected with plasmids encoding the
-,
-,
-, and
-subunits, permeabilized, and exposed to anti-
-subunit antibody
(mAb 210, a, b, green) and
anti-
-mannosidase II (
-ManII) antibody
(a, c, red).
-subunits, the
Arg313-Lys314 sequence was altered into
Arg313-Gln314, with the aim of conserving side
chain volume but neutralizing the charge of the lysine residue to
remove the potential trafficking signal. HEK cells were transfected and
processed for confocal microscopy in the same manner as the wild-type
-subunits displayed in Figs. 2B and 3. The confocal
microscope image of cells expressing K314Q
-subunits is displayed in
Fig. 4, with the
-subunits exhibited by FITC emission (Fig. 4b, green) and
-mannosidase II
displayed by rhodamine emission (Fig. 4c, red). The
yellow coloration corresponds to overlap between the
-subunits and
-mannosidase II (Fig. 4a). Evident
co-localization is observed between the K314Q
-subunits and
-mannosidase II (Fig. 4a), suggesting spatial overlap in the cell and strongly indicating that the K314Q
-subunit traffics to
and is retained in the Golgi compartment.
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Fig. 4.
The K314Q -subunit
expressed in the absence of the other acetylcholine receptor subunits
in HEK cells co-localize with
-mannosidase II
(a), demonstrating that altering the signal
Arg313-Lys314 to
Arg313-Gln314 promotes the trafficking of the
mutant
-subunit from the ER to the Golgi.
Plasmid encoding the mutant
-subunit was transfected into cells,
which were permeabilized and exposed to mAb 210 (b, green)
and antibody to
-mannosidase II (
-ManII)
(c, red). The merged image displays co-localization between
the
-subunits and
-mannosidase II, as evidenced by the
yellowish coloration (a).
-subunits are similar
(Fig. 5, lanes 3 and
4), overexpression and saturation of the quality control
machinery of the cell can be excluded as the cause for the acquired
trafficking characteristics of the K314Q
-subunit. Wild-type
-subunits, when expressed in the absence of other subunits, sediment
as monomers upon sucrose gradient centrifugation (13). Since residues
in the extracellular region govern subunit assembly, the K314Q mutation
is unlikely to promote assembly of the mutant
-subunit into a more
complex structure such as a homopentamer. The above results therefore suggest that altering the adjacent basic amino acid signal
Arg313-Lys314 into
Arg313-Gln314 directly promotes the
trafficking of the substituted
-subunit from the ER to the Golgi.
The Arg313-Lys314 sequence therefore appears
to be a major determinant that modulates the ER to Golgi trafficking of
the
-subunit. Since this sequence is largely conserved among mouse
nAChR subunits (Fig. 1B), it can be postulated that it also
plays a similar role in the trafficking of the other subunits.
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Fig. 5.
The K314Q mutation in the
-subunit, as well as subunit assembly, diminish the
interaction of the
-subunit with
-COP, a component of the COP-I-mediated ER
retrieval machinery. Separate experiments are displayed in
A and B. Wild-type (wt)
-subunits
expressed alone show an evident association with
-COP (A
and B, lanes 1), whereas K314Q
-subunits expressed alone
(A, lane 2), and wild-type
-subunits co-expressed with
-,
-, and
-subunits (B, lane 2) display minimal
associations. Cells were transfected as indicated, and samples were
immunoprecipitated with antibody to
-COP (A and B,
lanes 1 and 2). Equivalent volumes of lysates removed
before immunoprecipitation were also diluted in Laemmli sample buffer
and resolved in gels (A and B, lanes 3 and
4). Similar numbers of cells, amounts of plasmids, and
sample volumes were employed in the experimental work-up for each
lane.
