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The Journal of Neuroscience, May 1, 2001, 21(9):3000-3008
Rearrangement of Nicotinic Receptor 
Subunits during Formation
of the Ligand Binding Sites
Mirna
Mitra,
Christian P.
Wanamaker, and
William N.
Green
Department of Neurobiology, Pharmacology, and Physiology,
University of Chicago, Illinois 60637
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ABSTRACT |
Muscle nicotinic acetylcholine receptors (AChRs) are pentamers that
contain two 
subunits a 
, 
(or 
), and 
subunit.
In this paper, we have characterized subunit processing and folding events leading to formation of the two AChR ligand binding sites. subunit residues, 187-199, which are part of overlapping ACh and
-bungarotoxin (Bgt) binding sites on AChRs, were assayed using a
monoclonal antibody (mAb) specific for these residues. We found that
this region was inaccessible to the mAb early during AChR assembly but
became accessible as the first of two Bgt binding sites formed later
during assembly, indicating that the region changes conformation as the
Bgt binding site appears. Without previous reduction, 20% of the subunits could be alkylated by bromoacetylcholine bromide as the
first ACh binding site formed, which further indicated that the
disulfide bond between cysteines 192 and 193 does not form until the
first ACh binding site appears soon after Bgt binding site formation.
When subunits were mutated to add a glycosylation site at residue
187, the number of Bgt binding sites increased threefold, AChRs
assembled more efficiently, and 2.5-fold more AChRs reached the cell
surface. Our results indicate that binding site formation involves a
rate-limiting rearrangement of the subunit that exposes the
187-199 region to the endoplasmic reticulum lumen and
determines when cysteines 192 and 193 disulfide bond.
Key words:
nicotinic receptor; Torpedo; muscle; -bungarotoxin; protein folding and assembly; acetylcholine binding
site
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INTRODUCTION |
Signal transduction by members of a
family of neurotransmitter-gated ion channels, which include the
acetylcholine (ACh), GABAA, glycine, and
5-HT3 receptors, is initiated by binding of neurotransmitters to specific sites on the receptors. Muscle-type nicotinic ACh receptors (AChRs), which include the receptor in fish
electric organs, have long served as a model for the family (for
review, see Changeux, 1995
; Karlin and Akabas, 1995). AChRs are
composed of four homologous subunits, , , (or ), and ,
that assemble into pentamers with a stoichiometry of
2. There are two ligand binding sites
per receptor. Affinity labeling and mutagenesis of the subunit have
identified six residues, Tyr-93, Trp-149, Tyr-190, Cys-192, Cys-193,
and Tyr-198, in the vicinity of the binding site and have shown that
the adjacent cysteines, Cys-192 and Cys-193, are disulfide-bonded.
Residues on the and subunits have also been identified as
contributing to the binding site, from which it was concluded that the
two binding sites are found near the interfaces between an subunit and either the or subunits (for review, see Karlin and Akabas, 1995; Tsigelny et al., 1997; Changeux and Edelstein, 1998).
Venom-derived -peptide neurotoxins are potent AChR competitive
antagonists and bind with high affinity to regions overlapping ACh
binding sites. The best characterized of these neurotoxins is
-bungarotoxin (Bgt), for which the major region contributing to the
binding site has been localized to subunit residues 173-204 (Wilson et al., 1985; Tzartos and Remoundos, 1990). Neither Bgt nor ACh
sites are present on nascent subunits and only appear during AChR
assembly (Merlie and Lindstrom, 1983; Green and Wanamaker, 1998).
During AChR assembly, subunits first rapidly associate into
trimers. Trimers slowly assemble with subunits into
tetramers and then with a second subunit into
2 pentamers (Green and Claudio,
1993). The first Bgt binding sites form on trimers just before subunits associate, after which the first ACh sites appear at the
- subunit interface on tetramers. The second ACh
and Bgt sites appear later at the - interface on
2 pentamers. This sequence of events
suggests that the subunits undergo posttranslational changes after
their assembly so that the Bgt and ACh binding sites form.
In the experiments presented, we have probed subunit residues
187-199, which contribute to the Bgt and ACh binding sites, looking
for posttranslational changes during AChR assembly. We demonstrate that
this region is initially inaccessible to a monoclonal antibody (mAb)
specific for the region and becomes accessible only as the Bgt binding
site forms, implying that an intervening conformational change
involving this region occurs during AChR assembly. Evidence is also
presented that disulfide bonding of cysteine 192 and 193 requires the
same conformational change. Finally, addition of an N-linked glycan to
this region increases binding site formation and the efficiency of
assembly. Our data suggest that the added glycan expedites a
conformational change of residues 187-199 that is rate limiting,
thereby increasing binding site formation and the assembly process.
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MATERIALS AND METHODS |
AChR subunit and mutant cDNAs. Torpedo
, , , and subunit cDNAs and mouse , , , and subunit cDNAs subcloned into the pRBG4 expression vector (Lee et al.,
1991) were a gift from Dr. S. Sine (Mayo Clinic, Rochester, MN). The
mouse subunit N-glycosylation site mutation cDNA,
187-189 or W187N/F189T (Kreienkamp et al.,
1994) also cloned into pRBG4, was a gift from Dr. P. Taylor (University
of California, San Diego, San Diego, CA).
Cell lines. Mouse L fibroblasts, stably transfected with the
Torpedo subunit cDNAs under the control of SV 40 promoters
(Claudio et al., 1987), were maintained in DMEM plus 10% calf
serum and HAT (15 mg/ml hypoxanthine, 1 mg/ml aminopterin, and 5 mg/ml
thymidine) at 37°C in 5% CO2. To enhance
subunit expression, the DMEM was supplemented with 20 mM sodium butyrate (NB medium) 36 hr before the experiment.
