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The Journal of Neuroscience, August 1, 1999, 19(15):6298-6308
Nicotinic Receptor Assembly Requires Multiple Regions throughout
the  Subunit
Alison L.
Eertmoed and
William N.
Green
Department of Pharmacological and Physiological Sciences,
University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Assembly of ionotropic neurotransmitter receptors typified by
acetylcholine receptors (AChRs) is thought to be directed by an
N-terminal extracellular domain of a subunit. Consistent with this
hypothesis, chimeras with the  subunit N-terminal domain fused to
the rest of the  subunit can substitute for , but not ,
subunits during AChR assembly. However, chimeras with the subunit
N-terminal domain fused to the rest of the subunit cannot substitute for or subunits during assembly. Furthermore,
expression of this chimera with the four wild-type subunits prevents
the formation of  -bungarotoxin (Bgt) binding sites. Instead of AChR pentamers, complexes are assembled containing only the chimera and
either or  subunits. Based on the results of additional -
chimeras, there are at least two different regions within the
C-terminal half of the chimera required for the dominant-negative effect. Our results indicate that the N-terminal domain of the subunit mediates the initial subunit associations, whereas signals in
the C-terminal half of the subunit are required for subsequent subunit interactions.
Key words:
protein folding; conformational changes; assembly; acetylcholine; -bungarotoxin; nicotinic receptors
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INTRODUCTION |
As large oligomeric and polytopic
proteins, the folding and assembly of ion channels are complex and must
occur with high fidelity because misassembly of even a few channels,
each controlling the flow of ~107 ions/sec, can be
disastrous for a cell. Moreover, folding and assembly are the
rate-limiting steps during channel biogenesis (Green and Millar, 1995 )
and are likely to play an important role in regulating the number and
targeting of functional channels. The muscle-type nicotinic
acetylcholine receptor (AChR) was the first and remains among the few
ion channels for which the subunit composition and stoichiometry are
established definitively. As a member of the family of
neurotransmitter-gated ion channels that includes neuronal AChRs,
5HT3, glycine, and GABAA and
GABAC receptors, the muscle-type AChR has long served as a
model for studies of ion channel assembly.
Muscle-type AChRs are composed of four homologous subunits (, ,
, and ) that assemble into 2 pentamers
(Karlin and Akabas, 1995; Lindstrom, 1995; Changeux and Edelstein,
1998). According to the "heterodimer" model of AChR assembly
(Blount et al., 1990; Gu et al., 1991; Saedi et al., 1991; Kreienkamp et al., 1995), subunits fold into a conformation that can bind -bungarotoxin (Bgt) before assembling with or subunits into heterodimers. ACh binding sites form on the heterodimers that associate
together with subunits into 2 pentamers.
Evidence for this model is based on complexes formed in cells
expressing less than the full complement of subunits. When cells
expressing all four AChR subunits are used (Green and Claudio, 1993;
Green and Wanamaker, 1997), it has been found that AChR subunits
rapidly assemble into trimers. Afterward, subunits are
added to form tetramers, and then a second subunit is
added to make 2 pentamers. Another significant
difference is that the first Bgt and ACh binding sites appear only
after trimer assembly, and the second Bgt and ACh binding
sites appear after 2 pentamers form (Green and
Wanamaker, 1998).
Regions within the N-terminal extracellular domain of AChR subunits are
required for associations between subunits (Yu and Hall, 1991; Sumikawa
and Gehle, 1992; Verrall and Hall, 1992; Kreienkamp et al., 1995). The
N-terminal regions of glycine (Kuhse et al., 1993) and
GABAC and GABAA (Hackam et al., 1998) receptors also are involved in receptor assembly. In support of these
studies, we find that the chimeric subunit, which contains the
N-terminal domain of the AChR subunit fused to the rest of the subunit, substitutes for , but not , subunits during AChR
assembly. However, the reverse chimera with the N-terminal domain of
the subunit fused to the rest of the subunit does not
substitute for or subunits. Instead, when coexpressed with the
four wild-type subunits, the chimera assembles with or subunits
and blocks the formation of all Bgt binding sites. This
dominant-negative effect is specific for the loss of subunit
regions, not the presence of subunit regions, and requires at least
two different subunit regions in the C-terminal half of the
subunit. Our results indicate that regions within an N terminal domain
of a subunit mediate the initial rapid subunit associations, whereas
regions within the C-terminal half of the subunit are required for
subsequent subunit interactions.
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MATERIALS AND METHODS |
cDNA constructs. The 215 and 221 chimeras
were constructed via standard PCR methods (Yon and Fried, 1989).
For 215, an oligonucleotide was obtained consisting of the 15 base pairs (bp) of the cDNA preceding the region coding for the
first transmembrane domain (M1), followed by the first 15 bp of M1 from
the cDNA. Amplification of cDNA using this primer resulted in a
product corresponding to the sequence up to the start of M1,
followed by 15 bp of sequence. This product was used in a second
PCR step to amplify the cDNA, with the short region of sequence serving to anneal the sequence to the template. The 221
chimera was made by the same method, except that both a forward and a reverse chimeric primer corresponding to the chimera junction were
used. The 215 chimera was constructed by using a shared BclI restriction site located before M1 of the and subunits to join the and cDNAs at the BclI site.
