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Next Article 
The Journal of Neuroscience, August 1, 1998, 18(15):5555-5564
Formation of the Nicotinic Acetylcholine Receptor Binding
Sites
William N.
Green and
Christian P.
Wanamaker
Department of Pharmacological and Physiological Sciences,
University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Nicotinic acetylcholine receptors (AChRs) are activated by
ACh binding to two sites located on different  subunits. The two subunits,  and  , are distinguished
by their interface with  and  subunits. We have characterized the
formation of the ACh binding sites and found, contrary to the current
model, that the sites form at different times and in a set order. The first site forms on subunits during the process of
subunit assembly. Our data are consistent with the appearance of this site on  subunit tetramers soon after the site for the
competitive antagonist -bungarotoxin has formed and subunits
have assembled with trimers. The second site is located on
subunits and forms after AChR subunits have
assembled into 2 pentamers. By determining the
order in which the ACh binding sites form, we have also identified the
sites in which the and second subunits associate during subunit
assembly.
Key words:
protein folding and assembly; acetylcholine; -bungarotoxin; nicotinic receptors; pharmacology; ligand binding
sites
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INTRODUCTION |
The binding of different ligands to
a diverse set of membrane receptors is the primary mechanism by which
information is transferred across cell membranes. For many different
membrane receptors, much progress has been made in determining the
structural details of ligand binding to its receptor. However, almost
nothing is known about the protein folding and assembly events that
give rise to these binding sites. Using the muscle-type nicotinic
acetylcholine receptor (AChR), we have examined how the ACh binding
sites form on this class of receptors. These receptors are composed of
four distinct yet homologous subunits, , , , and , which
assemble into AChR 2 pentamers. The muscle-type
AChR is the best characterized member of a family of
neurotransmitter-gated ion channels that includes neuronal AChRs,
5-HT3 receptors, glycine receptors, and GABAA
receptors. As such, the muscle-type AChR has long served as a model
system for the other receptors in the family (for review, see Unwin,
1993 ; Changeux, 1995; Karlin and Akabas, 1995; Lindstrom, 1995).
Several small ligands bind to AChRs. These ligands include agonists
such as nicotine and the neurotransmitter ACh and small competitive
antagonists such as curare. Amino acid residues located within three
different regions of the N-terminal, extracellular domain of the subunits have been identified as residues at the ACh binding sites.
Other experiments have identified additional amino acids on the and
subunits that contribute to the binding sites (for review, see
Galzi and Changeux, 1994; Karlin and Akabas, 1995; Tsigelny et al.,
1997). Furthermore, for the ACh binding site to form, disulfide bonding
and N-linked glycosylation within this N-terminal subunit domain
appear to be necessary (Mishina et al., 1985). The formation of the ACh
sites, thus, requires the coordination of several post-translational
events that draw together different regions of the subunit
N-terminal domain, and assemble the subunits with the other
subunits.
Processing, folding, and assembly of AChR subunits are slow events,
taking ~2 hr to complete (Merlie and Lindstrom, 1983). Despite the
slow kinetics of these events, intermediates with ACh binding sites
have been difficult to isolate. ACh binding sites form when subunits are expressed heterologously with either the or subunits in the absence of the other two subunits (Blount and Merlie,
1989). Based on these findings, it was proposed that the two ACh
binding sites form on either or heterodimers at the same
time during the early stages of subunit assembly (Blount et al., 1990;
Gu et al., 1991; Saedi et al., 1991). In this paper, we have
characterized the formation of the ACh binding sites using cells
expressing all four AChR subunits and present data at odds with the
current model describing ACh binding site formation. We find that the
first ACh binding site forms on subunits during
subunit assembly and that the second site, located on the subunit, forms after AChR subunits have assembled
into 2 pentamers. The data provide evidence for
the subunit arrangement of assembly intermediates and for the location
of the subunit interfaces in which the unassembled and second subunits assemble.
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MATERIALS AND METHODS |
Metabolic labeling and subunit precipitations. Mouse
L fibroblasts, stably transfected with the Torpedo subunit
cDNAs under the control of SV40 promoters (Claudio et al., 1987), were
maintained in DMEM plus 10% calf serum and HAT (15 µg/ml
hypoxanthine, 1 µg/ml aminopterin, and 5 µg/ml thymidine) in 5%
CO2. To enhance subunit expression, the DMEM was
supplemented with 20 mM sodium butyrate (NB medium)
36 hr before the experiment. Cultures in 10 cm plates were labeled as
described previously (Green and Claudio, 1993; Green and Wanamaker,
1997). Briefly, cultures were pulse-labeled in 2 ml of methionine- and
cysteine-free NB medium, supplemented with 333 µCi of a
35S-methionine 35S-cysteine mixture (NEN
EXPE35S35S). The labeling was
stopped with the addition of DMEM plus 5 mM methionine and
cysteine. To follow the subsequent changes in the labeled subunits, the
cells were "chased" by incubation for the indicated times in NB
medium at 20°C. The cells were solubilized in (in
mM): 150 NaCl, 5 EDTA, 50 Tris, pH 7.4, 2 PMSF, and
2 N-ethylmaleimide, plus 0.02% NaN3,
1.83 mg/ml phosphatidylcholine, and 1% Lubrol. Solubilized AChR
subunits were purified on an acetylcholine affinity column,
precipitated with a slurry of bungarotoxin (Bgt)-Sepharose and/or immunoprecipitated with anti-Bgt polyclonal antiserum or the
conformation-dependent monoclonal antibodies (mAbs) 14 (Tzartos and
Lindstrom, 1980), 35 (American Type Culture Collection, Manassas, VA)
or 247g (Mihovilovic and Richman, 1987). For the labeled AChR complexes
immunoprecipitated with anti-Bgt polyclonal antiserum (see Figs.