Transient transfections. For transient transfections, the
human embryonic kidney-derived tsA201 cells (Margolskee et al., 1993)
were cultured in DMEM supplemented with 10% calf serum. A calcium
phosphate protocol described previously (Eertmoed et al., 1998) was
used for transfection. Because Torpedo subunits exhibit a
temperature-dependent assembly, the temperature was dropped to 20°C
for 2 d before the experiment. Cells transfected with mouse
subunits were maintained at 37°C for 1 d before the experiment.
Metabolic labeling. Cultures (10 cm) of stably
transfected cells (see Fig. 1) or 6 cm cultures of transiently
transfected tsA201 cells (see Fig. 3) were labeled as described
previously (Green and Claudio, 1993; Green and Wanamaker, 1997).
Briefly, cultures were pulse labeled in 2 (see Fig. 1) or 1 (see Fig.
3) ml of methionine-cysteine-free medium, supplemented with 333 (see Fig. 1) or 111 (see Fig. 3) µCi of a
35S-methionine
35S-cysteine mixture (NEN
EXPE35S35S).
The labeling was stopped with the addition of DMEM plus 5 mM methionine. To follow the subsequent changes
in the labeled subunits, the cells were "chased" by incubation for
the indicated times in regular medium at 20°C (see Fig. 1,
Torpedo subunits) or 37°C (see Fig. 3, mouse subunits).
All subsequent steps were performed at 4°C to prevent further subunit
folding and assembly. The cells were solubilized in 1% LPC (1.83 mg/ml
phosphatidylcholine and 1% Lubrol) (see Fig. 1) or 1% Triton (see
Fig. 3) in lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH
7.4, and 0.02% NaN3) containing protease
inhibitors (2 mM phenylmethylsulfonyl fluoride, 2 mM n-ethylmaleimide, and 10 µg/ml each of
chymostatin, pepstatin, tosyl-lysine chloromethyl ketone, and leupeptin).
Immunoprecipitation and Bgt-Sepharose precipitation.
Solubilized AChR subunits were immunoprecipitated with mAb 383c (a gift from Dr. R. Fairclough, University of California, Davis, CA), which is
specific for Torpedo subunits, mAb P22 (a gift from Dr.
V. Lennon, Mayo Clinic), which is specific for mouse subunits, or a
polyclonal Ab specific for both Torpedo and mouse subunits (Ross et al., 1991). Immunoprecipitations were performed by
overnight incubation at 4°C, and Ab-subunit complexes were
precipitated by incubating with Protein G-Sepharose for 3 hr at 4°C.
Alternatively, solubilized subunits were precipitated with a slurry of
Bgt-Sepharose, which was prepared by coupling Bgt to cyanogen
bromide-activated Sepharose according to the manufacturer's
directions (Amersham Pharmacia Biotech, Arlington Heights, IL).
mAb-Protein G- or Bgt-Sepharose-precipitated subunits and complexes
were 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. To determine subunit band intensities, gels
were exposed to a phosphor screen, developed using a PhosphorImager and
quantified using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).
125I-Bgt binding. For cell surface
125I-Bgt-binding, the cells were washed
with PBS and incubated at room temperature in PBS containing 4 nM 125I-Bgt for 2 hr. This incubation time is sufficient to saturate the binding. The
cells were then washed three times in PBS and counted in a gamma
counter. For intracellular
125I-Bgt-binding, cell surface receptors
were preblocked by incubation with 10 nM
unlabeled Bgt in medium at 20°C for at least 1 hr before solubilization (see Fig. 2). Because Bgt dissociates more rapidly from
the 187-189 mutated receptors, cell surface
receptors were immunodepleted with mAb 35 [American Type Culture
Collection (ATCC)] before intracellular receptor binding instead of an
unlabeled Bgt preblock (see Fig. 3). For this, intact cells were
incubated with saturating amounts of mAb 35 in PBS overnight at 4°C.
Excess mAb was removed with three washes with PBS. The cells were then solubilized in 1% Triton X-100 in lysis buffer with protease
inhibitors, and the mAb 35-bound surface receptors in the lysate
were precipitated out with Protein G-Sepharose. The
intracellular receptors remaining in the lysate were then incubated
with 5 nM 125I-Bgt
and mAb 35 overnight at 4°C and precipitated with Protein G-Sepharose, followed by three washes with lysis buffer to remove excess Bgt. The 125I-Bgt bound was then
counted in a gamma counter. For transiently transfected cells,
125I-Bgt binding in parallel samples from
sham-transfected cultures for which no cDNAs were included in the
calcium phosphate solution estimated nonspecific binding. For stably
transfected cells, nonspecific 125I-Bgt
binding was measured by binding in the presence of 10 mM carbamylcholine.