All additional chimeras were constructed by an introduction of
restriction sites into the subunit cDNA by PCR, using a mutagenic primer with a 1-3 bp mismatch (Ho et al., 1989). The 215 chimera was produced by introducing the BclI site described above
into the homologous location in the subunit to join the and cDNAs at the BclI site. To construct 215467, we
introduced a unique StyI restriction site within M4
of into the homologous location of the subunit to replace the C
terminus of 215, starting at amino acid 467, with that of .
The 322 and 215329 chimeras were constructed by
introducing the unique BglII site of the subunit into
the corresponding location of the subunit. Full-length chimeric
subunits were created by cutting with the appropriate restriction
enzyme and ligating subunit fragments together at the common sites.
Regions of mutant subunits constructed by PCR methods were sequenced to
control for errors in the PCR products.
Transfections. The four Torpedo subunit cDNAs in
the pRBG4 expression vector (Lee et al., 1991) were transiently
transfected into 6 cm cultures of tsA201 cells (Margolskee et al.,
1993), using a calcium phosphate method (Eertmoed et al., 1998). Unless otherwise indicated, 1 µg of , 0.5 µg of , 2.5 µg of ,
and 7.5 µg of subunit cDNAs were used. Cells were maintained at 37°C with 5% CO2 in DMEM supplemented with 10%
fetal bovine serum. Because of the temperature dependence of
Torpedo AChR expression (Claudio et al., 1987), cells were
shifted from 37 to 20°C 24 hr after the transfection. Maximal
expression occurred 4 d after the temperature shift.
The 215 chimeric cDNA in pRBG4 was stably transfected into a cell
line already expressing the four Torpedo subunits (Claudio et al., 1987). Cells were maintained in DMEM supplemented with 10%
calf serum, HAT (15 µg/ml hypoxanthine, 1 µg/ml
aminopterin, and 5 µg/ml thymidine), and G418
(1 mg/ml) to maintain thymidine kinase and neomycin selection of the
stably transfected subunits. To enhance the expression of the four
wild-type subunits under the control of SV40 promoters, we also
supplemented the culture medium with 20 mM sodium butyrate
for 1-5 d before assay. Using calcium phosphate transfection, we
cotransfected each 10 cm culture with 15 µg of the 215 chimeric
cDNA and 500 ng per plate of the neomycin resistance gene construct
pSV2Neo. Clonal isolates of neomycin-resistant cells were obtained by
adding 0.6 mg/ml G418 to the culture medium and were screened for the
expression of AChR subunits by 125I-Bgt binding.
125I-Bgt binding. To assay cell-surface
125I-Bgt binding, we washed the cultures with PBS and
incubated them at room temperature in PBS containing 4 nM
125I-Bgt (140-170 cpm/fmol) for 2.5 hr on a shaker table.
Plates were washed again with PBS and solubilized; 125I-Bgt
counts were determined by counting. To assay 125I-Bgt
binding to solubilized AChRs (surface plus intracellular), we incubated
cell lysates with 10 nM 125I-Bgt overnight at
4°C, followed by immunoprecipitation with monoclonal antibody (mAb)
35. Nonspecific binding was measured by binding to sham-transfected
cells or by adding 10 mM carbamylcholine during binding.
Metabolic labeling and SDS-PAGE analysis. 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-cysteine-free DMEM,
supplemented with 333 µCi of a 35S-methionine
35S-cysteine mixture (NEN
EXPRE35S35S). The labeling was
stopped with the addition of DMEM plus 5 mM methionine. To
follow the subsequent changes in the labeled subunits, we "chased"
the cells 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 and 1.83 mg/ml phosphatydlcholine and 1% Lubrol (LPC). Solubilized AChR subunits and chimeras were immunoprecipitated with rabbit polyclonal antisera, anti-, anti- (Claudio and
Raftery, 1977), or anti- mAb 148, anti- mAb 168 (Gullick and
Lindstrom, 1983), conformational-dependent mAb 14 (Tzartos and
Lindstrom, 1980), or and subunit-specific mAb 88b (American
Type Culture Collection, Rockville, MD).
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 (Ann Arbor, MI). 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 that the darker signals
were not saturated, we took three to five exposures of each
autoradiograph; each was scanned to insure that the scanned bands
remained in the linear range of the film.
Sucrose gradients. Cell lysates were layered on a 5 ml
5-20% linear sucrose gradient prepared in LPC lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm to
 2t = 9.0 × 1011. Eighteen fractions of 300 µl each were
collected from the top of the gradient. Then the fractions were counted
in a gamma counter to determine the amount of 125I-Bgt
bound to each fraction.
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RESULTS |
The subunit N-terminal extracellular domain is sufficient to
direct subunit assembly
Of the four AChR subunits, and subunits are the most
similar. Nonetheless, subunits rapidly associate with and subunits, while subunits remain unassembled (Green and Claudio,
1993). To determine regions on the subunits responsible for these
differences, we constructed subunit chimeras in which homologous
regions of the and subunits were swapped. In the chimera
221, regions of the subunit were replaced by regions of the
subunit starting at amino acid 221, which is at the junction
between the extracellular N-terminal domain and the first transmembrane
region, M1 (Fig. 1A).