3A, 4B), the complexes were first bound
with Bgt. In Figure 3A, 10 nM cold Bgt was added
to each of the gradient fractions and maintained at 4°C rotated
overnight before the immunoprecipitation. In Figure
4A,B 10 nM 125I-Bgt was added
to the chase medium for the length of the chase time, after
which the cells were solubilized. Bromo-ACh affinity resin was prepared
by a scaled-down version of a previously described protocol (Chak and
Karlin, 1992), and Bgt-Sepharose was prepared by coupling Bgt to
cyanogen bromide-activated Sepharose according to the manufacturer's
directions (Pharmacia, Piscataway, NJ).
The bromo-ACh affinity column does not quantitatively bind all ACh
binding sites. Compared with Bgt-Sepharose precipitation or
immunoprecipitating Bgt-bound complexes with anti-Bgt antibodies, the
bromo-ACh affinity protocol isolated 30-40% of the total subunit complexes (compared with Bgt-Sepharose), which is not surprising given
the relatively weak affinity of ACh to its binding site. In contrast,
Bgt-Sepharose precipitation, immunoprecipitation of Bgt-bound complexes
with anti-Bgt antibodies, and immunoprecipitation with mAb 247g all
appear to precipitate most available complexes.
Antibody-subunit complexes were precipitated with protein G-Sepharose
and electrophoresed on 7.5% SDS polyacrylamide gels, fixed, treated 30 min with Amplify (Amersham, Arlington Heights, IL) to enhance the
signal, 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. A standard, provided by Bioimage,
with 21 bands ranging from 0.05 to 3.05 optical density units in 0.15 increments was used to calibrate the linearity of the densitometer
before using it. To ensure that quantified bands were in the linear
range and the darker signals were not saturated, three to five
exposures were taken of each autoradiograph, and each was scanned to
ensure that the scanned bands remained in the linear range of the
film.
125I--Bgt binding and inhibition by
curare. To measure cell surface 125I-Bgt binding,
cells grown at 20°C for 48 hr were incubated at room temperature in
PBS containing 4 nM 125I-Bgt (140-170
cpm/fmol) for 2 hr with or without mAb 247g. Cultures were washed and
scraped off the plates, and the cell surface counts were determined by
gamma counting.
To measure the inhibition of intracellular 125I-Bgt binding
by d-tubocurarine (dTC), 125I-Bgt binding was
measured after solubilization of the cells as described previously
(Wang and Claudio, 1993). Cell surface Bgt binding sites were blocked
with cold Bgt, after which the cells were solubilized and AChR subunit
complexes were immunoprecipitated with the indicated antibody.
125I-Bgt binding was assayed with the AChR subunit
complexes bound to antibody and protein G-Sepharose. The protein
G-Sepharose pellets were incubated in the indicated dTC concentration
for 15 min before the addition of 125I-Bgt. Ten nanomolar
125I-Bgt was added to the pellets and incubated at room
temperature for 15 min to measure the initial rate of Bgt binding. The
pellets were washed, and the counts were determined by gamma counting. The line through the data represents a least squares fit to the equation: fraction of maximum 125I-Bgt bound = A(1/(1 + ([dTC]/IC501)) + B(1/(1 + ([dTC]/IC502)), where IC501 and
IC502 are IC50 values for two separate binding sites, and A and B are the
fractions of total sites for each of the two sites.
Sucrose gradients. Cell lysates were layered on a 5 ml
5-20% linear sucrose gradient prepared in the lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm for 14.25 hr
( 2t = 9.0 × 1011). Three
hundred-microliter fractions were collected from the top of the
gradient. The linearity of the gradient was confirmed by measuring the
osmolality of each fraction of some of the gradients.
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RESULTS |
Formation of ACh binding sites
In this study, we have characterized the formation of the ACh
binding sites using cells stably expressing all four Torpedo AChR subunits (Claudio et al., 1987). Torpedo AChR subunit
folding and assembly is temperature-sensitive, which allows us to
isolate and characterize assembly intermediates because the kinetics of AChR subunit assembly are slowed by more than an order of magnitude at
20°C (Green and Claudio, 1993; Green and Wanamaker, 1997). AChR
subunits, pulse-labeled with 35S-methionine and -cysteine,
were first purified on a bromo-ACh affinity column and then
immunoprecipitated with subunit-specific mAb 35 (Fig.
1A,B) or with and
subunit-specific mAb 88b (Fig. 1B). No labeled
subunits were isolated immediately after the pulse label using this
bromo-ACh affinity protocol in Figure 1A,B, even though both unassembled and assembled subunits were present in the
cell lysate. The presence of labeled AChR subunits immediately after
the pulse label was shown by the mAb 35 precipitation of labeled
subunits (Fig. 1C). The ACh binding sites isolated by the
bromo-ACh affinity protocol appear a significant amount of time after
the synthesis of the subunits (Fig. 1A,B, at 3, 6, and 12 hr). The time course of the formation of the ACh binding sites
is shown quantitatively from the scanning densitometry of this and
other experiments (Fig. 1B). Initially, the subunits isolated by the bromo-ACh affinity protocol had a ~1:1:1:1 ratio of
::: subunits (Fig. 1B, at 3, 6, and 12 hr). If ACh binding sites existed on unassembled subunits, we would
have expected a larger ratio of to the other subunits. Also, we
would expect differences in the results, which were not observed, when
the and subunit-specific mAb was used instead of the subunit-specific mAb 35 after the bromo-ACh affinity purification (Fig.