Alkylation with bromoacetylcholine bromide. For
measuring bromoacetylcholine bromide (bromoACh) alkylation of
intracellular receptors, surface receptors in 10 cm cultures of cells
stably transfected with Torpedo receptor subunits were
blocked by incubating in medium containing 10 nM
Bgt for 2 hr at 37°C, and then the temperature was lowered to 20°C
to initiate assembly. After 6, 24, or 48 hr at 20°C, the unbound Bgt
was washed away. The cells were harvested, solubilized in 1% LPC in
lysis buffer with protease inhibitors, and immunoprecipitated with
saturating amounts of mAb 88b (ATCC), which recognizes the and subunits. If receptors needed to be reduced before bromoACh alkylation
(see Fig. 2A), the precipitated receptor complexes
were incubated with 0.5 mM dithiothreitol (DTT)
in lysis buffer for 30 min at room temperature and rapidly washed three
times. The precipitated receptor complexes were incubated with the
indicated concentration of bromoACh (see Fig. 2A) or
with the saturating concentration of 10
4
M (see Fig. 2B) for 30 min at
room temperature. For the experiment in Figure 2B,
104 M bromoACh was
also included in the solubilization buffer. Unalkylated bromoACh was
washed off the precipitated complexes with lysis buffer, and the
bromoACh-treated receptors were bound with 10 nM
125I-Bgt as described above, in
lysis buffer containing 0.5% BSA. To control for block of
125I-Bgt binding by unalkylated agonist,
an equal number of samples were treated in parallel with
104 M ACh instead
of 104 M bromoACh.
To measure bromoACh alkylation of surface receptors without DTT
reduction, mAb 35 was bound to surface receptors by incubating intact
cells in PBS with mAb 35 overnight at 4°C. The cells were solubilized
and incubated with Protein G-Sepharose to precipitate the surface
receptor-Ab complexes, which were then incubated with 104 M bromoACh or
104 M ACh, washed with lysis
buffer, and bound with 125I-Bgt. The
percentage of alkylated receptors was determined as above.
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RESULTS |
The subunit 187-199 region changes conformation during binding
site formation
A monoclonal antibody, mAb 383c (Gomez and Richman, 1983)
generated against the Torpedo nicotinic AChR, was used to
probe for conformational changes during formation of the AChR binding sites. The interaction between mAb 383c and Torpedo AChRs
has been well characterized. Its epitope has been mapped to subunit residues 187-199 (Fairclough et al., 1998a), a region that contributes to both the Bgt and ACh binding sites. In support of this region contributing to the Bgt binding site, mAb 383c binding specifically inhibits Bgt binding to native Torpedo AChRs (Mihovilovic
and Richman, 1987). To test whether mAb 383c recognizes
Torpedo AChRs heterologously expressed in mouse fibroblast
cells, its effect on cell-surface 125I-Bgt
binding was assayed. As shown in Figure
1A, saturating
concentrations of mAb 383c blocks virtually all surface
125I-Bgt binding to intact fibroblasts.
Shown for comparison in Figure 1A is the effect of
mAb 247g on cell surface 125I-Bgt binding.
mAb 247g binds to subunits and blocks surface Bgt binding at only
one of the two binding sites so that saturating concentrations inhibit
only 50% of binding (Mihovilovic and Richman, 1987; Green and
Wanamaker, 1998). Thus, mAb 383c appears to bind to subunits in
assembled receptors and block Bgt binding to both binding sites as
observed previously for the native Torpedo AChRs.
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Figure 1.
Accessibility of mAb 383c to subunit residues
187-199. A, Binding of mAb 383c, which specifically
recognizes subunit residues 187-199, prevents 125I-Bgt
binding to AChRs. Intact cells stably expressing Torpedo
AChRs were incubated with the indicated amounts of mAb 383c
(squares) or mAb 247g (circles) for 2 hr
before performing 125I-Bgt binding to cell surface
receptors. After 125I-Bgt labeling, cells were washed and
counted. Points on the graph represent the mean of
duplicate samples. B-D, AChR subunits were
metabolically labeled for 30 min at 37°C and followed in culture for
the indicated times at 20°C. Equal amounts of solubilized, labeled
subunits were incubated with mAb 383c (B),
Bgt-Sepharose (C), or a polyclonal Ab
(D) and then precipitated subunits were
visualized by SDS-PAGE (7.5% gel) and autoradiography. The
band just above the subunit band
(' in D) in the subunit-specific AChR
precipitations has been shown previously to be an unprocessed form of
the subunit (Green and Claudio, 1993; Green and Wanamaker, 1997).
Both and subunits migrate as doublet bands as a result of
differential processing, possibly differences in disulfide bonding
(Gelman and Prives, 1996) or oligosaccharide trimming (Chang et al.,
1997). E, A comparison of mAb 383c epitope and Bgt
binding site formation. Subunits precipitated with mAb 383c or
Bgt-Sepharose were analyzed on SDS-PAGE as in B and
C and quantified using phosphorimaging. The values for
and subunits from both 383c and Bgt-Sepharose experiments are
shown. The values for and subunits are displayed in Table 1.
For the and subunits, both doublet bands were included in the
analysis.
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The slow, temperature-dependent assembly of Torpedo AChRs
was used to determine when during assembly mAb 383c recognizes subunits. At 37°C, subunit assembly is halted at the earliest stages.
Assembly can be initiated by lowering the temperature to 20°C and
proceeds at this temperature at a rate more than an order of magnitude
slower than for the mammalian subunits (Green and Claudio, 1993).
Subunits were labeled at 37°C, the temperature was lowered to 20°C,
and the labeled subunits were followed for the indicated times (Fig.
1B-D). mAb 383c immunoprecipitated subunits only
after a delay of 3-6 hr (Fig. 1B). At the 3 and 6 hr
time points, the ratio of /// subunits precipitated by mAb
383c was ~1:1:1:1 (Fig. 1E, Table
1). This result indicates that the
epitope is accessible to mAb 383 only after subunits have assembled
into complexes with the other three subunits. After the 3-6 hr delay,
mAb 383c precipitated progressively more of the assembled complexes
until ~24 hr after the temperature shift, at which time the ratio of
the to the other subunits almost doubled (Fig.