-subunit, K314Q
-subunits as well as wild-type
-subunits co-expressed with the full complement of receptor subunits
should be expected to display minimal interactions with the COP I
components, since these proteins traffic to the Golgi. Pull-down
experiments were performed in HEK cell lysates employing an antibody to
the COP I component protein,
-COP. Cells were transfected to express K314Q
-subunits, wild-type
-subunits, or co-express wild-type subunits with the
-,
- , and
-subunits. Similar numbers of cells, volumes of buffer, and amounts of samples loaded into the gels
were maintained in all steps. Proteins were subsequently resolved in
10% gels, transferred to nitrocellulose, and probed with an antibody
to the
-subunit (mAb 210). The immunoprecipitated samples (Fig. 5,
lanes 1 and 2) reflect the extent of COP protein recognition of the
-subunits when compared with the respective expression levels of the
-subunits displayed in the whole cell lysates (Fig. 5, lanes 3 and 4). Presumably, due
to a low affinity interaction that becomes more evident with
cross-linking (21, 22), only a fraction of
-subunits remained
associated with
-COP after completion of the immunoprecipitation
procedures employed in these experiments.
-subunits
expressed alone have the most pronounced association with
-COP
(Figs. 5, A and B, lane 1), whereas the
K314Q
-subunit (Fig. 5A, lane 2) and wild-type
-subunits co-expressed with the
-,
-, and
-subunits (Fig.
5B, lane 2) display minimal associations. Note also that in
the sample that displays the
-subunits co-expressed with the
-,
-, and
-subunits (Fig. 5B, lane 2), a minor
interaction of the
-subunits with
-COP is apparent; this likely
reflects the population of unassembled
-subunits present in these
cells (13, 24).
-subunits with
-COP, association with
-COP, another component of COP I coats
(21-23), was not detected. By employing the same buffer and conditions
for immunoprecipitation, the
-COP antibody did not co-immunoprecipitate the
-subunits, although
-subunits were abundant in these cells, and
-COP was immunoprecipitated in the samples (data not shown). Taken together, the experimental findings suggest that recognition of unassembled
-subunits by the COP I
complex is mediated through the Arg313-Lys314
signal, which potentially directly interacts with
-COP. Subunit assembly diminishes the interaction between the
-subunits and the
COP 1 complex, which is reflected in the trafficking of the receptor
beyond the ER compartment.
-Subunit Maintains the Capacity to Fold and
Assemble, as Detected with 125I-
-Bungarotoxin--
To
examine whether the K314Q
-subunit maintains the capacity to fold
and assemble into the mature receptor pentamer, ligand binding assays
were performed by protecting receptor-binding sites with
carbamylcholine from 125I-
-bungarotoxin. Carbamylcholine
recognizes and binds to the
-subunits associated with the
- or
-subunits but does not bind to unassembled
-subunits in the
concentration range employed (25). In contrast,
125I-
-bungarotoxin recognizes both unassembled and
assembled
-subunits (25). Therefore, prior exposure of cells to
carbamylcholine followed by the later addition of
125I-
-bungarotoxin provides a means to distinguish
whether the
-subunits are assembled. Since the addition of the
-subunit in the transfection results in transport of the associated
-,
-, and
-subunits to the cell surface, detection of the
receptor by carbamylcholine inhibition of
125I-
-bungarotoxin binding demonstrates complete
assembly of the subunits and expression of the receptor at the cell
surface. Equivalent numbers of cells were transfected with plasmid DNAs
encoding wild-type
-subunits with
-,
-, and
-subunits, or
K314Q
-subunits with
-,
-, and
-subunits, and cells were
subjected to the carbamylcholine protection assay. As displayed in Fig.
6, K314Q
-subunits are assembled and
expressed at the cell surface, since virtually all 125I-
-bungarotoxin counts are blockable by
carbamylcholine. Although the apparent expression of the assembled
wild-type
-subunits is approximately twice that of the assembled
K314Q
-subunits, this assay suggests that the K314Q
-subunit
maintains a sufficient capacity to fold, assemble, and traffic to the
cell surface. However, the lower expression observed for the K314Q
-subunit suggests that the cationic lysine 314 residue in the
cytoplasmic loop might facilitate interactions that modulate the
assembly of the subunits. Moreover, escape of the K314Q subunit from
the ER prior to subunit assembly might decrease the pool of fully
assembled receptor at the cell surface.
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Fig. 6.