As expected, the molecular weight of the chimera appears to be between
that of the and subunits (Fig. 1A).
Cotransfection of the 221 chimera with wild-type , , and
subunits yielded expression of Bgt binding receptors on the cell
surface at levels similar to those seen with all four wild-type
subunits (Fig. 1B). Consistent with previous studies
(Kurosaki et al., 1987; Sine and Claudio, 1991), surface Bgt binding
receptors were observed with the transfection of the wild-type subunit
cDNAs without either the or subunit cDNAs (Fig.
1B). However, the levels of surface Bgt receptors
lacking or subunits were very low, ~10% or less of control,
reflecting very inefficient assembly without all four subunits. The
addition of 221 subunits to wild-type , , and subunits
resulted in little to no increase in the number of Bgt binding sites
above what was observed with the expression of , , and subunits alone (Fig. 1B).
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Figure 1.
The subunit N-terminal extracellular domain
directs subunit assembly. A, The 221 chimera.
The N-terminal half of the subunit (filled
bar) up to amino acid 221 was fused to the C-terminal half of
the subunit (open bar) to create the 221
chimera. The subunit domain is entirely extracellular. The subunit domain is composed of three membrane-spanning regions, M1, M2,
M3 (broadened areas), followed by a cytoplasmic domain,
a fourth membrane-spanning region, M4 (broadened area),
and the extracellular C terminus. Cells were transiently transfected
with 2.5 µg of , , or 221 cDNA per 6 cm plate,
metabolically labeled, and immunoprecipitated with mAb 88b. SDS-PAGE
analysis of the 221 chimera relative to the and subunits
is displayed. B, The 221 chimera can substitute
for the subunit during AChR assembly. The indicated amounts of
221 chimera cDNA were cotransfected with set amounts of the ,
, and (filled squares) or , , and
(open squares) subunit cDNAs, and cell-surface
125I-Bgt binding was assayed. The addition of the chimera
to , , and subunit cDNAs did not increase cell-surface
125I-Bgt binding. When cotransfected with , , and subunits, 125I-Bgt binding increased to the level obtained
with the transfection of all four wild-type subunit cDNAs. Data are
expressed as the percentage of the binding observed for cells
expressing the four wild-type subunits (20 fmol of Bgt binding sites
for this experiment). Filled squares represent the mean
of two plates from a single experiment. Similar results were obtained
in three other experiments. Open squares are a mean of
four plates from two experiments. Error bars ± SEM are smaller
than the symbols. C, Sedimentation of 221 AChRs.
Surface 125I-Bgt binding was performed on cells expressing
, , , and 221 subunits (filled
symbols; + 221); the 125I-Bgt-bound
221 AChRs were size-fractionated on 5-20% linear sucrose
gradients. The results are plotted as the fraction of the maximum value
to facilitate comparison with cells expressing wild-type AChRs
(open symbols; ). The fraction number runs
from the top to the bottom of the
gradient, and the arrows mark the peak fractions of the
standards: alkaline phosphatase (5.4 S), catalase (11 S), and
2 AChRs (9 S). D, 221
AChRs have two ligand binding sites. Intact cells expressing the
221 chimera and the , , and subunits were incubated
with mAb 247G for 3 hr before cell-surface 125I-Bgt binding
was assayed. Data are expressed as a percentage of 125I-Bgt
binding to cells not incubated with mAb 247G (94 fmol of Bgt binding
sites). For receptors containing the 221 chimera, ~50% of the
binding sites remain with saturating amounts of mAb 247G. Values
represent the mean ± SEM of four plates from two experiments.
Because mAb 247G blocks Bgt binding to the  , but not
the  , ligand binding site on AChRs, the results
indicate that both sites are present on 221 AChRs.
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Further experiments tested whether AChRs containing the 221
chimera were pentamers with two ACh binding sites. The size of surface
receptors was examined by using sucrose gradients (Fig. 1C).
A single peak, at 9 S, was indistinguishable from the location where
AChRs composed of the four wild-type subunits migrated. This finding
indicates that 221-containing AChRs are pentamers with a
stoichiometry of 2(221), with 221
replacing the subunit in the Torpedo, wild-type
2 complexes. To test whether the
221-containing AChRs have two distinguishable ACh binding sites
like wild-type AChRs, we used mAb 247G, which recognizes and blocks Bgt
binding to one ACh site, but not the other (Mihovilovic and Richman,
1987; Green and Wanamaker, 1998). When cells expressing 221-containing AChRs were incubated with increasing amounts of
mAb 247G, ~50% of the Bgt binding sites were blocked, confirming the
presence of two different ACh binding sites. Similar results were
obtained previously by using a mouse 221 chimera (Sine, 1993)
and, together with our results, indicate that the 221 chimera can
substitute for the subunit, but not the subunit, during AChR assembly.
The C-terminal half of the subunit is required for Bgt binding
site formation
The chimera 215 was constructed by replacing the subunit
N-terminal domain with that of the subunit (Fig.
2A). Like the 221
chimera, 215 migrates as expected between the and subunits on SDS-PAGE gels (Fig. 2A). Unlike the
221 chimera, the 215 chimera does not substitute for the
subunit during AChR assembly because the addition of 215 to
, , and subunits did not increase AChR expression (data not
shown). However, the 215 chimera is not inert during assembly, as
evidenced by a complete loss of surface AChRs when 215 chimera
cDNAs are transfected with the four subunit cDNAs (Fig.
