(Received for publication, November 6, 1996, and in revised form, April 24, 1997)
From the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637
Nicotinic acetylcholine receptors (AChRs) are
composed of ,
,
, and
subunits, assembled into
2
pentamers. A highly conserved feature
of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino
acid cystine "loop." We find that an intact cystine loop is
necessary for complete AChR assembly. By preventing formation of the
loop with 5 mM dithiothreitol, AChR subunits assemble into
trimers, but the subsequent steps in assembly are blocked.
When
subunit loop cysteines are mutated to serines, assembly is
blocked at the same step as with dithiothreitol. In contrast, when
subunit loop cysteines are mutated to serines, assembly is blocked at a
later step, i.e. after assembly of
tetramers
and before the addition of the second
subunit. After formation of
the cystine loop, the
subunit undergoes a conformational change,
which buries the loop. This conformational change is concurrent with
the step in assembly blocked by removal of the disulfide bond of the
cystine loop, i.e. after assembly of
trimers and before the addition of the
subunit. The data indicate that the
subunit conformational change involving the cystine loop is key to a
series of folding events that allow the addition of unassembled subunits.
The molecular events involved in subunit folding and assembly of
large oligomeric proteins remain largely uncharacterized. The subunit
folding and oligomerization events that take place during the assembly
of ion channels are particularly complex, since the finished product
requires the correct oligomeric arrangement and subunit stoichiometry
for proper function (1). In terms of their structure, the best
characterized ion channels are the muscle-type nicotinic acetylcholine
receptors (AChRs),1 which are
the neurotransmitter receptors responsible for rapid signaling between
motor neurons and skeletal muscle. Muscle-type AChRs are composed of
four distinct, homologous subunits, ,
,
, and
, which
assemble into pentamers with the subunit stoichiometry of
2
.
Although AChR assembly is a slow process that takes ~2 h to complete
(2), assembly intermediates have been difficult to isolate. A number of
laboratories turned to expression of less than the full complement of
subunits in different heterologous expression systems to isolate
assembly intermediates (3-6). Based on their findings, the
"heterodimer" model was proposed, where the subunit must first
fold or "mature," as assayed by the formation of the
-bungarotoxin (BuTx) binding site and antigenic epitopes, before
assembling with other subunits. The mature
subunit assembles with
or
subunits in parallel to form
and
heterodimers, and the heterodimers associate together and with
subunits to form
2
pentamers.
We have developed techniques that have allowed isolation of assembly
intermediates in cells stably expressing all four AChR subunits (7, 8)
and have obtained results at odds with the heterodimer model. Instead
of heterodimers, two partially assembled complexes, trimers
and
tetramers, were isolated.
trimers, which
assemble extremely rapidly, were assembled first into
tetramers and then into
2
pentamers. Our data
demonstrated that assembly occurs sequentially, each step being the
addition of an uncomplexed subunit. We also demonstrated that the
subunit maturation steps, which were thought to precede its assembly, occurred after assembly into
trimers but prior to the
addition of the
subunit. These folding events require a specific
combination of subunits and correlate in time with the
subunit
addition. The data led us to suggest that the
subunit maturation
steps are folding events forming the
subunit recognition site,
i.e. the site where the
subunit associates with the
trimer.
Our goal has been to identify posttranslational processing sites and
regions on the AChR subunits involved in AChR subunit folding and
assembly. A good candidate is the highly conserved region defined by a
pair of cysteine residues separated by a stretch of 13 amino acids,
which is found on the neurotransmitter-binding, extracellular domain of
the subunits (Fig. 1, A and B). The two cysteines
form a disulfide bridge on all four Torpedo AChR subunits (9, 10). Analogous cystine "loops" appear to form on other neurotransmitter-gated ion channel subunits, which include all muscle
and neuronal AChR subunits and all GABAA, glycine, and 5HT3 receptor subunits. Other residues in the loop are
identically conserved across species from Caenorhabditis
elegans to mammals and are even conserved on some of the glutamate
receptor subunits (11). Site-directed mutations of the cysteines in the
subunit prevented BuTx binding site formation and reduced AChR
expression (12), which suggested that the cystine loop is critical for subunit conformational stability or assembly. A recent study where the
conserved proline in the cystine loop (see Fig.
