The Role of the Cystine Loop in Acetylcholine Receptor Assembly*

(Received for publication, November 6, 1996, and in revised form, April 24, 1997)

William N. Green Dagger and Christian P. Wanamaker

From the Department of Pharmacological and Physiological Sciences, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Nicotinic acetylcholine receptors (AChRs) are composed of alpha , beta , gamma , and delta  subunits, assembled into alpha 2beta gamma delta pentamers. A highly conserved feature of ionotropic neurotransmitter receptors, such as AChRs, is a 15-amino acid cystine "loop." We find that an intact cystine loop is necessary for complete AChR assembly. By preventing formation of the loop with 5 mM dithiothreitol, AChR subunits assemble into alpha beta gamma trimers, but the subsequent steps in assembly are blocked. When alpha  subunit loop cysteines are mutated to serines, assembly is blocked at the same step as with dithiothreitol. In contrast, when beta  subunit loop cysteines are mutated to serines, assembly is blocked at a later step, i.e. after assembly of alpha beta gamma delta tetramers and before the addition of the second alpha  subunit. After formation of the cystine loop, the alpha  subunit undergoes a conformational change, which buries the loop. This conformational change is concurrent with the step in assembly blocked by removal of the disulfide bond of the cystine loop, i.e. after assembly of alpha beta gamma trimers and before the addition of the delta  subunit. The data indicate that the alpha  subunit conformational change involving the cystine loop is key to a series of folding events that allow the addition of unassembled subunits.


INTRODUCTION

The molecular events involved in subunit folding and assembly of large oligomeric proteins remain largely uncharacterized. The subunit folding and oligomerization events that take place during the assembly of ion channels are particularly complex, since the finished product requires the correct oligomeric arrangement and subunit stoichiometry for proper function (1). In terms of their structure, the best characterized ion channels are the muscle-type nicotinic acetylcholine receptors (AChRs),1 which are the neurotransmitter receptors responsible for rapid signaling between motor neurons and skeletal muscle. Muscle-type AChRs are composed of four distinct, homologous subunits, alpha , beta , gamma , and delta , which assemble into pentamers with the subunit stoichiometry of alpha 2beta gamma delta .

Although AChR assembly is a slow process that takes ~2 h to complete (2), assembly intermediates have been difficult to isolate. A number of laboratories turned to expression of less than the full complement of subunits in different heterologous expression systems to isolate assembly intermediates (3-6). Based on their findings, the "heterodimer" model was proposed, where the alpha  subunit must first fold or "mature," as assayed by the formation of the alpha -bungarotoxin (BuTx) binding site and antigenic epitopes, before assembling with other subunits. The mature alpha  subunit assembles with gamma  or delta  subunits in parallel to form alpha gamma and alpha delta heterodimers, and the heterodimers associate together and with beta  subunits to form alpha 2beta gamma delta pentamers.

We have developed techniques that have allowed isolation of assembly intermediates in cells stably expressing all four AChR subunits (7, 8) and have obtained results at odds with the heterodimer model. Instead of heterodimers, two partially assembled complexes, alpha beta gamma trimers and alpha beta gamma delta tetramers, were isolated. alpha beta gamma trimers, which assemble extremely rapidly, were assembled first into alpha beta gamma delta tetramers and then into alpha 2beta gamma delta pentamers. Our data demonstrated that assembly occurs sequentially, each step being the addition of an uncomplexed subunit. We also demonstrated that the alpha  subunit maturation steps, which were thought to precede its assembly, occurred after assembly into alpha beta gamma trimers but prior to the addition of the delta  subunit. These folding events require a specific combination of subunits and correlate in time with the delta  subunit addition. The data led us to suggest that the alpha  subunit maturation steps are folding events forming the delta  subunit recognition site, i.e. the site where the delta  subunit associates with the alpha beta gamma trimer.