2B). In addition to blocking the expression of
surface AChRs, 215 chimeras blocked the formation of all
intracellular Bgt binding sites (Fig. 2B). The loss
of surface Bgt binding sites because of the 215 chimera thus is caused by a failure of the sites to form and not by a block of their
transport to the cell surface. The dominant-negative effect of the
215 chimera differs from the inhibition of expression observed
when a single AChR subunit is overexpressed relative to the other three
subunits (Gu et al., 1991). Overexpression of the subunit, which
most affected AChR expression, did not alter significantly the number
of intracellular Bgt binding sites (Gu et al., 1991), unlike the effect
of adding the 215 chimera (Fig. 2B).
Additionally, overexpression of the subunit caused, at most, a
40-50% decrease in expression (Gu et al., 1991). In our
experiments, transfection of an additional 10 µg of the subunit
cDNA caused a <20% decrease in AChR expression, whereas transfection
of the same amount of the 215 chimera cDNA decreased expression
by >90% (Fig. 2B).
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Figure 2.
The C-terminal half of the subunit is required
for Bgt site formation. A, The 215 chimera. The
N-terminal extracellular domain of the subunit (open
bar) up to amino acid 215 was fused to the C-terminal half of
the subunit (filled bar) to create the
215 chimera. The subunit domain is entirely extracellular,
whereas the subunit domain extends from the first membrane-spanning
region, M1 (broadened area), to the C terminus. SDS-PAGE
analysis of the 215 chimera was performed as in Figure
1A. B, The 215 chimera
inhibits AChR surface expression and Bgt binding site formation.
Cultures were transfected with the four wild-type subunit and the
indicated amount of 215 cDNA. 125I-Bgt binding to
both intact cells (triangles) and cell lysates
(circles) was assayed. Also assayed was the effect of
additional subunit expression on surface 125I-Bgt
binding. The indicated amounts of cDNA were added to the 7.5 µg
that yielded maximum AChR expression (squares). Values
are expressed as a percentage of the 125I-Bgt binding
observed in the absence of chimera or additional subunit
expression. Each point represents the mean of 10 6 cm
plates from five experiments (Surface) and six plates
from three experiments (Total). Error bars ± SEM are smaller than the symbols for all 215 values.
C, The 215 and 215 chimeras. The N-terminal
extracellular domain of the subunit (open bar) up to
amino acid 215 was fused to the C-terminal half of the (striped bar) or (shaded bar) subunit
to create the 215 and 215 chimeras, respectively. SDS-PAGE
analysis of the 215 and 215 chimeras was performed as in
Figure 1A except that and 215 were
immunoprecipitated with polyclonal anti- Ab, and and 215
were immunoprecipitated with polyclonal anti- Ab. D,
215 and 215 chimeras inhibit AChR expression. Cells were
transfected with all four subunit cDNAs plus the indicated amount of
each chimera cDNA and assayed for surface 125I-Bgt binding
as in B. Values are expressed as a percentage of the
125I-Bgt binding observed in the absence of chimera (43 and
45 fmol for the experiments with 215 and 215,
respectively). Each point represents the mean of six
cultures from three experiments (215) or the mean of seven
cultures from three experiments (215). For comparison,
cell-surface 125I-Bgt binding to cells transiently
transfected with all four subunit cDNAs plus 215 is repeated from
B. E, The 215 chimera cannot
substitute for the subunit. The indicated amounts of 215
chimera were cotransfected with a set amount of the , , and subunit cDNAs (open diamonds), and cell-surface
125I-Bgt binding to the cells was assayed. Also displayed
are results from cells transfected with varying amounts of subunit
cotransfected with a set amount of the , , and subunit cDNAs
(open squares). Data are expressed as the percentage of
the maximum binding for cells expressing the four wild-type subunits,
which occurred at 5 µg of subunit cDNA, yielding 146 fmol of Bgt
sites. Each point represents the mean ± SEM
determined from four cultures from two separate experiments. Note that
the 215 chimera error bars are smaller than the symbols.
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Two more chimeras were constructed to test whether the
dominant-negative effect of the 215 chimera is caused by the loss of subunit regions or by the addition of subunit regions. The
chimeras 215 and 215 contained the N-terminal domain of the
subunit and the C-terminal half contributed by either the or
subunit (Fig. 2C). The translated product of both
chimera cDNAs appeared to be full-length, because 215 migrates on
gels between the and subunits and 215 migrates between
the and subunits (Fig. 2C). When cotransfected with
all four wild-type subunits, both the 215 and 215 chimeras
blocked the formation of surface AChRs (Fig. 2D). The
dominant-negative effect of the 215 chimera was less pronounced
than that of the 215 or 215 chimeras. This difference was
not caused by the assembly of any 215-containing AChRs, because
the transfection of 215 chimeras with , , and subunits
did not increase the surface expression of AChRs (Fig.
2E). In summary, a dominant-negative effect occurs regardless of whether the replaced regions are from the , , or
subunits. Regions from these subunits, therefore, do not contribute, and the dominant-negative effect results from a loss of
signals within the C-terminal half of the subunit.