The K314Q -subunit
maintains the capacity to assemble into the receptor complex and
express at the cell surface, as evidenced by carbamylcholine protection
of 125I-
-bungarotoxin-binding
sites on the surface of cells. Similar numbers of HEK cells were
co-transfected with plasmids encoding the wild-type (wt)
-subunit with the
-,
-, and
-subunits or the K314Q
-subunit with the
-,
-, and
-subunits and exposed or
unexposed to carbamylcholine (Carb.) prior to the addition
of 125I-
-bungarotoxin. Each histogram bar
represents a single sample tube, with the percent of maximum counts
calculated among the tubes.
-Subunit at the
Cell Surface--
We next examined whether the K314Q
-subunit
expressed in HEK cells can be detected at the cell surface, by first
employing selective biotinylation of surface proteins and their
pull-down with streptavidin beads, and second, by immunofluorescence
using an antibody that recognizes the extracellular domain of the
-subunit in non-permeabilized cells. Although wild-type
-subunits
co-expressed with the
-,
-, and
-subunits in HEK cells are
readily detected by both of these procedures (Fig.
7, A and B),
wild-type and K314Q
-subunits expressed in the absence of the other
subunits were not observed at the cell surface following extensive and
repetitive attempts (not shown). Although the K314Q alteration promotes
trafficking of the unassembled
-subunit from the ER to the Golgi in
HEK cells, it does not appear to be sufficient to bring the mutated
-subunit into the secretory pathway that proceeds to the cell
surface. Our experimental observations therefore suggest that
additional mechanisms other than COP I-mediated retrieval modulate the
trafficking of the K314Q
-subunit beyond the Golgi compartment.
View larger version (18K):
[in a new window]
Fig. 7.
Selective biotinylation (A)
and exposure of non-permeabilized cells to antibody (mAb 210) that
recognizes the extracellular domain of the
-subunit (B) detects the
-subunits co-expressed with the
-,
-, and
-subunits on the surface of HEK cells.
A, cells were transfected with the listed subunits,
permeabilized, and subunits were co-immunoprecipitated with
anti-
-subunit antibody (mAb 111, lane 1, T;
total); this sample displays the cellular
-subunit pool and the
associated
-subunits. Other cells, transfected with the same
mixture, were subjected to selective biotinylation of cell surface
proteins, lysis, and pull-down with streptavidin beads (lane 2, S; surface); this sample displays acetylcholine receptor subunits
on the cell surface. The resultant Western blot was developed with a
mixture of anti-
- and
-subunit antibodies (mAbs 111 and 210). The
protein banding densities in lane 2 reflect the selective
stoichiometry of the fully assembled receptor, with two
-subunits
incorporated for each
-subunit. B, intact cells were
exposed to mAb 210, followed by incubation with FITC-labeled secondary
antibody, and visualized with a confocal microscope.
-subunit can be postulated. First, the subunit might traffic beyond
the trans-Golgi network to the lysosomes and subsequently be degraded.
Second, the subunit could reach the cell surface but undergo rapid
endocytosis. As with the ER retrieval sequences, the assembly of the
subunits might occlude other targeting signals that potentially
intercept and sort proteins away from the cell surface. Our earlier
study demonstrated that
-subunits are ubiquitinated (5), and
ubiquitination has been shown to modulate trafficking in post-Golgi
compartments by targeting proteins to the lysosome (26-29). We
therefore examined whether ubiquitination potentially functions as a
signal that further interferes with the expression of the unassembled
subunit at the cell surface.
-subunits were
expressed transiently by gene transfection in the hamster fibroblast
cell line ts20, which encodes for a mutant ubiquitin-activating enzyme.
This enzyme is inactive when cells are grown at 40 °C (16). To
detect expression of the
-subunits at the cell surface, non-permeabilized cells were exposed to an antibody that recognizes the
extracellular domain of the
-subunit (mAb 210). Other plates of
cells, transfected with aliquots of the same transfection mixture, were
permeabilized to detect the
-subunits in intracellular locations. Confocal microscope images are shown in Fig.