1B). In agreement with previous studies (Blount and
Merlie, 1988; Paulson et al., 1991), we conclude, therefore, that no
ACh binding sites appear on unassembled subunits, and ACh binding
sites form on complexes containing and other AChR subunits.
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Figure 1.
Formation of the ACh binding sites.
A, Purification of labeled AChR subunits with bromo-ACh
affinity gel. Displayed is an autoradiogram of the labeled subunits
analyzed on a 7.5% SDS-PAGE gel. Mouse L fibroblasts, stably
expressing all four Torpedo AChR subunits (Claudio et
al., 1987), were pulse-labeled with a
35S-methionine-cysteine mixture for 30 min at 37°C and
chased for the indicated times at 20°C. Cell lysates were passed
through a bromo-ACh affinity column two times; the subunits were eluted
from the column and immunoprecipitated with the subunit-specific
mAb 35. Similar results were obtained when the bromo-ACh
affinity-purified subunits were immunoprecipitated with the and subunit-specific mAb 88b instead of mAb 35 (Fig.
1B). B, Scanning densitometry of
the bromo-ACh affinity-purified AChR subunits. The time course of the
formation of the ACh binding site is shown quantitatively from the
scanned values of the experiment shown in A and other
experiments in which mAb 35 or mAb 88b were used to precipitate the
subunits eluted from the bromo-ACh affinity column. No differences in
the results were observed when mAb 88b was substituted for mAb 35. All
scanned values are displayed as the fraction of the subunit value
at 48 hr. The 0, 6, 24, and 48 hr time points represent the mean ± SD for five separate experiments. The 3 hr time point represents the
mean ± SD for three separate experiments, and the 12 and 72 hr
time points represent the mean for two separate experiments. At the 6 hr time point, the values for the subunits were , 0.30 ± 0.07;
, 0.30 ± 0.04; , 0.27 ± 0.05; and , 0.32 ± 0.10, yielding a ::: subunit ratio of 1.0:1:0.9:1.1. Taking
into account differences in subunit cysteine and methionine content and
the ratio of 35S-methionine and 35S-cysteine
during labeling of the subunits, the subunit ratio should be
1.3:1.0:0.8:1.0 for a subunit stoichiometry of 1:1:1:1. At the 48 hr
time point, the values were , 2.1 ± 0.3; , 1.0; ,
0.80 ± 0.13; and , 0.94 ± 0.09, and the ratio of the subunit to the other subunits doubled from the 6 to the 48 hr time
point. C, Immunoprecipitation of labeled subunits with
subunit-specific mAb 35. Cells were treated as in A
except that immunoprecipitation using mAb 35, which recognizes both
unassembled and assembled subunits (Green and Claudio, 1993),
replaced the bromo-ACh affinity purification step. The band labeled
' has previously been shown to be different from the subunit and
related to the subunit, although it migrates just above the subunit (Green and Claudio, 1993; Green and Wanamaker, 1997).
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An important feature of the time course of ACh binding site formation
was that, 12 hr after subunit labeling, the number of subunits in
the isolated complexes continued to increase and eventually more than
doubled whereas the number of , , and subunits remained
constant (Fig. 1A,B). We have found previously that
the ratio of subunit doubles relative to the other subunits as a
second subunit assembles with the other subunits to form 2 pentamers (Green and Claudio, 1993). The
doubling of subunits relative to the other subunits, thus,
indicates that the AChR subunit complexes isolated by the bromo-ACh
affinity protocol initially contain a single subunit. Based on the
time course shown in Figure 1B, subunit complexes
with ACh binding sites isolated 3-12 hr after subunit labeling have a
single subunit, and the second subunit is added 12-48 hr after
subunit labeling. Because subunits can have at most a single ACh
binding site, one of the two ACh binding sites is present before the
addition of the second subunit. Furthermore, because ACh binding
sites do not form on unassembled subunits, the second ACh binding
site must appear after the addition of the second .
Formation of Bgt binding sites
Previous studies suggested that the formation of the binding sites
for Bgt, a high-affinity competitive antagonist of ACh, precedes the
formation of the ACh binding sites (Merlie and Lindstrom, 1983; Blount
and Merlie, 1988; Paulson et al., 1991). To test when the Bgt binding
sites form relative to the ACh binding sites, labeled AChR subunits
were isolated using Bgt-Sepharose (Fig. 2A), and the results of
this and other experiments were quantified by densitometry (Fig.
2B). Many features of Bgt binding site formation are
similar to ACh binding site formation. As with the bromo-ACh affinity
purification, no labeled AChR subunits were precipitated by
Bgt-Sepharose immediately after the subunit label. The AChR subunits
initially precipitated by Bgt-Sepharose had a ~1:1:1:1 ratio of
::: subunits (Fig. 2B, at 3, 6, and 12 hr), and the ratio of to the other subunits doubled by the 48 hr
time point.
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Figure 2.
Formation of the Bgt binding sites.