1E, Table 1). The lack of subunit precipitation by mAb 383c at early time points is not caused by an absence of labeled
subunits. Other Abs that recognize subunits, such as the subunit-specific polyclonal Ab used in Figure 1D,
precipitate an excess of subunits relative to the other subunits at
the early time points. Because mAb 383c binds to denatured subunits or linear stretches of the subunit virtually as well as it binds to
fully assembled receptors (Fairclough et al., 1998b), the delayed recognition of subunits by mAb 383c indicates that the mAb 383c epitope is inaccessible at the early stages of AChR assembly. Thus,
during AChR assembly, subunits appear to change conformation from a
state in which residues 187-199 are inaccessible to mAb 383c to a
state in which this region is accessible to the mAb.
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Table 1.
Quantitation and comparison of the time course of formation
of the mAb 383c epitope and Bgt binding site during assembly of the
Torpedo AChR
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Because Bgt binding sites appear on subunits during AChR assembly
(Green and Claudio, 1993) and the mAb 383c epitope contributes to the
Bgt binding site, we tested whether Bgt binding site formation parallels the appearance of the mAb 383c epitope. The formation of Bgt
binding sites was assayed using Bgt-Sepharose, which can be used to
recognize and precipitate assembling receptor complexes (Fig.
1C) (Green and Wanamaker, 1998). Saturating amounts of
either mAb 383c or Bgt-Sepharose were used to precipitate equal
aliquots of the pulse-labeled subunits. We observed no significant
difference in the amount of labeled subunits precipitated by mAb 383c
or Bgt-Sepharose at any of the chase times (Fig.
1B,C). The results from six
different pulse-chase experiments, performed as in Figure 1,
B and C, were quantified, and the means and
SD of each subunit gel band are displayed in Figure
1E and Table 1. The similar intensities of the
subunit bands precipitated with mAb 383c or Bgt-Sepharose indicate that
the mAb 383c epitope becomes accessible at approximately the same time
as the first Bgt binding site appears during AChR assembly. Initially,
the ~1:1:1:1 ratio of /// subunits precipitated by both
mAb 383c and Bgt-Sepharose occurs because tetramers are
rapidly assembled immediately after the appearance of the first Bgt
binding site on trimers (Green and Wanamaker, 1998). Later,
the ratio of the to the other subunits almost doubles as
2 pentamers assemble (Green and
Claudio, 1993). Because mAb 383c binds specifically to the 187-199
region (Fairclough et al., 1998a) and this region comprises a major
part of the Bgt binding site, it appears that the 187-199 region
becomes accessible to mAb 383c as part of the subunit folding that
forms the first Bgt binding site. This interpretation of our data can explain why the Bgt binding site is not initially present after subunit
synthesis, although Bgt can bind to the 173-204 peptide alone and to
denatured subunits (Wilson et al., 1988; Chaturvedi et al.,
1992).
Affinity alkylation of subunit cysteines 192 and/or 193 without
previous reduction
Significant evidence indicates that most, if not all, of AChR
assembly occurs before the subunits are released from the endoplasmic reticulum (ER) (Smith et al., 1987; Gu et al., 1989; Ross et
al., 1991; Gelman et al., 1995). If the 187-199 region is in fact initially buried, as suggested by the inaccessibility of the mAb 383c
epitope, then the disulfide bond between cysteines 192 and 193 would
not be expected to form until this region changes conformation and
becomes accessible to the oxidizing ER lumen (Hawkins et al., 1991;
Hwang et al., 1992). Affinity alkylating agents, such as bromoACh, bind
to the ACh binding site and specifically alkylate the
Torpedo subunit 192 and 193 cysteines after their
reduction (Silman and Karlin, 1969; Damle et al., 1978; Kao and Karlin, 1986). BromoACh was used to determine when the 192-193 disulfide bond
forms by testing at different times whether bromoACh could alkylate the
cysteines without previous reduction of the disulfide bond. When
solubilized intracellular AChR complexes were reduced with 0.5 mM DTT, bromoACh alkylated the binding sites and
blocked all 125I-Bgt binding to the
complexes in a dose-dependent manner (Fig. 2A). Intracellular
AChRs were separated from cell surface receptors by first blocking
surface receptors with cold Bgt before solubilization. Without previous
DTT reduction, bromoACh was unable to alkylate and block Bgt binding to
cell surface receptors (Fig. 2B). However, a
significant number of intracellular AChR complexes were alkylated by
bromoACh in the absence of reduction (Fig. 2B). The
ability to alkylate with bromoACh without reduction depended on when
the cells expressing AChRs were harvested after a temperature shift from 37 to 20°C. Six hours after initiating assembly, a saturating concentration of bromoACh (104
M) alkylated 20% of the intracellular complexes
with no previous reduction of disulfide bonds (Fig.
2B). At lower bromoACh concentrations, fewer
complexes were alkylated without previous reduction, consistent with
the dose dependence observed with reduction (Fig. 2A,
open symbols, B). Six hours after the temperature
shift, assembling subunit intermediates are either trimers
that lack ACh binding sites or tetramers on which the
first of two ACh sites forms (Green and Wanamaker, 1998). At this time,
if alkylation by bromoACh occurs with the specific binding of bromoACh
to ACh binding sites, then we can conclude that bromoACh is alkylating
subunits assembled into tetramers. Almost all newly
assembled tetramers continue on to assemble into pentamers and are
transported to the surface (Green and Wanamaker, 1998), indicating that
the 20% of the Bgt binding sites alkylated by bromoACh in the absence
of DTT are not on misfolded subunits.
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Figure 2.