The effect of the 215 chimera on AChR assembly
Because Bgt binding sites form during AChR assembly (Merlie and
Lindstrom, 1983; Green and Claudio, 1993; Green and Wanamaker, 1998),
the block of Bgt binding site formation by the 215 chimera appears to be altering AChR assembly. To determine more precisely how
the 215 chimera affects AChR assembly, we stably transfected the
215 chimera into a AChR-expressing cell line we have used previously to characterize AChR assembly ( cells; Claudio et al., 1987). Eighteen cell lines were isolated after stable transfection of the 215 cDNA into cells. All
isolated cell lines displayed a significant reduction in surface AChR
expression as assayed by Bgt binding. A single cell line was chosen for
characterization in which Bgt binding was not totally eliminated but
was greatly reduced. As displayed in Figure
3A, cell-surface Bgt binding
to these cells was reduced sevenfold as compared with the parent cell
line. There was also an approximately twofold decrease in the levels of
the wild-type subunits relative to the cells as determined
by metabolically labeling the subunits (Fig. 3B). The
decrease in wild-type subunit levels, apparently caused by the added
215 chimera synthesis, may be contributing to the loss of Bgt
binding, but it is unlikely to be the major factor. The reduction in
the subunit levels is much less than the decrease in Bgt binding.
Moreover, we have shown in Figure 2B that the 215 chimera causes a specific inhibition of AChR expression that
is not duplicated by the overexpression of other AChR subunits.
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Figure 3.
The effect of the 215 chimera on AChR
assembly. A, 125I-Bgt binding to cells
stably expressing 215 in addition to the four AChR subunits
( +215 cells). The 215 chimera cDNA was stably
transfected into a cell line expressing the four AChR subunits
( cells). Surface 125I-Bgt binding to
cells was 77 fmol per plate, whereas binding to the
+215 cells was reduced to 11 fmol (mean ± SEM
of seven plates from three experiments). B, Subunit
synthesis and coprecipitation. The and + 215 cells were labeled for 30 min, solubilized to preserve
subunit associations, and immunoprecipitated with -specific
polyclonal antibodies (lanes 1 and 2),
-specific polyclonal antibodies (lanes 3 and
4), or - and -specific mAb 88b
(lanes 5 and 6), which also
recognizes the 215 chimera. The precipitated subunits from
one-half of a 10 cm plate were loaded onto each lane. C,
Assembly of AChR subunits in cells. The cells
were labeled metabolically as in B and then followed for
the indicated times at 20°C before being immunoprecipitated with
either an -specific polyclonal antibody (left) or the
conformation-dependent mAb 14 (right). The precipitated
subunits from a 10 cm plate were loaded onto each lane. The band
labeled ' just above the band previously was
shown to be different from the subunit (Green and Claudio, 1993;
Green and Wanamaker, 1997). The band that migrates between the and
subunit bands is actin. D, The subunit
associations with the 215 chimera inhibit its folding and
assembly. The + 215 cells were labeled metabolically
and followed for the indicated times; the subunits were
immunoprecipitated as in C. Only the chimera
coprecipitates with the subunit (left) and prevents
formation of the mAb 14 epitope (right). Qualitatively
similar results were obtained two times with the + 215 cell line shown and two more times with a second + 215 cell line. E, 215 chimeras associate
with or subunits. The + 215 cells were
labeled metabolically and followed as in C. Subunits
were immunoprecipitated with -specific mAb 148 (left)
or - and -specific mAb 88b (right), which also
recognizes the 215 chimera. Qualitatively similar results were
obtained two times with the + 215 cell line shown
and two more times with a second + 215 cell
line. F, The rate of subunit degradation in + 215 cells. The time course of subunit degradation is shown
quantitatively from the scanned values of the experiment shown in
C (left) for the subunits and in
D (left) for 215
(filled circles) and subunits
(filled triangles). The data are plotted on a
semilog scale. The decay of the subunit signal from the
cells (filled squares) is biphasic.
The fast component corresponds to degrading unassembled subunits.
To estimate the unassembled rate of decay, we subtracted the 48 hr
value from the other values and plotted it as open
squares. The half-life was estimated to be 8.1 hr, based on a
least-squares fit of an exponential function to the three data
sets.
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Subunit assembly was assayed by metabolically labeling the subunits for
30 min and following changes in the labeled subunits for the indicated
times (Fig. 3C-E). When labeled subunits from the
cells were immunoprecipitated with subunit-specific polyclonal antibodies, and subunits coprecipitated with subunits consistent with the assembly of trimers during the label period (Fig. 3C, left). Initially, the subunit
signal was larger than that of the other subunits because both
assembled and unassembled subunits were precipitated by the
anti- antibodies. Excess unassembled subunits degraded with a
half-life of 8-10 hr at this temperature (Fig. 3F)
(Green and Claudio, 1993; Green and Wanamaker, 1997), whereas the
assembled subunits were stable. Labeled subunits also were
immunoprecipitated with mAb 14 (Fig. 3C, right), which
recognizes a conformation-dependent epitope formed as subunits are
added to trimers, and only precipitates complexed subunits
well after the assembly of (Green and Claudio, 1993). Subunit
assembly was changed significantly by the addition of 215
chimeras to the parent cell line (Fig. 3D). When the labeled
subunits were immunoprecipitated with the subunit-specific antibodies, only 215 chimeras significantly coprecipitated (Fig. 3B,D). Also, subunit-specific mAb coprecipitated
predominantly 215 chimeras, and the / subunit-specific mAb
coprecipitated and subunits with 215 chimeras (Fig.