A, Purification of labeled AChR subunits with
Bgt-Sepharose. Cells were treated as in Figure
1A, except that precipitation using Bgt-Sepharose
replaced the bromo-ACh affinity purification steps. B,
Scanning densitometry of AChR subunits precipitated by Bgt-Sepharose.
The time course of the formation of Bgt binding sites is shown
quantitatively from the scanned values of the experiment shown in
A and other experiments. All scanned values are
displayed as the fraction of the subunit value at 48 hr. The 0, 6, 24, and 48 hr time points represent the mean ± SD for six
separate experiments. The 3 hr time point represents the mean ± SD for five separate experiments, and the 12 and 72 hr time points
represent the mean for two separate experiments. At the 6 hr time
point, the values for subunits were , 0.71 ± 0.28; ,
0.57 ± 0.31; , 0.50 ± 0.18; and , 0.62 ± 0.20, yielding an ::: subunit stoichiometry of 1.2:1:0.87:1.1. At
the 48 hr time point, the values were , 2.2 ± 0.3; , 1.0;
, 1.0 ± 0.07; and , 1.3 ± 0.5. As with the bromo-ACh
affinity purification, the ratio of the subunit to the other
subunits doubled from the 6 to the 48 hr time point. The reason that
the ratio of the subunit to the other subunits is somewhat larger
than expected is attributable to the occasional presence of a
contaminating band, ', related to the subunit that migrates on
gels close to the subunit (Fig. 1C) and will merge
with the subunit.
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Although the time course of Bgt binding site formation was similar to
that of ACh binding site formation, there were differences at the 6 and
24 hr time points. During the formation of both binding sites, the 12 hr time point is the point at which the number of , , and subunits saturated and after which the number of subunits doubled.
As discussed above, these data indicate that the formation of the first
ACh and Bgt binding sites occurs during the first 12 hr on complexes
containing a single subunit, whereas the second binding sites form
in the period between the 12 and 48 hr time points after the addition
of a second subunit. For the Bgt-Sepharose precipitation at the 6 hr time point, the scanned values (mean ± SD for six gels) were
0.71 ± 0.28, 0.57 ± 0.31, 0.50 ± 0.18, and 0.62 ± 0.20 for the , , , and subunits, respectively. For
bromo-ACh affinity purification at the 6 hr time point, the normalized
scanned values (mean ± SD for five gels) were half that of the
Bgt-Sepharose precipitation: 0.30 ± 0.07, 0.30 ± 0.04, 0.27 ± 0.05, and 0.32 ± 0.10 for the , , , and subunits, respectively. These data suggest that formation of the first
ACh binding site lags behind formation of the first Bgt binding site.
Furthermore, at the 24 hr time point the scanned value of the
Bgt-Sepharose subunit was 2.1 ± 0.7 compared with 1.6 ± 0.48 for the bromo-ACh affinity-purified subunit. Again, the ACh
binding site, in this case the second site, appears to form after the
second Bgt binding site.
The first ACh site forms on tetramers after the Bgt
binding site forms
In an earlier study, we presented data that Bgt binding sites and
the conformation-dependent epitope to mAb 14 first appear on
trimers just before the addition of subunits to the trimers to
assemble tetramers (Green and Claudio, 1993). If ACh
binding sites form after Bgt binding sites, it raises the possibility that the first ACh binding site forms on tetramers. To
characterize in more detail the subunit complexes on which the first
ACh and Bgt binding sites form, cells were solubilized 6 hr after the pulse label, and labeled subunits were size-fractionated on sucrose gradients before isolation (Fig. 3).
After the sucrose gradient, subunit complexes were isolated by three
different methods. In Figure 3A, complexes were bound by Bgt
and isolated by anti-Bgt antibodies. In Figure 3B, complexes
were isolated by bromo-ACh affinity purification. In Figure
3C, complexes were isolated by mAb 14 immunoprecipitation.
Bgt binding sites as well as the conformation-dependent mAb 14 epitope
are evident on complexes that migrate at 6-7 S and that contain
predominantly , , and subunits (Fig. 3A,C, boxed
fractions), consistent with the presence of these sites on
trimers. The trimers are absent when the labeled
subunits are isolated using bromo-ACh affinity purification (Fig.
3B).
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Figure 3.
The first ACh site forms just after the Bgt
binding site forms on tetramers. A-C,
Sedimentation of AChR subunit complexes containing Bgt binding sites
(A), ACh binding sites (B),
or the mAb 14 epitope (C). Cells were
pulse-labeled with 35S-methionine-cysteine for 30 min at
37°C and cultured for 6 hr at 20°C. Solubilized subunits were
fractionated on a 5-20% linear sucrose gradient. AChR subunits in
gradient fractions 4-15 were precipitated by three different methods.
In A, Bgt-bound subunit complexes were precipitated
using anti-Bgt antibodies. In B, subunit complexes were
bromo-ACh affinity-purified as in Figure 1A. In
C, subunit complexes were immunoprecipitated with
conformation-dependent mAb 14. The arrows above
A mark the peak fractions of each of three standards:
alkaline phosphatase (5.4S), cell surface Torpedo AChRs
(9S, as in Fig. 4C), and catalase (11S), which were run
on parallel gradients. Consistent with the results in Figures
1A and 2A, the majority of
subunit complexes precipitated 6 hr after the
35S-methionine-cysteine label are tetramers.