1, A and B) was
mutated also suggested that the cystine loop may play a role in subunit
assembly (13). However, mutated
or
subunits lacking the cystine
loop disulfide bond assembled with other wild type subunits (14, 15)
and produced functional receptors (15). Based on these data, it was
suggested that formation of the cystine loop is not required for
subunit assembly but instead plays an important role in the rate that
subunits are degraded and in the efficiency of transport of AChRs to
the cell surface. Contrary to this viewpoint, we demonstrate in this
paper that an intact cystine loop is essential for proper AChR subunit
assembly. Elimination of the cystine loop separately on the
and
subunits blocks different steps during assembly. For the
subunit,
the cystine loop undergoes a conformational change, which appears to be
an event required for assembly to continue.
The cystine loop mutations, 128,
142, and
128 (15), where the subscript
indicates which cystine residues were replaced by serine residues, were
a generous gift from Dr. Sumikawa. The cystine loop mutation,
128/142, was created by inserting the
142
BclI fragment back into
128.
128/142 and
128 were subcloned into the
EcoRI site of pSVDF4 (16). The
128/142
and
128
cell
lines were established by stably transfecting each of the two cystine
loop mutations along with the appropriate wild type subunit constructs
plus the thymidine kinase gene (tk) into mouse fibroblast L
cells deficient in tk as described previously (7). To
establish the
128 cell line, mouse NIH3T3 cells were
infected with the retroviral recombinant, pDOJ, which contained the
128 subunit cDNA in the EcoRI site as
well as the neomycin resistance gene. The
and
cell lines, which stably express the indicated Torpedo AChR
subunits, were previously described (7, 8).
The assembly of the Torpedo subunits is
temperature-dependent (7, 17). To allow assembly to occur,
the temperature was dropped from 37 to 20 °C. The transfected
Torpedo subunit cDNAs in the cell line are
under the control of SV40 promoters (7). To enhance expression of
Torpedo AChR subunits, the medium (Dulbecco's modified
Eagle's medium (DMEM; JRH Scientific) plus 10% calf serum (Hyclone)
and HAT (15 µg/ml hypoxanthine, 1 µg/ml aminopterin, and 5 µg/ml
thymidine), was replaced with fresh DMEM supplemented with 20 mM sodium butyrate (NB medium; Baker) 36-48 h prior to the
experiment.
To pulse-label
the subunits, 10-cm cultures were first washed with PBS and starved of
methionine for 15 min in methionine-free DMEM. Cultures were incubated
at 5% CO2 and labeled in 2 ml of methionine-free DMEM,
supplemented with 20 mM sodium butyrate and 333 µCi of
[35S]methionine for 30 min at 37 °C. The labeling was
stopped with the addition of DMEM containing 5 mM
methionine and, if not "chased," two washes of ice-cold PBS. To
follow the subsequent changes in the labeled subunits, the cells were
chased. Specifically, these cells were washed two more times with DMEM
containing 5 mM methionine and incubated for various times
in NB medium at 20 °C in the absence or presence of 5 mM
dithiothreitol (DTT). After a [35S]methionine pulse
chase, cultures were rinsed with ice-cold PBS, scraped, and pelleted by
centrifugation at 5,000 × g for 3 min, and the pellets
were resuspended in lysis buffer: 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02%
NaN3, 2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide plus 1% solubilizing agent.
In some of the experiments, 1 mM MgATP replaced the 5 mM EDTA to reduce the amount of actin that nonspecifically
precipitated with the subunits. To solubilize the labeled subunits, a
Lubrol-phosphatidylcholine mixture composed of 1.83 mg/ml
phosphatidylcholine and 1% Lubrol was used. Antibody-subunit complexes
were precipitated with Protein G-Sepharose and electrophoresed on 7.5%
SDS-polyacrylamide gels, fixed, enhanced for 30 min, dried on a gel
dryer, and exposed to film at 70 °C with an intensifying screen.
Autoradiographs were quantified by scanning densitometry using a
flatbed scanner and analyzed with the Intelligent Quantifier software
from BioImage.