8 displaying images that exhibit the
approximate frequencies of cells showing evidence for expression of the
transfected gene. Two images from a plate are shown to emphasize the
representative expression patterns for the fraction of cells exhibiting
a positive immunoreactivity; significantly bright emission relative to
background was interpreted to display receptor subunit expression. As
shown in Fig. 8c, wild-type
-subunits were not found to
show expression on the surface of ts20 cells under conditions that
repress ubiquitination. Fluorescent emission distinguishable from
background was not observed on the surface of cells expressing
wild-type
-subunits, indicating that suppressing ubiqutination is
not sufficient to promote the trafficking of this unassembled subunit
to the cell surface. By contrast, K314Q
-subunits, expressed alone,
are observed on the cell surface when ubiquitination is suppressed at a
frequency approximately comparable to that detected in the
permeabilized cells (Figs. 8, a and b),
suggesting that the mutated
-subunit traffics to the cell surface.
The observed immunoreactivity on the surface of cells expressing the
K314Q
-subunits was clearly discernible over that detected for the
wild-type
-subunits in multiple fields; exact quantitative estimates
would simply show the number of cells transfected and demonstrating
expression relative to those showing no expression, respectively. By
comparing the trafficking characteristics of the K314Q
-subunit
relative to the wild-type
-subunit, these data strongly suggest that
ubiquitination directly influences the sorting of the subunit into
post-Golgi compartments, in contrast to modulating expression through
degradation.
View larger version (88K):
[in a new window]
Fig. 8.
Inhibition of ubiquitination by expression of
the K314Q -subunit in ts20 cells at 40 °C
enables detection of this mutant
-subunit on
the cell surface (a). ts20 cells were transfected
with plasmids encoding K314Q
-subunits or wild-type (wt)
-subunits, grown for 24 h at 30 °C, switched to 40 °C for
15 h to inhibit ubiquitination, and then fixed in
paraformaldehyde. The surface preparation refers to antibody
added to non-permeabilized cells, whereas the inside
preparation reflects antibody added to permeabilized cells; mAb 210, which recognizes an epitope in the extracellular domain of the
-subunit (15), was employed in all panels. Two representative
microscope fields are presented for each experiment to display the
typical ratio of cells that express the
-subunit.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-subunits to the cell surface, as diagrammed schematically in Fig.
9. The first mechanism regulates
trafficking from the ER to the Golgi and is modulated through
recognition of the ER retrieval sequence
Arg313-Lys314 by the COP I protein complex.
Altering this sequence reduces the interaction of the mutant
-subunit with the COP I complex and corresponds to the trafficking
of the mutant
-subunit from the ER to the Golgi; this is labeled
path I in Fig. 9. However, removal of the ER retrieval
sequence is not sufficient to place the mutant
-subunit at the cell
surface in detectable levels. Sorting beyond the Golgi is modulated by
ubiquitination. Trafficking from the Golgi to the cell surface is
evident by detection of the K314Q
-subunit on the cell surface when
ubiquitination is inhibited, which is labeled as path II in
Fig. 9. The experimental observations therefore demonstrate that
simultaneous inhibition of both
-COP recognition and ubiquitination
are sufficient to place unassembled
-subunits on the cell surface.
The requirement for full assembly of the subunits of the receptor for
expression at the cell surface is likely due to the masking of the
trafficking signals and potential ubiquitination sites exposed in
the unassembled subunit.
View larger version (14K):
[in a new window]
Fig. 9.
Proposed checkpoints in the trafficking
pathway of acetylcholine receptor
-subunits. Path I, ER to Golgi
trafficking, which is modulated by the adjacent dibasic signal;
path II, Golgi to plasma membrane (P.M.) sorting,
which is regulated by ubiquitination.
As the subunits are oriented and assembled into dimers and
larger complexes, exposure of adjacent basic amino acids at an unassembled interface to the COP I machinery should continue to sequester the subunits in the ER and facilitate the assembly of additional subunits into the ion channel configuration. Introduction of
the adjacent basic sequences might therefore be an evolutionary adaptation that facilitates the ordered assembly of the subunits into a
completely enclosed and functional pentameric ion channel receptor. The
roles for basic amino acid sequences in the maturation and trafficking
of potassium channel subunits have also been observed (30), suggesting
that their eventual occlusion during folding and assembly might be a
general phenomenon in the processing of multisubunit channel proteins.