BromoACh alkylates assembling AChR subunits
without previous reduction. A, Dose dependence of
bromoACh alkylation of intracellular AChR complexes that were reduced
with (filled diamonds) or without (open
squares) DTT. Intracellular AChR complexes were
immunoprecipitated with the and subunit-specific mAb 88b.
Samples were then treated with or without 0.5 mM DTT and
incubated with the indicated concentrations of bromoACh. The bromoACh
alkylation was determined by measuring the loss of 125I-Bgt
binding caused by alkylation. For the DTT-treated samples
(y-axis on the left), the
percentage of bromoACh alkylation was determined as the percentage of
125I-Bgt binding blocked by bromoACh alkylation in the
alkylated samples compared with samples not treated with bromoACh but
treated with DTT. Shown are the data obtained 6 and 48 hr after
initiation of assembly. Points on the graph represent
the mean ± SD of four measurements, two at 6 hr and two at 48 hr.
For the samples not treated with DTT (y-axis on
the right), the bromoACh alkylation was performed 6 hr
after initiation of assembly. The percentage of bromoACh alkylation was
determined as the percentage of 125I-Bgt binding blocked by
bromoACh alkylation compared with samples treated with
104 M ACh instead of bromoACh and was
maximally 20% of the total 125I-Bgt binding (for details,
see Fig. 2B). B, BromoACh
alkylation of intracellular and surface receptors without previous
reduction. At the indicated times after the temperature shift from 37 to 20°C, intracellular AChR complexes were alkylated using the
indicated concentration of bromoACh without previous reduction with
DTT. In parallel, cell surface AChRs were incubated with
104 M bromoACh before
125I-Bgt binding. The percentage of bromoACh alkylation was
determined as the percentage of 125I-Bgt binding blocked by
bromoACh alkylation compared with samples treated with
104 M ACh instead of bromoACh. For
each experiment, the bromoACh and ACh treatment was performed in
triplicate. The values shown represent the mean ± SEM of
n experiments. The values determined using
104, 106, and
107 M bromoACh at 6 hr are replotted
(open squares) in A. Statistical
significance of the differences between the means for
104 M bromoACh was estimated using the
t test and the Mann-Whitney rank sum test. The 6 hr
intracellular mean was significantly different from all those at 24 and
48 hr (t values ranging from 2.62 to 2.89; of 0.05).
The 24 and 48 hr values did not differ significantly from each
other.
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Twenty-four and 48 hr after the temperature shift to initiate AChR
assembly, a much smaller percentage of the complexes were alkylated
without previous reduction by a saturating concentration of bromoACh
(Fig. 2B). Our ability to affinity alkylate cysteines 192 and 193 without reduction was transitory and was largest early in
assembly when the first ACh binding site appears. The second ACh
binding sites appear 24-48 hr after the temperature shift (Green and
Wanamaker, 1998), and the much smaller percentage alkylated by bromoACh
at these times could indicate that the second site forms differently in
that the 192 and 193 cysteines are oxidized more rapidly than at the
first site. However, an alternative explanation for the data are that,
for assembling AChR subunits, events occur more synchronously early in
assembly than later. The time when the 192 and 193 cysteines can be
affinity alkylated without reduction occurs after the cysteines become
accessible to the ER lumen and before the disulfide bond forms.
Therefore, the ability to observe a significant amount of alkylation
requires that the change in cysteine accessibility occur on a
significant number of AChR complexes at approximately the same time.
Because the formation of the second Bgt and ACh binding sites occurs
24-48 hr after the temperature shift, these events should be less
synchronous and fewer cysteines would be expected to be available for
alkylation at the same time.
Addition of an N-linked glycan to subunit residue 187 increases
assembly and binding site formation
To alter the initial conformation of the subunit region
187-199, an N-linked glycosylation consensus site was added so that residue 187 was glycosylated. N-Linked glycosylation of this residue is
of interest because it, in part, causes the resistance to Bgt binding
that is observed in certain snakes and mongoose (Neumann et al., 1989;
Barchan et al., 1992). We wanted to test whether the oligosaccharide
would prevent the amino acids 187-199 from being buried within the subunit and thereby expedite formation of the Bgt site. For the
Torpedo subunit N-linked glycosylated at residue 187 and
coexpressed with the other subunits, the dissociation rate for Bgt from
the Torpedo mutant was too rapid to quantitatively characterize the Torpedo mutant using our standard Bgt
binding techniques (data not shown). As an alternative, a mutant mouse muscle subunit with the glycosylation site at residue 187 was obtained (187-189; a kind gift from Dr. P. Taylor). As with Torpedo receptors, Bgt dissociates more
rapidly from receptors containing the mouse
187-189 mutant compared with wild-type receptors (Kreienkamp et al., 1994) but with a dissociation rate slower
than for the Torpedo receptor. The slower dissociation of
Bgt from the mouse 187-189 subunit makes it
feasible to determine whether the rate at which Bgt binding sites form is affected by glycosylation of residue 187.
187-189 mutant or wild-type mouse subunits along with mouse , , and subunits were transiently
expressed in the human embryonic kidney cell line tsA201 to assay
125I-Bgt binding. In parallel,
125I-Bgt binding was performed on cell
lysates to measure intracellular sites (Fig.