This conclusion is based on the ~1:1:1:1 stoichiometry of
::: subunits and the fact that the peak fractions of all
three gradients occur at fractions 9 and 10 or at ~8S, which is where
tetramers would be expected to sediment (Green and
Claudio, 1993). A shoulder to this peak is observed at fractions 6-8
on the Bgt binding site and mAb 14 epitope gradients (highlighted by
the boxes) but not on the ACh binding site gradient.
Complexes containing predominantly , , and subunits are
observed in this shoulder, which migrate at 6-7S and appear to be
trimers. Although many of the trimers and tetramers migrate
where expected, large numbers of both complexes extend farther into the
gradient than expected. We do not fully understand the broad size
distribution displayed by trimer and tetramers. However, this property
has been observed previously for the intracellular AChR complexes, both
for the Torpedo subunits at reduced temperature (Ross et
al., 1991; Saedi et al., 1991; Green and Wanamaker, 1997) and for the
mouse subunits at 37° (Blount et al., 1990; Gu et al., 1991; Green
and Claudio, 1993; Kreienkamp et al., 1995).
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The properties of the bromo-ACh affinity-purified subunit complexes in
Figure 3B are consistent with tetramers. These complexes contain all four subunits with a ~1:1:1:1 stoichiometry among the subunits, and the peak fractions for these complexes, fractions 9 and 10, migrate at ~8 S, between where trimers at 6-7S
and surface 2 pentamers at 9S migrate on the
gradient. The properties of the Bgt-Sepharose-precipitated subunit
complexes in Figure 3A are consistent with a combination of
trimers and tetramers. In the 6-7S region,
there are predominantly trimers. At the 8-9S region, the
complexes have a subunit stoichiometry of ~1:1:1:0.6 for
:::, attributed to the presence of both trimers and
tetramers. In contrast, in the 10-11S region, the subunit
stoichiometry is ~1:1:1:1, consistent with only tetramers. The
results with the gradients indicate that the first ACh binding site,
unlike the Bgt binding site, does not form on trimers. Instead, the first ACh binding site forms on tetramers
after the formation of the first Bgt binding site and addition of the subunit to trimers.
The second ligand binding sites form on 2
pentamers that are transported to the surface
Up to 12 hr after the pulse label, both the Bgt and ACh binding
sites exist on subunit complexes with a ~1:1:1:1 stoichiometry of
::: subunits. In the later stages of ligand binding site formation, the AChR complexes purified by Bgt-Sepharose and bromo-ACh affinity resin undergo the same change in which the amount of subunit doubles (Figs. 1B, 2B). If
the doubling of the subunit is actually a measure of the addition
of a second subunit during assembly of 2
pentamers, then the second ACh and Bgt binding sites must form after
the assembly of 2 pentamers. To confirm that
the doubling of the subunit in Figure 2B
corresponds to the assembly of 2 pentamers,
subunit complexes isolated using Bgt-Sepharose were size-fractionated
on sucrose gradients. In Figure
4A, cells were
solubilized 48 hr after the pulse label, and cell surface AChRs were
selectively removed from the cell lysate to obtain intracellular
complexes (see Materials and Methods for details). The distribution of
subunit complexes on the gradients has clearly changed during the
period between 6 hr (Fig. 3A) and 48 hr (Fig.
4A). By 48 hr, the trimers observed at 6 hr
in the 6-7S region have disappeared, and the majority of the complexes migrate in the 9S region of the gradient, as shown by the scanned values displayed in Figure 4D. Cell surface receptors
in these cells consist of a single population of
2 pentamers (Hartman et al., 1990), and the
surface 125I-Bgt-bound AChRs, which were size-fractionated
on parallel gradient, migrate precisely at 9S (Fig. 4C). In
addition, the intracellular complexes at 48 hr in the 9S region have a
subunit ratio of ~2:1:1:1 for ::: (see Fig.
4D legend for details), which differs from that at 6 hr in which the subunit ratios were consistent with trimers
and tetramers. These findings are all consistent with
predominantly 2 pentamers precipitated by
Bgt-Sepharose at 48 hr.
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Figure 4.
The second Bgt binding site forms on 9S
complexes that are transported to the cell surface. A,
Metabolically labeled, intracellular AChRs. Cells were pulse-labeled
with 35S-methionine-cysteine for 30 min at 37°C and
cultured for 48 hr at 20°C. During the chase, the cells were
surface-bound with 125I-Bgt and solubilized. The number of
cell surface 125I-Bgt sites was then determined, after
which the surface AChRs were immunoprecipitated with anti-Bgt
antibodies to separate surface and intracellular AChR complexes. After
the immunoprecipitation, the number of precipitated
125I-Bgt sites was again determined as well as the number
of sites remaining in the cell lysate after the immunoprecipitation.
Both were consistent with precipitation of 94% of the surface counts.