To measure cell surface
125I-BuTx binding, cultures were grown at 37 °C for
36 h in NB medium and then shifted to 20 °C for 48 h.
Cultures were washed with PBS and incubated at room temperature in PBS
containing 4 nM 125I-BuTx (140-170 cpm/fmol)
for 2 h, which results in the saturation of binding. Cultures were
washed again and solubilized, and the cell surface counts were
determined by -counting, or counts were immunoprecipitated with
appropriate antibodies and counted. Total cell 125I-BuTx
binding was measured after solubilization. In this case, cell lysates
were incubated in 10 nM 125I-BuTx at 4 °C
overnight to saturate binding at this lower temperature. The
125I-BuTx in the lysates was then immunoprecipitated with
the indicated antibodies.
To distinguish subunit complexes on the
basis of their size, solubilized subunits (labeled with
[35S]methionine and/or bound by 125I-BuTx)
were sedimented on sucrose gradients. For this procedure, lysates were
layered on a 5-ml 5-20% linear sucrose gradient prepared in the
appropriate lysis buffer. Gradients were centrifuged in a Beckman SW
50.1 rotor at 40,000 rpm to 2t = 9.0 × 1011. ~17 fractions (300 µl) were collected from the
top of the gradient, and the appropriate antibodies were then added to
the fractions to be assayed.
When
added extracellularly to cultured cells, DTT reaches the endoplasmic
reticulum and prevents the formation of protein disulfide bonds without
altering most other cellular functions (18). Cells stably expressing
the four Torpedo AChR subunits were subjected to an
[35S]methionine pulse-chase protocol in the absence or
presence of 5 mM DTT (Fig. 2,
A and B). Labeled subunits were
immunoprecipitated with either a subunit-specific monoclonal
antibody (mAb 168) or
subunit, conformation-dependent
mAb 14, to assay for subunit assembly and formation of the mAb 14 epitope. During a 30-min pulse of [35S]methionine,
trimers formed as shown by the coprecipitation of
predominantly
and
subunits with the
subunits (Fig.
2A). During the chase in the absence of DTT, progressively
more
subunits are added to the trimers, followed by the addition of
the second
subunit as shown by the doubling in the amount of
subunit relative to the other coprecipitated subunits. These two
subunit additions are better resolved by the mAb 14 immunoprecipitations (Fig. 2, A and C). Since the
mAb 14 epitope forms on
trimers just prior to the addition of
the
subunit (8), these immunoprecipitations show the complete time
course of both the addition of the
and second
subunits to the
trimers.
In the presence of DTT, the subunits clearly retain the ability to
assemble into trimers and with approximately the same efficiency as occurs in the absence of DTT (Fig. 2B). This
result is at odds with a recent study (19), which suggested that 5 mM DTT completely blocks AChR subunit assembly. There are
several differences between this and the other study that explain the conflicting results. Probably the most important difference in terms of
the ability to observe
trimers was that their only assay for
measuring the assembly of
subunits with other subunits was an
immunoprecipitation with
subunit-specific antibodies. Obviously,
trimers could never be observed with this assay. Another difference was the protocol used to solubilize the AChR subunit
complexes. We have shown previously that solubilization in 1% Triton
X-100 causes the assembly intermediates to dissociate and that the
subunit associations are stable when solubilized with a combination of
Lubrol PX and phosphatidylcholine (8).
After trimers assemble in the presence of 5 mM
DTT, the ensuing subunit associations, the addition of the
and
second
subunits, fail to occur, and the
trimers that had
assembled were degraded 48 h after the
[35S]methionine pulse (Fig. 2B).
subunits
in the presence of DTT degrade at about the same rate as unassembled
subunits in the absence of DTT (Fig. 2D). Subunit
complexes degrade more rapidly in the presence of DTT because the
complexes are no longer stabilized by events subsequent to trimer
formation. Although there are almost as many
trimers present
with DTT at the times when
subunits normally assemble with
trimers (Fig. 2, A and B; 3- and 6-h chase), the formation of
tetramers and
2
pentamers is inhibited in the presence of
DTT. Furthermore, the BuTx binding site (Figs. 2E and
3A) and the mAb 14 epitope
(Fig. 2B) do not form on the
trimers in the
presence of DTT. We conclude that the addition of DTT blocks subunit
assembly after the association of the
,
, and
subunits and
before the BuTx binding site and mAb 14 epitope form on the
trimers. Since formation of the BuTx binding site and mAb 14 epitope
precede the addition of the
subunit (8), the data suggest that a
block of these folding events by DTT prevents subsequent subunit
associations.