3A) and on intact cells to
measure surface sites (Fig. 3B). Because Bgt dissociates
more rapidly from the 187-189 mutant subunit,
cold Bgt could not be used to quantitatively block 125I-Bgt binding to surface receptors as
in Figure 2. To separate intracellular Bgt binding sites from the
surface sites, saturating amounts of mAb 35 were bound to intact cells
and the mAb 35-bound receptors were precipitated after solubilization.
mAb 35, which is a conformation-specific mAb (Merlie and Lindstrom,
1983), was used because it appears to recognize and precipitate AChRs
containing 187-189 subunits just as well as
wild-type subunits. When metabolically labeled,
187-189 subunits were precipitated by mAb 35 just as well as wild-type subunits, and the same percentage of
125I-Bgt-bound surface AChRs was
precipitated by mAb 35 whether they contained
187-189 subunits or wild-type subunits (data not shown). Intracellular 125I-Bgt binding
sites, precipitated with mAb 35 appeared 2 hr after the transfection,
and their formation saturated after 24 hr. Over this time period,
N-linked glycosylation of the mouse 187-189 subunit resulted in a threefold increase in the formation of
intracellular Bgt binding sites compared with wild-type subunits.
As observed previously (Kreienkamp et al., 1994), a 2.5-fold increase
in 125I-Bgt binding to the cell surface
was observed for AChRs containing 187-189
subunits compared with AChRs containing wild-type subunits. The
appearance of 125I-Bgt binding sites on
the surface lagged behind the appearance of intracellular sites by ~2
hr. Because only fully assembled AChRs are released from the ER and
transported to the surface, the lag in surface expression is consistent
with the time for assembly and transport to the surface (Merlie and
Lindstrom, 1983).
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Figure 3.
Glycosylation of subunit residue 187 alters
AChR assembly. A, Effect of the glycosylation of subunit residue 187 on formation of Bgt binding sites. Mouse mutated or
wild-type subunits were transiently transfected along with the
mouse , , and subunits into tsA201 cells, and
125I-Bgt binding to intracellular AChR subunits was
determined at the indicated times. The 125I-Bgt binding was
plotted as the fraction of maximum binding for wild-type -containing
AChRs at 24 hr. All points on the graph represent the
mean ± SD of three samples. Maximum binding was 69 ± 1.3 fmol. B, Effect of the glycosylation of subunit
residue 187 on AChR surface expression. In parallel to the experiments
in Figure 3A, 125I-Bgt binding to surface AChRs was
determined at the indicated times. The 125I-Bgt binding was
plotted as in A. Again, the points on the
graph represent the mean ± SD of three samples. Maximum binding
was 132 ± 5.3 fmol. C, D, Subunit
synthesis and formation of Bgt binding sites. Mouse mutated or
wild-type subunits transiently transfected with the , , and
subunits were metabolically labeled for 5 min and followed in
culture for the indicated times at 37°C. Equal amounts of
solubilized, labeled subunits were incubated with mouse -specific
mAb P22 (C) or Bgt-Sepharose
(D) and analyzed by SDS-PAGE (7.5% gel) and
autoradiography. E, Quantitation of the metabolically
labeled mutated and wild-type subunits. Metabolically labeled
mutated and wild-type subunits were precipitated with the -specific
mAb, mAb P22, analyzed on SDS-PAGE as in C, and
quantitated using a PhosphorImager. Points on the graph
represent the mean ± SEM band intensities from five
experiments.
|
|
To further characterize the effects of the
187-189 mutation, subunits were metabolically
labeled and precipitated with either subunit-specific Abs (Fig.
3C) or Bgt-Sepharose (Fig. 3D), which
precipitates Bgt-binding subunits.
187-189 or wild-type mouse subunits were
expressed along with mouse , , and subunits and metabolically
labeled for 5 min, after which maturation of the labeled subunits was
followed in culture for the indicated times (Fig.
3C,D). The 187-189
subunits had an apparent molecular weight larger than wild-type
subunits because of the addition of the oligosaccharide at
residue 187. The addition of the glycan occurred as soon as the
subunits were labeled, consistent with the cotranslational addition of
the oligosaccharide. subunit-specific Abs precipitated comparable
amounts of labeled wild-type and mutated subunits at all of the
time points as shown from the quantitation of wild-type and mutated subunit bands from five experiments (Fig. 3E). The rate of
synthesis and the stability of the 187-189
subunits are thus indistinguishable from that of the wild-type
subunits. Furthermore, there were no differences in the levels of
labeled , , and subunits when coexpressed with the
187-189 subunits compared with their expression with the wild-type subunits (data not shown).
Significant differences were observed when
187-189 mutant and wild-type subunits
were precipitated with Bgt-Sepharose (Fig. 3D). Under
identical conditions, Bgt binding sites formed more readily on the
187-189 subunits compared with wild-type subunits. As shown in Figure 3D, wild-type Bgt binding site
formation reached saturating levels by 1 hr of chase, at which point
the 187-189 still had threefold greater Bgt
sites. Because the 187-189 subunit affinity
for Bgt is less than that of wild-type, this estimate of the increase
in Bgt binding site formation is perhaps an underestimate. However, the
increase observed with the 187-189 subunit
for Bgt-Sepharose precipitation and
125I-Bgt binding to the total cell lysate
was similar. The intensities of the subunit gel bands in Figure
3D plus four other separate experiments were estimated.
These values were used to determine the ratio of the
187-189 subunit to wild-type averaged over
all the time points. This ratio was 3.1 ± 0.3-fold (mean ± SEM) for Bgt-Sepharose precipitation of the
187-189 to wild-type subunits. The ratio
of 187-189 to wild-type subunits for
125I-Bgt binding to AChRs in the total
cell lysate was also determined. The ratios were measured over four
time points between 0 and 24 hr after transfection (i.e., until
saturation of binding) in three separate experiments, and a ratio of
3.1 ± 0.8 (mean ± SEM), which is approximately the same as
for Bgt-Sepharose binding, was observed. Because there was no apparent
difference in subunit protein levels between
187-189 and wild type (Fig. 3E),
the difference in Bgt binding site formation must have resulted from an
increased number of mutated subunits with Bgt binding sites.