Other factors, such as chaperones, might assist in the maturation
process by promoting folding, stabilizing the intermediates from
degradation or by contributing to the retention of unassembled subunits
prior to their assembly (1, 31). However, our experimental observations
suggest that the basic amino acid trafficking signal is a major factor
that governs the trafficking of the nAChR -subunit to proceed beyond
the ER compartment.
Studies of type 1 transmembrane proteins have revealed that ER retrieval signals modulate protein trafficking from the ER to the cell surface and that addition of an adjacent basic amino acid retrieval sequence to a protein that otherwise traffics constitutively to the surface restrict this protein to the ER (23, 32). The polytypic configuration of acetylcholine receptor subunits or the presence of additional signals such as those that recruit the ubiquitination machinery add additional levels of complexity to events that modulate the post-Golgi sorting of the receptor. An unassembled subunit that proceeds beyond the Golgi might follow a pathway that sorts the protein from the trans-Golgi network to the lysosomes when specific sites are ubiquitinated. A similar trafficking route has been identified in yeast (28), and it can be expected that related pathways are manifested in mammalian cells. In the absence of ubiquitination, as in the case for nAChR subunits expressed in ts20 cells at the non-permissive temperature, the subunit might proceed from the Golgi and deposit at the cell surface.
Rapid endocytosis signaled by ubiquitination is another
plausible pathway that could interfere with the placement of the
unassembled subunit at the cell surface. The precedence for this
mechanism comes from the down-regulation of surface receptors by
ubiquitination and subsequent endocytosis (25, 29, 33, 34). Expression of growth hormone receptor in ts20 cells has shown that endocytosis of
this protein is blocked at the non-permissive temperature for ubiquitination, suggesting that ubiquitin is the tag that promotes internalization of the receptor (35, 36). Moreover, the ubiquitin moiety itself has recently been demonstrated to contain an endocytosis signal (37). However, our preliminary data indicate that the K314Q
-subunit expressed in the absence of the other subunits does not
follow a route of transient placement at the cell surface and rapid
endocytosis, since the K314Q
-subunit does not co-localize with
fluorescently labeled transferrin, a protein that follows an
endocytotic pathway (data not shown). Therefore, a plausible route for
the post-Golgi sorting of ubiquitinated K314Q
-subunits might
involve interception at the trans-Golgi network and subsequent targeting to the lysosomes. Subunit assembly presumably occludes the
key ubiquitination sites and allows the subunit to sort into an
alternative pathway that leads to the cell surface. Although beyond the
scope of this study, further investigations examining whether
ubiquitination modulates the targeting of the subunit from the
trans-Golgi network to the lysosomes should be revealing.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Marilyn Farquhar
(University of California, San Diego) for providing antibody to
-mannosidase II; Dr. Rogen Tsien (University of California, San
Diego) for the plasmid encoding the GFP-Golgi protein; Dr. Ron Kopito
(Stanford University) for providing the ts20 cells; and Tom Deerinck
for assistance with the confocal microscope. Part of this work utilized
the facilities of the National Center for Microscopy and Imaging
Research; this facility was supported in part by National Institutes of
Health NCRR P41-04050 (to M. H. E.).
![]() |
FOOTNOTES |
---|
* This work was supported by an American Heart Association Fellowship and a grant from the Cystic Fibrosis Foundation (to S. K.), National Institutes of Health Grant NS11323, a grant from the Smokeless Tobacco Research Council, Inc. (to J. L.), and National Institutes of Health 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.
§ Present address: Dept. of Medicine 0693, University of California, San Diego, La Jolla, CA 92093.
¶ To whom correspondence should be addressed. Tel.: 858-822-3386; E-mail: shkeller@ucsd.edu.
Published, JBC Papers in Press, February 15, 2001, DOI 10.1074/jbc.M100691200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
nAchR, nicotinic
acetylcholine receptors;
ER, endoplasmic reticulum;
PBS, phosphate-buffered saline;
FITC, fluorescein isothiocyanate;
mAb, monoclonal antibody;
GFP, green fluorescent protein;
Endo-H, endo--N-acetylglucosaminidase H.
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