Elimination of Cystine Loop Disulfide Bond on the
The addition of DTT in the above experiments
prevents disulfide bond formation for all AChR subunits as well as for
other proteins that might affect AChR subunit assembly. To test whether the cystine loop on AChR subunits is involved in the DTT block of
assembly, we obtained mutations of the Torpedo AChR and
subunits in which cysteines forming the cystine loop were replaced by serines (15). Of the four AChR subunits, only
subunits contain
an additional cystine formed between adjacent cysteines (cysteines 192 and 193) located at the ACh binding site (20) (Fig. 1B).
Deletion mutations eliminating that cystine cause the loss of ACh
binding but have no effect on BuTx binding, subunit assembly, or cell
surface expression (12, 14). An
subunit construct was created,
128/142, where both cystine loop cysteines were replaced
by serines to avoid the possibility that aberrant disulfides form
between either cysteine 192 or 193 and the remaining cystine loop
cysteine. For the
subunit mutation,
128, the
formation of the cystine loop was eliminated by replacing only cysteine 128 with serine. Each of the two mutated subunits, along with the other
three wild type subunits, was stably transfected into L fibroblasts.
Cell lines were isolated that expressed each of the mutated
and
subunits and the corresponding three wild type subunits (Figs.
4A, lane 1, and
5A, lane 1).
Elimination of the cystine loop disulfide on
the subunit. A, AChR subunit assembly in the
128
cell line.
128
cells were pulse-labeled with [35S]methionine and chased
for the indicated times. Labeled subunits were immunoprecipitated with
a mixture (cocktail) of
,
,
, and
subunit-specific antibodies (lane 1), the
subunit-specific mAb 148 (lanes 2-6), or mAb 14 (lane
7). As in Fig. 2, A and B, the band labeled
as
coprecipitates with the other subunits. The band (~43 kDa)
that migrates between the
and
subunit bands is believed to be
actin. B, the rate of
128 subunit
degradation. Displayed are the scanned values for the
128 subunit bands and the
subunits that
coprecipitated with the
128 subunits in Fig. 5A. The data are plotted on a semilog scale. The half-life
was estimated to be 15.4 h for
128 subunits based
on a least squares fit of an exponential function to the data, which is
within the range of values found for the wild type subunits under the
same conditions. C, sedimentation of
128
subunit complexes.
128
and
cells were bound with
125I-BuTx as in Fig. 3B and size-fractionated on
a 5-20% linear sucrose gradient. Shown are 125I counts
from the 125I-BuTx-bound intracellular complexes in
fractions 4-14, which were immunoprecipitated with the
subunit-specific mAb 148.
cell surface complexes were
first bound with cold BuTx to block 125I-BuTx binding to
the surface AChRs. Also displayed on the figure are the
standards, alkaline phosphatase (5.4 S), catalase (11 S), and surface
2
complexes (9 S; dashed line). The
128
complexes peak at 8 S as estimated from a
least squares linear regression fit to the S values of the standards.
The shape of the intracellular
profile can be duplicated
by the sum of the intracellular
128
profile
reduced by 60% plus the 9 S peak cell-surface
2
complexes. This indicates that intracellular
128
complexes are similar in size and composition to intracellular
complexes with the exception of the
2
complexes
in the 9 S peak. The broad profile observed for the intracellular AChR complexes relative to the cell surface
2
9 S
peak has been seen in other studies both for the Torpedo
subunits at reduced temperature (4, 8, 40) and the mouse subunits at
37 °C (3, 5, 6, 8). D, the effects of the
128/142 and
128 subunits on subunit
assembly are consistent with the model shown. The
128/142 subunit and DTT block assembly after the
formation of trimers but before the formation of the BuTx binding site
and mAb 14 epitope. The
128 subunit blocks assembly
after the addition of the
subunit but before the addition of the
second
subunit.