3B,E). Thus, instead of AChR assembly, 215 chimeras
associated with or subunits, and complexes with all three
subunits were not observed.
The 215-containing complexes were not stabilized like normal
assembly intermediates and were degraded at the same rate as unassembled wild-type subunits (Fig. 3F).
Furthermore, subunit folding events that normally occur on partially
assembled complexes during AChR assembly were not observed for the
215-containing subunit complexes. None of the
215-containing complexes was precipitated by mAb 14 (Fig.
3D, right) and thus lacked the mAb 14 epitope. In the
215-expressing cells, a small number of subunit complexes were
precipitated by mAb 14 (see last lane in Fig.
3D). Importantly, these complexes contained only the four
wild-type subunits and appear to be the small number of AChRs that do
assemble and are transported to the cell surface (Fig. 3A).
Because the addition of 215 chimeras also blocks the formation of
Bgt binding sites (Figs. 2B, 3A), neither
the mAb 14 epitope nor the Bgt binding site forms on
215-containing complexes. All together, our data indicate that
associations between either or subunits and 215 chimeras
prevent the assembly of trimers, the first stable assembly
intermediate. The mAb 14 epitope and Bgt binding sites fail to form on
these -215 and -215 complexes and, as misfolded
partially assembled complexes, they are degraded at a rate similar to
unassembled subunits.
Multiple assembly signals within the C-terminal half of the
subunit
Additional chimeras were constructed to identify discrete regions
within the C-terminal domain important for assembly. It was reported
previously that a chimera composed of the  subunit N-terminal and
subunit C-terminal domains substituted during assembly for the subunit, which is the mammalian adult isoform of the subunit (Yu
and Hall, 1991). The / chimera differed from the 215
chimera in that its short extracellular C terminus was from the subunit, not the subunit. To test whether the C terminus
contributes to the dominant-negative effect of 215, we replaced
the C terminus of 215 by that from the subunit to construct
215467 (Fig.
4A). Transfection of
the 215467 chimera along with the four wild-type subunits
completely blocked AChR expression (Fig. 4B), and the
addition of the subunit C terminus does not reverse the
dominant-negative effect. It is unclear why our results differ from
those with the / chimera.
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Figure 4.
Multiple assembly signals within the C-terminal
half of the subunit. A, The 215467 chimera.
The C terminus of the 215 chimera starting at amino acid 467 was
replaced by the corresponding region from the subunit (open
bar) to create the 215467 chimera. The C-terminal subunit domain extends from the end of the fourth membrane-spanning
region, M4 (broadened area), to the C terminus.
Immunoprecipitation and SDS-PAGE analysis of the 215467
chimera were performed as in Figure 1A.
B, The 215467 chimera blocks the formation of
Bgt binding sites when cotransfected with all four wild-type subunits.
Cells were transiently transfected with and the indicated
amount of chimera cDNA as in Figure 2B and were
assayed for cell-surface 125I-Bgt binding. Values are a
mean ± SEM of four plates from two experiments, expressed as a
percentage of the amount of 125I-Bgt bound in the absence
of the chimera, which was 66 fmol. Results with the 215 chimera
from Figure 2B are shown also. C,
The 215329 and 322 chimeras. The 215 chimera
starting at amino acid 329 was replaced by the corresponding region
from the subunit (open bar) to create the
215329 chimera. This part of the subunit extends from just
after the third membrane-spanning region (broadened
area) to the C terminus. The subunit starting at amino acid
322 was replaced by the corresponding region from the subunit
(filled bar) to create the 322 chimera.
Immunoprecipitation and SDS-PAGE analysis of the chimeras were
performed as in Figure 1A. D, The
215329 and 322 chimeras block the formation of Bgt
binding sites when cotransfected with all four wild-type subunits.
Cells were transiently transfected with and the indicated
amount of chimera cDNA as in Figure 4B and were
assayed for cell-surface 125I-Bgt binding. Values are a
mean ± SEM of seven plates from three experiments, expressed as a
percentage of the amount of 125I-Bgt bound in the absence
of the chimera, which was 57 fmol. Results with the 215 chimera
from Figure 2B are shown also.
|
|
Two more chimeras were constructed and characterized. The subunit
region that contains the first three transmembrane domains was replaced
by the homologous region of the subunit to produce the
215329 chimera, and the subunit region just after the third transmembrane domain to the C terminus was replaced by the homologous subunit region to construct the 322 chimera (Fig. 4). Surprisingly, expression of either the 215329 or 322 chimera along with the four wild-type subunits again completely blocked
expression of cell-surface AChRs (Fig. 4D). On the
basis of these results, we conclude that multiple regions within the subunit C-terminal half are required to overcome the
dominant-negative effect observed when this domain is replaced.