Note also that any Bgt-bound AChRs that escaped precipitation, at most
6%, would not be precipitated by the Bgt-Sepharose used to precipitate
the intracellular sites in Figure 4A. The
intracellular AChR complexes that remained in the supernatant after the
immunoprecipitation were fractionated on a 5-20% linear sucrose
gradient and precipitated using Bgt-Sepharose. The
arrows mark the peak fractions of each of three
standards: alkaline phosphatase (5.4S), cell surface
Torpedo AChRs (9S, Fig. 4C), and catalase
(11S), which were run on parallel gradients. B,
Metabolically labeled, surface AChRs. Cells were pulse-labeled with
35S-methionine-cysteine for 30 min at 37°C, cultured for
48 hr at 20°C, and then cultured for 2 hr at 37°C to increase the
transport of AChRs to the cell surface (Ross et al., 1991). During the
chase, the cells were surface-bound with 125I-Bgt and
solubilized. Solubilized subunits were fractionated on a 5-20% linear
sucrose gradient, and the surface, Bgt-bound AChRs were
immunoprecipitated with anti-Bgt antibodies. C,
125I-Bgt-bound, surface AChRs. After 48 hr in culture at
20°C, the cells were surface-bound with 125I-Bgt and
solubilized. Solubilized Bgt-bound AChRs were fractionated on a 5-20%
linear sucrose gradient. The 125I-Bgt-bound AChRs
sedimented in a single peak, which has previously been shown to be
centered at 9S (Claudio et al., 1987). The location of
arrow for the 9S standard in Figure
4A is based on the sedimentation of the peak
fractions (10 and 11) on this gradient. D, Scanning
densitometry of intracellular AChR subunit. The scanned values of
the 35S-labeled AChR subunits in Figure
4A are displayed. The signal in fraction 8 was
too weak to quantify. The subunit ratio, :::, was
1.6:1:0.90:0.95 in fraction 9, 2.4:1:0.80:0.94 in fraction 10, 1.8:1:0.85:0.98 in fraction 11, 1.6:1:0.91:1.0 in fraction 12, 1.2:1:1.1:1.0 in fraction 13, 1.2:1:1.2:1.0 in fraction 14, and
1.3:1:0.70:1.1 in fraction 15. The scanned values are in arbitrary
units. E, Scanning densitometry of surface AChRs. The
scanned values of the 35S-labeled AChR subunits in Figure
4B are displayed. The subunit signal could be
quantified in fractions 9-14, the subunit signal in fractions
10-12, and the and subunit signal only in fractions 10 and 11. The subunit ratio, :::, was 2.6:1:0.70:1.0 in fraction 10, 2.6:1:0.70:1.0 in fraction 11, and 3.1:1:0:0 in fraction 12. The
scanned values are in arbitrary units but are scaled relative to the
values in Figure 4D taking into account the
difference in the exposure time of the autoradiograms (6 d in Fig.
A and 5 d in Fig. B).
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In Figure 4B, metabolically labeled, cell surface
AChRs were also characterized on sucrose gradients. Our ability to
isolate the labeled surface AChRs in Figure 4B
demonstrates that the labeled complexes purified by Bgt affinity
methods (Fig. 2A,B) are transported to the cell
surface. These AChR subunit complexes differ from the intracellular
complexes (Fig. 4A,D) in several respects. The surface complexes (Fig. 4B,E) migrate in a tighter
peak centered at 9S on the sucrose gradient than the intracellular
complexes. Almost all of the surface complexes are found in fractions
10, 11, and 12 of the gradient, whereas the intracellular complexes migrate in a broader peak encompassing fractions 9-14. For the surface
receptors, the subunit ratio is on average 2.7:1:0.7:0.9 (see Fig.
4E legend for details). This subunit ratio is almost precisely the ratio obtained taking into account differences in subunit
cysteine and methionine content and the ratio of
35S-methionine and 35S-cysteine during
labeling, which is 2.7:1.0:0.8:1.0 for a subunit stoichiometry of
2:1:1:1 for :::. The ratio of the to the other subunits
is smaller for the intracellular complexes, ranging from ~2 for the
complexes in the 9S region (fractions 10-12) to ~1 in the 11S region
(fractions 13-15). The results indicate that the intracellular
complexes include tetramers with a ~1:1:1:1 subunit
ratio and a broad distribution across the gradient (fractions 9-15) as
well as 2 pentamers at 9S. The presence of both
pentamers and tetramers explains why the ratio of the to the other
subunits for intracellular complexes is ~2 at 48 hr (Figs.
1B, 2B, 4D) as
opposed to the expected value of 2.7. Altogether, our results characterizing subunit complexes on sucrose gradients (Figs. 3, 4)
support our previous findings that AChR subunits assemble first as
trimers, next into tetramers, and finally into
2 pentamers, after which the complexes are
transported to the cell surface (Green and Claudio, 1993; Green and
Wanamaker, 1997).
The first ACh binding site forms on the
subunit
Differences in the affinity of the two ACh binding sites for small
ligands are caused by the association of the two subunits with
different subunits, either the or subunits (Blount and Merlie,
1989; Pedersen and Cohen, 1990; Sine and Claudio, 1991). To test which
of the two ACh binding sites forms first, the AChR antagonist
dTC was used to inhibit the initial rate of 125I-Bgt
binding (Fig. 5). dTC binds to the two
ACh binding sites with ~100-fold difference in affinity (Neubig and
Cohen, 1979; Sine and Taylor, 1981), and the high- and low-affinity
sites are located on the and
subunits, respectively (Blount and Merlie, 1989; Pedersen and Cohen,
1990). When intracellular AChR complexes are isolated 48 hr after the
cells are shifted from 37 to 20°C, the isolated complexes are
predominately 2 pentamers (Fig.
4B). At this time, the inhibition by dTC is
consistent with two different binding sites on the two subunits
with a 70-fold difference in affinity for dTC, as calculated by the fit to the data of Figure 5 (see also Claudio et al., 1987). Different results were obtained for AChR complexes isolated 6 hr after the temperature shift, at which time assembled complexes consist of trimers
and tetramers (Fig. 3; see Green and Claudio, 1993). The inhibition by
dTC then was consistent with AChR complexes that predominantly contain
a single binding site with high affinity for dTC (Fig. 5). The data
together indicate that the first ACh binding site formed, the site
found on tetramers, is the high-affinity curare site
located on the subunit.