125I-BuTx binding experiments were performed on the stably
transfected cells to characterize the effect of the mutations on AChR expression (Fig. 3, A and B). No
125I-BuTx binding was detected on the cell surface of
either the 128/142
or the
128
cell lines (Fig. 3A) or for the
intracellular compartments of the
128/142
cells
(Fig. 3B). However, intracellular 125I-BuTx
binding sites were expressed in the
128
cells
(Fig. 3B). The intracellular 125I-BuTx binding
sites were immunoprecipitated using
subunit-specific antibodies,
which demonstrates that these sites in the
128
cells contained the mutated
subunits.
Since the 128/142
cells failed to express any
BuTx sites, we tested whether the mutated
subunit assembled with
other subunits. [35S]methionine pulse-chase experiments
were performed on the
128/142
cell line to
characterize the assembly of the mutated subunit with the three wild
type subunits. Displayed in Fig. 4A are the [35S]methionine-labeled subunits from the
128/142
cells immunoprecipitated with
subunit-specific polyclonal antibodies (lanes 2-6). As shown by the coprecipitation of the wild type
and
subunits with
the
128/142 subunit, these two wild type subunits
assemble with the mutated
subunits in approximately a 1:1 ratio. No
assembly was observed between
subunits and the mutated
subunits. These results indicate that
128/142
trimers assemble, but neither the
nor second
128/142subunits subsequently assemble with the
128/142
trimers. We were unable to precipitate any
[35S]methionine-labeled subunits with conformation
dependent mAb 14 (data not shown); thus, the mAb 14 epitope
does not form on the
128/142
complexes during
assembly. Furthermore, BuTx binding sites failed to form on the
128/142
complexes (Fig. 3, A and B). Subunit assembly in the
128/142
cells is thus blocked at the same step as the block of assembly by DTT,
i.e. after assembly of the
trimers and before the
BuTx binding site and mAb 14 epitope form.
Similar to the trimers assembled in the presence of DTT, the
assembled
128/142
complexes degrade more rapidly
than
complexes in the absence of DTT. The faster rate of
degradation appears to occur because the complexes are no longer
stabilized by events subsequent to trimer formation (Fig.
2D). As shown in Figs. 4C and 5B, the
rate of
128/142 and
128 subunit
degradation is in the range found for the unassembled wild type
subunits (8). Therefore, the failure to complete the assembly process
is not caused by an increased rate of degradation of the mutated
subunits.
In addition to the block of assembly, the efficiency of assembly of the
128/142
trimers was reduced 2-3-fold relative to wild type subunit assembly (see Fig. 2A, lane 2,
and Fig. 6C, lane
1). A similar reduction in assembly efficiency was observed for
subunit complexes containing the
128 subunit (Fig.
5A). The decrease in assembly efficiency could be caused by
misfolding of some of the mutated subunits, which has been observed for
other proteins where cysteines have been mutated (21). To examine whether
128/142 subunits are misfolded, we tested
whether the
128/142 subunits are recognized by mAb 35. mAb 35 is a conformation-dependent antibody specific for
the
subunit (2). mAb 35 differs from mAb 14 in that its epitope
forms on unassembled
subunits, and it forms well before the mAb 14 epitope (8). As shown in Fig. 4B, the results obtained with
the mAb 35 precipitation are similar to the results with
subunit-specific polyclonal antibodies (Fig. 4A, lanes
2-6). mAb 35 recognizes a large percentage of unassembled
128/142 subunits as well as the
128/142
complexes, which indicates that the
unassembled
128/142 subunits are not grossly
misfolded.
Elimination of the Disulfide Bond on the
The finding that the BuTx binding site
forms on complexes that contain the 128 subunit (Fig.