Minimally, two separate regions are required: one that contains the
first three transmembrane domains and a second that extends from the
cytoplasmic loop just after the third transmembrane domain to the C terminus.
| |
DISCUSSION |
A large number of studies have identified regions and specific
residues within the AChR subunit N-terminal domain that mediate subunit
associations during assembly (Yu and Hall, 1991, 1994a; Sumikawa and
Gehle, 1992; Verrall and Hall, 1992; Sumikawa and Nishizaki, 1994;
Kreienkamp et al., 1995). These studies did not assay the process of
receptor assembly directly. Instead, assembly was assayed indirectly,
either by monitoring cell-surface expression or by studying
associations between subunits and/or subunit fragments in isolation of
the other subunits. Thus, it remains to be determined when N-terminal
regions participate during assembly and whether other subunit regions
also could be involved in assembly (see also Yu and Hall, 1994b).
We found that the membrane-spanning and the cytoplasmic domains of the
subunit are essential for complete AChR assembly. When these
regions of the subunit are replaced by the homologous regions of
the , , or subunits, AChRs no longer fully assemble, as shown
by a complete block of AChR surface expression and Bgt binding site
formation. Our approach differed from previous work in that methods
were used that directly assayed full AChR assembly and allowed us to
determine which steps in assembly were altered. We found that, by
altering the C-terminal half of the subunit, assembly was blocked
at a step that prevented the appearance of trimers. Instead
of assembling into trimers, 215 chimeras associated with and
subunits, but not with and subunits. These results indicate
that the initial subunit associations required for the assembly of
trimers, i.e., the recognition of and by the subunit, are preserved when 215 chimeras replace subunits.
Thus, the N-terminal domain of the subunit in the 215 chimera
appears to mediate the initial subunit associations. Importantly,
215 chimeras only interact with or subunits, not both
together. This finding further indicates that 215 chimeras block
AChR assembly at a step in which heterodimers specifically recognize a
third subunit for assembly into trimers.
The results from this paper, together with data from other studies,
indicate that the first steps of AChR assembly occur as shown in Figure
5. We have shown previously that ,
, and subunits rapidly associate into trimers during
or just after subunit synthesis, and interactions between N-terminal
domains may be in part cotranslational (Green and Claudio, 1993). The
data from this study further indicate that and
heterodimers precede the rapid assembly of trimers, which
suggests that assembly is initiated when the N-terminal domains of two
subunits interact. The space between ribosomes on the endoplasmic
reticulum (ER) membrane is ~500 Å, too large a distance to allow
associations between two partially synthesized subunits (Hurtley and
Helenius, 1989) but, as shown in Figure 5, would allow the N-terminal
domain of a subunit undergoing synthesis to interact with a subunit no longer associated with the translocation machinery. A similar process
also may initiate K+ channel assembly in which a
region of the subunit N-terminal domain is required for rapid, perhaps
cotranslational, interactions between subunits (Deal et al., 1994).
According to the "heterodimer" model of AChR assembly (Blount et
al., 1990; Gu et al., 1991; Saedi et al., 1991), and the other
subunits remain unassembled for a considerable time, during which the
subunits fold and the Bgt binding site is created on the subunits.
This feature of the assembly model is difficult to reconcile with our
results, in particular our finding that subunit chimeras prevent
the formation of Bgt binding sites when added to wild-type
subunits.
View larger version (39K):
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Figure 5.
The early steps in AChR assembly are diagrammed,
consistent with the results of this paper. Regions within the
N-terminal domain of the subunit mediate rapid, perhaps
cotranslational, and promiscuous associations. Productive subunit
associations, as shown in A, result from a subset of the
subunit pairings: the , , or heterodimers. These
heterodimers undergo further folding, which strengthens the
association, allows for the assembly of trimers, and thereby
the subsequent steps in AChR assembly. Unproductive subunit
associations, shown in B, result from the other subunit
pairings: , , , , , , or
heterodimers. These heterodimers fail to undergo further folding and
are free to associate with other subunits after dissociation or are
degraded. The and subunit associations with the 215
chimera, shown in C, result in tightly associated
complexes that fail to assemble further. These results suggest that the
C-terminal half of the subunit is required for subsequent folding
and assembly.
|
|
Interactions between subunits show a lack of subunit specificity,
because homomeric associations occur when each of the four AChR
subunits is expressed alone (Paulson et al., 1991) or when any
heteromeric pair of subunits is expressed (Green and Claudio, 1993).
These subunit associations also appear to form rapidly (Green and
Claudio, 1993) and involve the N-terminal domain of the subunit
(Verrall and Hall, 1992). The lack of subunit specificity, together
with the rapidity by which associations occur, suggests that the
function of the initial subunit associations is to protect critical
subunit domains from exposure to the membrane or the aqueous
environment and to prevent the misfolding of these domains. As shown in
Figure 5, A and B, a lack of subunit specificity
also can help to explain why AChRs assemble inefficiently such that only 20-30% of the synthesized subunits assemble into AChRs and the
rest of the subunits are degraded (Merlie and Lindstrom, 1983; Ross et
al., 1991). As modeled in Figure 5A, productive subunit associations occur with the assembly of , , or
heterodimers. If each potential dimer assembles with equal probability,
only 30% of the initial interactions would form productive subunit pairs. Dimers assembled from other subunit pairs, i.e., , , , , , , or , would not continue into
trimers. Although these dimers can form, they are not
detected when all four subunits are present (Green and Claudio, 1993;
Green and Wanamaker, 1998), indicating that the associations are
relatively weak and short-lived. The transient nature of these
subunit interactions could allow some of the subunits to
reassociate with other subunits, perhaps to form a productive
pairing. However, many of the subunits that formed unproductive pairs
would be expected to misfold and be rapidly degraded (Fig.