Because the half-life for turnover of the surface receptors is 21 hr
(Gu et al., 1990), the kinetics of surface expression are too fast
(Fig. 3B) for a decrease in the mutated AChR turnover rate
to contribute to the difference in surface expression, and we conclude
that the difference in surface expression is caused by an increase in
the efficiency of subunit assembly.
| |
DISCUSSION |
Little is known about how AChR subunits are folded and
processed as ligand binding sites form. Here, we have examined how one
of the major determinants of ligand binding, subunit amino acids
187-199, changes conformation as Bgt and ACh binding sites emerge
during AChR assembly and how this change affects the disulfide bonding
of cysteines 192 and 193. We presented evidence that this region
changes conformation by showing that the region initially could not be
bound by mAb 383c and became accessible only as the first Bgt binding
site formed. The first Bgt binding site forms just before the addition
of subunits to trimers to make tetramers
(Fig. 4) (Green and Wanamaker, 1998). The
timing of the 187-199 conformational change, thus, indicates that it occurs while the subunit is part of a trimer and precedes association of subunits to trimers. Before the formation of the Bgt binding site, a conformational change occurs on trimers in which the subunit amino acids 128 to 142, initially accessible to a mAb specific for this region, becomes inaccessible (Green and Wanamaker, 1997). The conformational change of amino acids
128-142 occurs with a time course similar to the appearance of the
first Bgt binding site but precedes it because the mAb does not
recognize Bgt binding subunits. Moreover, cysteines 128 and 142 are
disulfide bonded before this conformational change can occur. As
depicted in Figure 4, the conformational change of subunit amino
acids 187-199 (Fig. 4D) appears to be preceded first
by the assembly of trimers and disulfide bonding of subunit cysteines 128 and 142 (Fig. 4B) and second by
the rearrangement of the 128-142 region (Fig. 4C). The
series of events in Figure 4 are the simplest interpretation of our
results. Other proteins, such as the ER chaperone proteins
immunoglobulin heavy chain binding protein (Blount and Merlie,
1991; Paulson et al., 1991; Forsayeth et al., 1992) and calnexin
(Gelman et al., 1995; Keller et al., 1996, 1998), can associate with
AChR subunits. It is possible that the accessibility of the subunit
187-199 region to mAb 383c changes because a chaperone protein that is
bound to the region unbinds. This possibility would alter the model in
Figure 4 in that the appearance of the mAb 383c epitope and the Bgt
binding site would not result directly from a conformational change but from the unbinding of a chaperone protein.
View larger version (35K):
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|
Figure 4.
Sequence of subunit conformational changes and
processing events that occur during formation of the ligand binding
sites. The model proposed in the figure is based on the interpretation
of data presented in this paper together with previous data on AChR
assembly and formation of the ligand binding sites. A,
The subunit rapidly folds during synthesis. B, Soon
after, (or ) trimers assemble and the 128-142 cystine
loop forms. At this point, the 187-199 region is inaccessible to mAb
383c and Bgt, whereas the 128-142 region is accessible to its
region-specific mAb. C, A conformational change causes
the 128-142 cystine loop to become inaccessible to its region-specific
mAb. D, A conformational change exposes the 187-199
region during formation of the Bgt binding site so that Bgt and mAb
383c can bind. E, The ACh binding site forms as the subunit assembles at the interface between the and (or )
subunits, and the and (or ) subunits fold bringing together
all of the residues lining the ACh binding site. F, In
the final step, cysteines 192 and 193 disulfide bond.
|
|
During AChR assembly, a significant delay occurs before subunits
associate with trimers to assemble into
tetramers (Green and Claudio, 1993; Green and Wanamaker, 1998). The
delay occurs despite an abundance of subunits and suggests that
what is rate limiting are the folding events that precede the addition of the subunits. The effect of the
187-189 glycosylation provides additional
evidence that a conformational change of the 187-199 region is part of
a rate-limiting step at this stage of AChR assembly. We found that the
187-189 glycosylation increased Bgt binding
site formation threefold and the efficiency of AChR assembly by at
least twofold. Based on this and other results in this paper, it
appears that the added oligosaccharide lowers the energy barrier for
the 187-199 region conformational change, thereby expediting formation
of the Bgt binding site and, as a consequence, the entire assembly process.
If the glycan added to residue 187 is, in fact, promoting the
conformational change of the 187-199 region, we might expect Bgt
binding sites to appear more rapidly on the mutated subunits. We
attempted to measure the kinetics of Bgt binding site formation during
AChR assembly by briefly metabolically labeling the subunits and
measuring Bgt binding site formation by subunit precipitation with
Bgt-Sepharose (Fig. 3D). Based on quantitation of the bands from several experiments such as the ones shown in Figure
3D, there was no significant difference in the rate in which
the Bgt binding sites formed (data not shown), although Bgt binding
sites did form on many more of the 187-189
subunits compared with wild-type subunits. In another study, we found
that transiently transfected AChR subunits assembled into receptors
much less efficiently than when the subunits were stably transfected
into cells (Eertmoed et al., 1998). The results of this study suggested
that the low efficiency of assembly was caused by cell-to-cell
variations in the amounts of each of the four subunits expressed, which
resulted in much larger amounts of misfolded subunits and misassembled complexes. It is possible that differences in the kinetics of Bgt
binding site formation are masked by the more rapid formation of
misfolded subunits on which Bgt binding sites can form. Consistent with this possibility is that Bgt binding sites formed more rapidly in
the transiently transfected cells (Fig. 3D) than in other
studies in which cells stably expressing the subunits were used (Merlie and Lindstrom, 1983; Blount and Merlie, 1988; Green and Claudio, 1993).