3B) suggests that subunit assembly with the
128 subunit progresses to a later step than assembly with the
128/142 subunit. The assembly of the
128 subunit with the wild type
,
, and
subunits was characterized using an [35S]methionine
pulse-chase protocol with the
128
cells as
shown in Fig. 5A, lanes 1-7. In contrast to
subunit assembly with the
subunit mutation or after DTT treatment,
assembly continued after the formation of
trimers.
Immunoprecipitation of the labeled subunits with
subunit-specific
antibodies (lanes 2-6) coprecipitated the
subunit as
well as the
and
subunits. The mAb 14 epitope forms on the
assembled complexes as shown by precipitation of the
[35S]methionine-labeled subunits by mAb 14 (Fig.
5A, lane 7), and as with the
subunit-specific
antibodies, all four subunits are precipitated. The BuTx binding site
also forms on these complexes as shown by the
125I-BuTx-bound complexes precipitated by
subunit-specific antibodies (Fig. 3B).
The assembly of 128
complexes differs from the
assembly of the four wild type subunits in that the second
subunit
is not added to the
128
complexes. The
pulse-chase experiments demonstrate that the amount of
subunit
coprecipitated with
128 subunits (Fig. 5B) is
constant throughout the pulse-chase protocol, in contrast to the
doubling observed with wild type subunit assembly. To further
investigate the nature of the
128
subunit
complexes formed, their sedimentation on a linear sucrose gradient was
determined (Fig. 5C). The
128
complexes, bound with 125I-BuTx and immunoprecipitated with
subunit-specific antibodies as in Fig. 3B, migrated in a
peak at a value of 8.3 S. The size of the
128
complexes is consistent with tetramers (8), which sediment at ~8 S
and are smaller than the cell-surface
2
complexes, which sediment at 9 S.
The effects of the two subunit mutations and DTT on subunit assembly
are summarized in Table I and are
consistent with the model displayed in Fig. 5D. Based on the
results of both the pulse-chase experiment and the sedimentation of the
mature 128
complexes, the end product of
subunit assembly in the
128
cells is an
128
tetramer. Thus, the
128
subunit, like the
128/142 subunit, causes a block in
subunit assembly. However, the
128 subunit blocks
assembly at a later step, after the formation of
tetramers and before the addition of the second
subunit. The failure of
128
complexes to fully assemble into pentamers
provides an explanation for why
128
complexes
are not transported to the cell surface (Fig. 3A). Since
128
complexes never fully assemble, most likely
they are retained and degraded in the endoplasmic reticulum (22).
|
To address when the subunit cystine loop forms
during assembly, we made use of a mAb (mAb 259) that selectively
recognizes the
subunit only when the cystine loop is intact (23).
This specificity of the mAb is demonstrated in Fig. 6A,
where the mAb failed to recognize either the reduced
subunit or the
mutated
subunit with the cystine loop eliminated. In Fig. 6,
B and C, the
cells were
pulse-labeled with [35S]methionine and chased to test at
what point in assembly the
subunit is recognized by the cystine
loop mAb. Immediately following the half-hour
[35S]methionine pulse, the cystine loop mAb precipitated
about the same amount of labeled
subunit as our
subunit-specific polyclonal antibodies (Fig. 6B, compare
lanes 1 and 3). The
subunit cystine loop,
thus, must form shortly after the subunit is synthesized. Since the
events blocked by DTT and by the
subunit mutation occur several
hours after the synthesis of the
subunit, the block occurs after
the formation of the cystine loop.
The ability of the cystine loop mAb to precipitate subunits
diminished with time. The loss of the epitope occurs during subunit
assembly as shown by the difference in the subunits precipitated by the
cystine loop mAb compared with the subunits precipitated with the
subunit-specific polyclonal antibodies (Fig. 6, B, lanes 3-7, and C, lanes 1-5). During
the chase, the amount of
subunit precipitated by the cystine loop
mAb is progressively reduced. By the last chase time, the cystine loop
mAb precipitated very little
subunit (Fig. 6B,
lane 7) and also was unable to precipitate any significant
amount of cell surface AChRs (Fig. 7A). The mAb epitope, although
inaccessible to the mAb, is still present on the
subunit as
demonstrated by the cystine loop mAb precipitating as much
subunit
as the
subunit-specific antibodies after the subunits were
denatured with SDS (Fig. 6, B and C, compare lanes 8 and 5). The loss of the epitope thus
results from a conformational change that buries the epitope.