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Figure 5.
The first ACh site formed has a high affinity for
curare. Displayed is the inhibition of the initial rate of
125I-Bgt binding to AChR intracellular subunit complexes by
dTC. Intracellular subunit complexes were isolated 6 (filled circles) or 48 (open
squares) hr after cells were shifted from 37 to 20°C by
immunoprecipitation with mAb 14. For the AChR subunit complexes
isolated 48 hr after the temperature shift, the data were well fit by
assuming an equal number of two binding sites with a ~70-fold
difference in the IC50, 4.5 ± 1.4 × 10 7 versus 3.0 ± 0.7 × 105 M, for dTC, as indicated by the
line through the data. For the AChR subunit complexes
isolated 6 hr after the temperature shift, the data could not be fit by
the same model used to fit the 48 hr data. Instead, most of the
inhibition by dTC (~80%) was well fit assuming a single binding
site, as indicated by the line through the data. The
IC50 for this site, 8.5 ± 1.7 × 107 M, was approximately the same as
the high-affinity value estimated from the two-site model fit to the 48 hr data. The rest of the sites (~20%) were well fit by assuming a
single low-affinity binding site for dTC. These data indicate that the
first ACh binding site formed, the site found on
tetramers, is the high-affinity curare site. Each point
represents the mean ± SD of three determinations, with each
determination the mean from 125I-Bgt binding to two 6 cm
cultures.
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As another test of which of the two ACh binding sites forms first, we
obtained a conformational-dependent mAb, mAb 247g, which has been
reported to specifically recognize and block the high-affinity dTC site
on the subunit (Mihovilovic and Richman, 1987). At saturating
concentrations, mAb 247g blocked ~50% of the surface Bgt binding
sites (Fig. 6A).
Furthermore, when the inhibition of 125I-Bgt binding by dTC
was measured for subunit complexes precipitated by mAb 247g 48 hr after
the temperature shift, the inhibition by dTC was consistent with AChR
complexes that predominantly contain a single binding site with the low
affinity for dTC (Fig. 6B). Together, the data
indicate that mAb 247g binds to and blocks the binding site on the
subunit but not the other binding site. When mAb
247g was used to immunoprecipitate labeled subunits undergoing assembly
(Fig. 6C), we obtained results similar to those obtained
using the bromo-ACh (Fig. 1A) and Bgt (Fig.
2A) affinity resin. This similarity is shown
quantitatively in Figure 6D, in which the scanned
values for all four subunits are displayed. These data demonstrate that
the mAb 247g epitope forms at approximately the same time as the first
ACh site, well before the second ACh site forms, and is further
evidence that the ACh binding site is the first ACh
binding site formed.
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Figure 6.
The first ACh binding site forms on the
subunit. A, mAb 247g binds to and
blocks one of the two Bgt binding sites. The indicated amounts of mAb
247g were added to the medium of intact cells for 2 hr at 20°C, after
which intact cells were bound by 125I-Bgt to determine the
number of cell surface Bgt sites. At saturating concentrations, mAb
247g blocked ~50% of the cell surface Bgt sites. Each
point represents the mean from 125I-Bgt
binding to two 6 cm cultures. B, mAb 247g binds to the
high-affinity curare binding site. Displayed is the inhibition of the
initial rate of 125I-Bgt binding to AChR intracellular
subunit complexes by dTC. Forty-eight hours after cells were shifted
from 37 to 20°C, intracellular subunit complexes were isolated by
immunoprecipitation with mAb 247g (filled
squares). Also displayed for comparison are the data from
Figure 5 in which intracellular subunit complexes isolated by
immunoprecipitation with mAb 14, 48 hr after cells were shifted from 37 to 20°C (open squares). The inhibition by dTC obtained
for the complexes precipitated by mAb 247g was fit by the two-site
model used to fit the mAb 14 data. Most of the inhibition by dTC
(~75%) was well fit, assuming a single binding site as indicated by
the line through the data. The IC50 for this site, 3.2 ± 0.6 × 105 M, was
approximately the same as the low-affinity value estimated from the
two-site model fit to the 48 hr data. The rest of the sites (~25%)
were well fit by assuming a single high-affinity binding site for dTC.
Each point represents the mean ± SD of three
determinations, with each determination the mean from
125I-Bgt binding to two 6 cm cultures. C,
mAb 247g precipitation of labeled subunits. Cells were treated as in
Figures 1A and 2A, except
that AChR subunits were immunoprecipitated with mAb 247g.
D, Scanning densitometry of AChR subunits precipitated
by mAb 247g. The time course of the formation of the mAb 247g epitope
is shown quantitatively from the scanned values of the experiment shown
in C.
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DISCUSSION |
ACh binding sites form on subunit complexes other than and
heterodimers
The data presented in this paper contradict the current model in
which it is proposed that the two ACh sites form at the same time early
in the process of subunit assembly. According to this model, the
binding sites appear after subunits have assembled with or subunits into and heterodimers. Differences in the two
binding sites occur because one binding site forms in the vicinity of
the subunit interface on the resulting heterodimers, and
the other site forms in a homologous location on heterodimers.