Subsequent to the association of subunits with trimers,
the first ACh binding sites appear on tetramers (Green and
Wanamaker, 1998). Because other subunit residues in addition to
187-199 lie within the ACh binding pocket, rearrangement of the subunit to create ACh binding sites involves a large portion of the subunit extracellular domain (Fig. 4E). The first ACh binding sites formed during assembly are located at or near the interface between and subunits (Green and Wanamaker, 1998) and
residues on the subunits also contribute to the sites (Karlin and
Akabas, 1995; Tsigelny et al., 1997; Changeux and Edelstein, 1998;
Chiara et al., 1999). subunits associate between the and subunits in the trimer (Green and Wanamaker, 1998) and could
alter the interface between and subunits, as depicted in Figure
4, D and E. The assembly of subunits with
trimers may lower the energy barrier for and thus drive the
formation of the ACh binding site. For bromoACh to bind and
specifically alkylate cysteines 192 and 193, much of the first ACh
binding site must be intact. Our ability to block
125I-Bgt binding by bromoACh alkylation
early in assembly indicates the 192-193 disulfide bond is not formed
until after and subunits have rearranged to bring together the
residues that serve as the binding pocket for ACh, as depicted in
Figure 4E. The last step in the formation of the
first ACh binding sites appears to be the disulfide bonding of
cysteines 192 and 193. Initially, this disulfide bonding may be
prevented when the 187-199 region is inaccessible to the oxidizing
potential of the ER lumen. Once this region is exposed to the ER lumen,
disulfides should rapidly form. Our ability to alkylate these cysteines
and block 20% of Bgt binding indicates that the cystine 192-193 does
not form rapidly. What might slow the disulfide bonding of cysteines
192 and 193, and thus allow their alkylation by bromoACh, is the strain
put on the peptide backbone by disulfide bonding adjacent cysteines (Kao and Karlin, 1986). Possibly, cysteines 192 and 193 are not placed
into the proper positions to overcome the strain of disulfide bonding
until after they are positioned in the ACh binding site, as shown in
Figure 4, E and F.
Our experiments have only assayed changes involving the first ligand
binding site, which forms at the - (or ) subunit interface (Green and Wanamaker, 1998). The second ligand binding site on the AChR
appears to differ structurally from the first site. The second ligand
binding site, which forms at the - subunit interface, differs in
its affinity for competitive antagonists (Neubig and Cohen, 1979; Sine
and Taylor, 1981; Blount and Merlie, 1989; Pedersen and Cohen, 1990;
Sine and Claudio, 1991) and is recognized by Abs different from those
that recognize the first site (Mihovilovic and Richman, 1984; Dowding
and Hall, 1987; Fairclough et al., 1998b). However, the same regions of
the subunit and regions on the subunit analogous to those on
the (or ) subunit appear to contribute to the second site. It is
likely, therefore, that a series of events similar to those we
have described for the first site occurs during formation of the second site.
The data presented in this paper suggest that formation of the 192-193
disulfide bond requires the occurrence of a conformational change so
that cysteines 192 and 193 are exposed to the oxidizing ER lumen and
that formation of this bond is one of the final steps in binding site
formation. When cysteines 192 and 193 are mutated to serines, agonists
bind with lower affinity, but the receptors are nonfunctional (Mishina
et al., 1985). In other words, the ACh binding sites form in the
absence of the 192-193 cystine, but ACh binding no longer activates
the receptor. This finding further suggests that the ACh binding site
is not functional until a series of steps is completed ending with the
disulfide bonding of the 192 and 193 cysteines. A similar situation
occurs during assembly of hen lysozyme (van den Berg et al., 1999). Of
the four disulfide bonds present in the native state of hen lysozyme,
one bond, between cysteines 76 and 94, is the last to form. After formation of the first three disulfide bonds of hen lysozyme, a
native-like intermediate structure is formed without an active site.
This intermediate then requires an additional structural rearrangement,
which causes cysteines 76 and 94 to be repositioned and allows
oxidizing agents to access Cys-76 and Cys-94. After this
structural rearrangement and the consequent formation of the
Cys-78-Cys-94 bond, a functional active site is formed. Thus, in hen
lysozyme, as may be occurring in the ACh receptor, acquisition of
functionality depends on the occurrence of a conformational change that
allows formation of a disulfide bond. Formation of the disulfide bond
constitutes one of the final events in the assembly process and plays
the crucial role of conferring functionality to the protein.
| |
FOOTNOTES |
Received Sept. 13, 2000; revised Feb. 15, 2001; accepted Feb. 15, 2001.
This work was supported by grants from the National Institutes of
Health (W.N.G) and the Brain Research Foundation (W.N.G.) and a
training grant from the National Institute on Drug Abuse (M.M. and
C.P.W.). We thank Dr. S. Sine for the AChR subunit cDNAs in pRBG4, Dr.
P. Taylor for the subunit mutation 187-198, Dr. R. Fairclough
for mAb 383c, and Dr. V. Lennon for mAb P22. We also thank John C. Christianson, Dr. R. Fairclough, and members of the Green laboratory
for discussions about this work, and Dr. D. Patneau for comments on
this manuscript.
Correspondence should be addressed to William N. Green, Department of
Neurobiology, Pharmacology, and Physiology, University of Chicago, 947 East 58th Street, Chicago, IL 60637. E-mail:
wgreen{at}midway.uchicago.edu.
| |
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Ar
TOP
ABSTRACT
INTRODUCTION