The Conformational Change Occurs before Formation of the BuTx Binding Site and the Addition of the
The only AChR
subunit complexes that appear to be recognized by the cystine loop mAb
are trimers. This is most clearly observed at the times when
the
subunit is maximally assembled with the other subunits,
i.e. 24-48 h after assembly begins (Fig. 2, A
and C). At these times, only
and
subunits
coprecipitate with the
subunits recognized by the cystine loop mAb
(Fig. 6B, lanes 6 and 7), although
complexes containing
as well as the other three subunits are
present when all
subunits are precipitated (Fig. 6C,
lanes 4 and 5) or when the subunits are
precipitated by the
-specific mAb or mAb 14 (Fig. 2A). At
earlier times in the pulse-chase experiments of Fig. 6, B
and C, the
band, which is specifically recognized by
the cystine loop mAb and
subunit-specific antibodies, may be
obscuring the presence of any
subunit. The DTT block of assembly
was used to address whether
subunits are present at the earlier
times during assembly. As shown in Fig. 2B, the addition of
5 mM DTT blocks assembly so that only
trimers
assemble. In Fig. 6D, subunit complexes were precipitated with the
-specific antibodies after cells were treated with 5 mM DTT. There are no significant differences between the
results in Fig. 6B (lanes 4-7) and those of Fig.
6D. From this we conclude that the complexes recognized by
the cystine loop mAb are identical to the
trimers
precipitated by the
-specific antibodies in Fig. 6D.
To further characterize the subunit complexes recognized by the cystine
loop mAb, we tested whether the BuTx binding site and the mAb 14 epitope form on these complexes. The cystine loop mAb precipitated only
about 12% of the intracellular 125I-BuTx binding sites
precipitated by either mAb 14 or the subunit-specific mAb alone
(Fig. 7B). To ascertain whether these 125I-BuTx
binding sites are on complexes containing
subunits and the mAb 14 epitope, 125I-BuTx binding sites were first precipitated
with either mAb 14 or the
subunit-specific mAb before using the
cystine loop mAb. The number of sites recognized by the cystine loop
mAb was not significantly reduced if samples were first depleted of
complexes containing
subunits or the mAb 14 epitope (Fig.
7B). Therefore, the
subunit-containing complexes
recognized by the cystine loop mAb do not contain the BuTx binding site
or the mAb 14 epitope. Since formation of the BuTx binding site and the
mAb 14 epitope precedes assembly of
tetramers (8), the
complexes recognized by the cystine loop mAb appear to be
trimers. The data further indicate that the cystine loop conformational
change occurs prior to the formation of the BuTx binding site and the
mAb 14 epitope.
The point during subunit assembly when the cystine loop conformational
change takes place is evident in the pulse-chase experiment of Fig.
6B. The loss of the cystine loop epitope during assembly, shown by decreasing amounts of ,
, and
subunits that are
precipitated by the cystine loop mAb, begins at the 3-h time point and
is almost completed by the 24-h time point (Fig. 6B,
lanes 4-6). This is the time period during which the BuTx
site and mAb 14 epitope form and are quickly followed by the addition
of
subunits to the trimers (Fig. 2, A and C;
see also Ref. 8). The similarity in the kinetics of the cystine loop
conformational change and these assembly events together with the block
of these events by
128/142 and DTT demonstrate a strong
correlation between the
subunit cystine loop conformational change
and the assembly events. As summarized in Table I, there is a close
identity between the subunit complexes precipitated by the cystine loop
mAb and those assembled in the presence of DTT and with the
subunit mutation. Altogether the data indicate that after the
subunit cystine loop forms, it undergoes a conformational change, which is
essential for the BuTx site and mAb 14 epitope to form and for the
subunit to associate with the
trimer.
The strong conservation of the cystine loop among the different
ionotropic neurotransmitter receptors and throughout evolution suggests
that this structure plays a vital role with respect to the receptors.