After ACh binding site formation, and heterodimers
assemble with subunits into 2 pentamers
(Blount et al., 1990; Gu et al., 1991; Saedi et al., 1991). The
evidence in support of this "heterodimer model" is based on
experiments in which less than the full complement of AChR subunits
were heterologously expressed. When subunits are expressed alone
(Blount and Merlie, 1988; Paulson et al., 1991) or with subunits
(Blount and Merlie, 1989), no ACh binding sites form. However, when subunits are expressed with either or subunits, ACh binding
sites with high affinity for curare form on subunit complexes,
and ACh binding sites with low affinity for curare form on the
subunit complexes.
It is important to note that there are no disagreements about the data
on which the heterodimer model is based. In fact, we obtained similar
results when the same combinations of Torpedo AChR subunits
were expressed (Green and Claudio, 1993). Contradictions with the
heterodimer model only arose when studying the assembly of all four
AChR subunits. Two features of our methods allowed us to overcome
difficulties previously encountered when isolating AChR assembly
intermediates. First, it was critical that AChR complexes were
solubilized in detergent other than Triton X-100, which causes the
dissociation of most partially assembled AChR subunit complexes. We
used a mixture of Lubrol PX and phosphatidylcholine to solubilize the
subunit complexes, and this mixture appears to preserve most of the
subunit associations (Green and Claudio, 1993; Green and Wanamaker,
1997). The second feature is the temperature sensitivity of the
Torpedo AChR subunit assembly. At 20°C, the temperature at
which experiments were performed in this paper, the rate of assembly is
slowed by more than an order of magnitude, greatly aiding the isolation
of subunit complexes on which the ACh binding sites form.
Within the error of our measurements, we find no evidence for ACh
binding to or heterodimers. All complexes isolated by
bromo-ACh affinity purification, Bgt-Sepharose precipitation, or mAb
247g immunoprecipitation contained subunits. In addition, when
size-fractionated on sucrose gradients, the isolated complexes are
larger than expected for heterodimer complexes, either trimers, tetramers, or 2 pentamers
(Figs. 3, 4). It is unlikely that that we missed and
heterodimers with ACh binding sites using these different techniques.
Even though the bromo-ACh affinity purification of the AChR subunits
does not quantitatively bind all AChR complexes, the and
heterodimers would have to bind ACh with a lower affinity than the
complexes purified for us to have missed these complexes. A number of
studies have demonstrated that and complexes bind
agonists and antagonists with the same affinity as the fully assembled
receptor (Blount and Merlie, 1989), thus eliminating this possibility.
Furthermore, although the bromo-ACh affinity purification is not
quantitative, precipitations using both Bgt-Sepharose and mAb 247g are
quantitative; that is, we precipitate just as much of the assembling
subunit complexes with the Bgt-Sepharose and mAb 247g as with any other subunit-specific antisera. Because Bgt binding sites and the mAb 247g
epitope appear to overlap with ACh binding sites on and
dimers, both techniques should have precipitated any and
dimers. Finally, previous studies from our laboratory using different
subunit-specific and conformation-dependent antibodies never found
evidence for the assembly of and heterodimers in cells
expressing the all four AChR subunits, neither Torpedo AChR
subunits at 20°C nor mouse AChR subunits at 37°C (Green and Claudio, 1993; Green and Wanamaker, 1997). Thus, the results do not
appear to be an artifact attributed to the temperature or species
specificity.
Subunit association sites for the and second subunits
Contrary to the heterodimer model, we found that the two
distinguishable ACh binding sites form at different times. While the
AChR is still assembling, the first ACh binding site forms on
subunits apparently in tetramers. After
completion of subunit assembly, the second site forms on
subunits in 2 pentamers.
These findings, together with our data that the first Bgt binding site
and the mAb 14 epitope appear on trimers (Fig.
3A,C), are in agreement with "the sequential model" of
AChR assembly that we previously proposed (Green and Claudio, 1993). As
shown in Figure 7A, the
sequential model is consistent with two different pathways, which can
be distinguished based on the order in which the two ACh binding sites
form. Evidence was presented that the first ACh binding site formed is
the site and the second ACh binding site is the
site. AChRs assemble, therefore, along path 2, with
the subunit assembling at the interface between the and subunits and the second subunit assembling at the interface between
the and subunits.
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Figure 7.
A, The order of ACh binding site
formation distinguishes between two different paths of AChR subunit
assembly. Displayed are two different paths of AChR subunit assembly
consistent with our previous studies (Green and Claudio, 1993; Green
and Wanamaker, 1997). The two paths differ where the and second subunits associate (marked by arrows) and where the two
different ACh binding sites, the high-affinity dTC site (marked as
dTC) or the low-affinity dTC site (marked as
ACh), form first. AChR subunit assembly along path 2 is
demonstrated by our finding that the ACh binding
site forms first. The subunit arrangement of the
2 pentamer shown in both paths is the favored
arrangement (Karlin et al., 1983; Blount and Merlie, 1989; Pedersen and
Cohen, 1990; Sine and Claudio, 1991; Machold et al., 1995). Another
subunit arrangement of the 2 pentamer has been
proposed (Kubalek et al., 1987), in which the positions of the and
subunits in the pentamer are switched. However, the positions of
the arrows do not change with this arrangement, even
though the positions of the and subunits change.
B, The subunit folding and oligomerization events that
precede ACh binding site formation. Diagrammed are the subunit folding
and oligomerization events that precede formation of both ACh binding
sites. Subunit folding events are denoted by the < |
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Abstract
Introduction