5B).
Because there appears to be little subunit specificity in the initial
subunit associations among AChR subunits, we would expect the ,
, and heterodimer associations to be no tighter than any
other combination. What then stabilizes the productive , ,
and heterodimers? In previous studies we showed that the addition of subunits to trimers is preceded by the
folding of the trimer subunits, as evidenced by the appearance of the Bgt binding site and mAb 14 epitope. Similarly, the addition of the
second subunits to tetramers is preceded by the
folding of the tetramer subunits (Green and Claudio, 1993; Green and
Wanamaker, 1997, 1998). We propose that a similar set of events occurs
after a productive heterodimer forms and is required for
trimer assembly (Fig. 5A). The correct subunit combination
in the dimer allows the subunits to fold into a conformation that
strengthens the association and also allows the appropriate third
subunit to bind tightly to the dimer (Fig. 5A).
The (215) and (215) heterodimers form a tightly
associated complex but fail to recognize the third subunit needed for assembly into trimers (Fig. 5C). This finding
indicates that the subunit C-terminal regions are involved in the
subunit folding that creates a new subunit recognition site and are not
necessarily part of the site where the third subunit associates with
the heterodimer. Expression of the 215 chimera has a
dominant-negative effect, therefore, because association with or
subunits allows for strengthening of the association, but the dimer
fails to fold into a conformation that will allow assembly of the next
subunit. It is possible that the chimera can associate properly with
or subunits but cannot undergo subsequent conformational
changes because the C-terminal half of the chimera is misfolded.
Although the difference between starting in a misfolded state versus a failure to fold correctly is subtle, several of our findings suggest that the 215 chimera is not misfolded. The chimera is not
recognized by the cells as misfolded because it is degraded at the same
rate as unassembled wild-type subunits (see Fig. 3F).
Furthermore, similar effects on surface expression were observed when
different regions of the C-terminal half of the subunits were
substituted by the corresponding subunit region (see Fig. 4). More
generally, it is possible that all of the nonproductive dimers as well
as the 215-containing dimers are unable to fold correctly and
contribute to the assembly of trimers because they lack the
appropriate C-terminal regions. This conclusion about the role of the
subunit C-terminal regions is supported by the dominant-negative
effect of all of the additional - subunit chimeras described in
Figure 4. We found that replacing any part of the C-terminal half of the subunit on the 215 chimera by the appropriate regions failed to prevent the dominant-negative effect. This failure to rescue
cell-surface expression by replacing piece-by-piece all of the regions by regions argues that there is no general C-terminal motif
that is part of the association site. Instead, it is consistent with
the idea that the site where the third subunit associates is created by
rearrangement of the heterodimer subunits, and the ability to undergo
the rearrangement requires the C-terminal regions of the subunit.
The picture that emerges from this work is that associations between
AChR subunits are at first weak and nondiscriminating. If the purpose
of the initial subunit associations is, indeed, to protect critical
subunit domains and prevent misfolding, then a relatively weak and
promiscuous type of association fits with an apparent chaperone role of
the subunit associations. Other proteins, such as
immunoglobulin-binding protein (BiP) and calnexin, that are
endogenous ER chaperones also transiently interact with newly
synthesized AChR subunits (Blount and Merlie, 1991; Paulson et al.,
1991; Forsayeth et al., 1992; Gelman et al., 1995; Keller et al.,
1996). Only the correct associations between AChR subunits subsequently
are strengthened and stabilized as the subunits continue to fold and
mature during assembly of the AChR. Because these assembly events occur
in the plane of ER membrane (Smith et al., 1987), cells must be
solubilized to purify assembling subunits. Previously, we showed that
the ability to purify intact assembly intermediates is dependent on the
detergent used and the presence of phospholipids during the
solubilization (Green and Claudio, 1993). These milder solubilization
conditions allow us to observe intact trimers and
tetramers, but we have never observed intact heterodimer
precursors. It is likely, therefore, that even the mildest of
solubilization protocols disperses the heterodimers because of the
weakness of the interaction and that subunit associations strengthen as
assembly continues from dimer to trimer to tetramer to the final
pentamer, which can be dispersed only by SDS.
| |
FOOTNOTES |
Received Feb. 9, 1999; revised May 10, 1999; accepted May 14, 1999.
This study was funded by grants from National Institutes of Health and
the Brain Research Institute to W.N.G. We are most grateful to Dr. J. Lindstrom for mAbs 14, 148, and 168 and to Dr. T. Claudio for the cell
line stably expressing the four Torpedo AChR subunits
and the anti- and antiserum. We also thank Dr. A. Fox and
members of the Green laboratory for discussion and comments about this paper.
Correspondence should be addressed to Dr. William N. Green, Department
of Pharmacological and Physiological Sciences, University of Chicago,
947 East 58th Street, Chicago, IL 60637.
| |
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TOP
ABSTRACT
INTRODUCTION