In this paper, we demonstrate that the cystine loop is essential for
different events that take place during subunit assembly. Events
occurring during the assembly of the AChR can be broadly classified as
either subunit folding or oligomerization. Previously, we observed that
AChR subunits continued to fold after associations with other subunits
(8). The data suggested a link between subunit folding and subsequent
subunit associations. We proposed that the folding events, BuTx binding
site and mAb 14 epitope formation, are part of the process to create a
recognition site for the incoming subunit. In support of this
hypothesis, we find that eliminating the
subunit cystine loop
blocks these folding events and also blocks the subsequent addition of
the
subunit to the
trimer. As shown in the model in Fig.
8, we suggest that a subunit recognition
site for the
subunit is created concurrently with the
subunit
cystine loop conformational change. Because elimination of the
subunit cystine loop blocks the addition of the second
subunit to
the
tetramer, we further propose that the recognition site
for the second
subunit is created in parallel with a change in the
conformation of the
subunit cystine loop.
Formation of the different subunit recognition sites is likely to
involve large rearrangements of the assembly intermediates. Such a
rearrangement occurs on the subunit. Three folding events, 1) the
cystine loop conformational change, 2) the formation of the BuTx
binding site, and 3) the formation of the mAb 14 epitope, occur at
about the same time and precede the addition of
subunits to
trimers. The three events occur on three separate regions that span the length of the N-terminal domain of the
subunit. The
mAb 14 epitope overlaps or is very near to the main immunogenic region
(24) at amino acids 67-76 (25, 26), while the BuTx binding site is at
the other end of this domain in the region of amino acids 185-196 (27,
28). Regions of the
subunits are also involved in these events,
since the presence of this subunit is necessary for the mAb 14 epitope
to form and greatly enhances formation of the BuTx binding site (8).
Since the
subunit cystine loop conformational change is part of a
large rearrangement of the
subunit, it is possible that the cystine loop itself does not directly associate with the
subunit during the
assembly of the
tetramer. Instead, other regions, involved in subsequent folding events and distant from the cystine loop, could
associate with the
subunit.
A feature of our model (Fig. 8) is that AChR subunit assembly is
controlled by the ordered formation of subunit recognition sites. These
events are rate-limiting, as shown by the kinetics of the and
second
subunit additions, and as such provide checkpoints during
assembly where the fidelity of the assembly process can be tested. Each
subunit recognition site would form only if assembly had proceeded
properly up to that point. Any misfolded or misassembled intermediates
would be prevented from participating in later stages of assembly, and
these improperly assembled subunits would be selectively retained and
targeted for degradation by the endoplasmic reticulum "quality
control" mechanisms (29). This paradigm, where the formation of
subunit recognition sites is rate-limiting and contributes to quality
control mechanisms during oligomer assembly, is likely to be found in
the assembly of other ionotropic neurotransmitter receptors and ion
channels (1) and may also apply in the assembly of other complex
oligomeric proteins. Key to the formation of subunit recognition sites
appears to be the cystine loop conformational change. As shown in Fig.
8, we envision the role of the cystine loop as a switch in subunit
assembly. In the "up position," the cystine loop blocks subunit
recognition site formation and assembly. In the "down position,"
subunit recognition site formation and assembly continue. In such a
role, the cystine loop conformational change is an essential part of
the assembly process, which allows the subunits themselves to guide
proper subunit folding and maintains the correctly ordered pathway by which subunits oligomerize.
We are most grateful to Dr. Katumi Sumikawa
for Torpedo and
subunit mutations
128,
142, and
128; to Drs.
S. Tzartos, J. Lindstrom, and T. Claudio for antibodies, in particular
Dr. J. Lindstrom for mAb 259; to Dr. T. Claudio for the
Torpedo
,
,
, and
subunit cDNAs, the
cell line, and pDOJ vector; and to Dr. D. Fambrough for
the vector pSVDF4. mAb 35 developed by J. Lindstrom was obtained from
the Developmental Studies Bank (Johns Hopkins University and University
of Iowa). We also thank Alison Eertmoed, Arjun Natesan, Justin Chen,
and Suleiman Salman for help with some of the subunit constructs and
Dr. A. Fluet for discussions at the early stages of this work.