Our goal has been to identify posttranslational processing sites and regions on the AChR subunits involved in AChR subunit folding and assembly. A good candidate is the highly conserved region defined by a pair of cysteine residues separated by a stretch of 13 amino acids, which is found on the neurotransmitter-binding, extracellular domain of the subunits (Fig. 1, A and B). The two cysteines form a disulfide bridge on all four Torpedo AChR subunits (9, 10). Analogous cystine "loops" appear to form on other neurotransmitter-gated ion channel subunits, which include all muscle and neuronal AChR subunits and all GABAA, glycine, and 5HT3 receptor subunits. Other residues in the loop are identically conserved across species from Caenorhabditis elegans to mammals and are even conserved on some of the glutamate receptor subunits (11). Site-directed mutations of the cysteines in the alpha  subunit prevented BuTx binding site formation and reduced AChR expression (12), which suggested that the cystine loop is critical for subunit conformational stability or assembly. A recent study where the conserved proline in the cystine loop (see Fig. 1, A and B) was mutated also suggested that the cystine loop may play a role in subunit assembly (13). However, mutated alpha  or beta  subunits lacking the cystine loop disulfide bond assembled with other wild type subunits (14, 15) and produced functional receptors (15). Based on these data, it was suggested that formation of the cystine loop is not required for subunit assembly but instead plays an important role in the rate that subunits are degraded and in the efficiency of transport of AChRs to the cell surface. Contrary to this viewpoint, we demonstrate in this paper that an intact cystine loop is essential for proper AChR subunit assembly. Elimination of the cystine loop separately on the alpha  and beta  subunits blocks different steps during assembly. For the alpha  subunit, the cystine loop undergoes a conformational change, which appears to be an event required for assembly to continue.


Fig. 1. The cystine loop region of AChR subunits. A, comparison of the cystine loop region in different neurotransmitter-gated ion channel subunits. Shown are sequences from the four Torpedo nAChR subunits (30), the rat brain nAChR alpha 2, beta 2 (31) and alpha 7 (32) subunits, the C. elegans nAChR subunit deg-3 (33), the calf brain GABAA receptor alpha 1 and beta 1 subunits (34, 35), the rat brain glycine receptor alpha 1 subunit (36), the mouse 5HT3 receptor subunit (37), and the rat brain glutamate receptor subunit Glu R1 (11). B, the cystine loop is located within the subunit's N-terminal, extracellular region. The putative secondary structure of AChR subunits is displayed, showing the four membrane-spanning regions, M1-M4 (for a recent review, see Ref. 38).
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EXPERIMENTAL PROCEDURES

Cystine Loop Mutations, Cell Lines, and AChR Subunit Expression

The cystine loop mutations, alpha 128, alpha 142, and beta 128 (15), where the subscript indicates which cystine residues were replaced by serine residues, were a generous gift from Dr. Sumikawa. The cystine loop mutation, alpha 128/142, was created by inserting the alpha 142 BclI fragment back into alpha 128. alpha 128/142 and beta 128 were subcloned into the EcoRI site of pSVDF4 (16). The alpha 128/142beta gamma delta and alpha beta 128gamma delta cell lines were established by stably transfecting each of the two cystine loop mutations along with the appropriate wild type subunit constructs plus the thymidine kinase gene (tk) into mouse fibroblast L cells deficient in tk as described previously (7). To establish the alpha 128 cell line, mouse NIH3T3 cells were infected with the retroviral recombinant, pDOJ, which contained the alpha 128 subunit cDNA in the EcoRI site as well as the neomycin resistance gene. The alpha beta gamma delta and alpha beta gamma cell lines, which stably express the indicated Torpedo AChR subunits, were previously described (7, 8).

The assembly of the Torpedo subunits is temperature-dependent (7, 17). To allow assembly to occur, the temperature was dropped from 37 to 20 °C. The transfected Torpedo subunit cDNAs in the alpha beta gamma delta cell line are under the control of SV40 promoters (7). To enhance expression of Torpedo AChR subunits, the medium (Dulbecco's modified Eagle's medium (DMEM; JRH Scientific) plus 10% calf serum (Hyclone) and HAT (15 µg/ml hypoxanthine, 1 µg/ml aminopterin, and 5 µg/ml thymidine), was replaced with fresh DMEM supplemented with 20 mM sodium butyrate (NB medium; Baker) 36-48 h prior to the experiment.

Metabolic Labeling and Immunoprecipitations

To pulse-label the subunits, 10-cm cultures were first washed with PBS and starved of methionine for 15 min in methionine-free DMEM. Cultures were incubated at 5% CO2 and labeled in 2 ml of methionine-free DMEM, supplemented with 20 mM sodium butyrate and 333 µCi of [35S]methionine for 30 min at 37 °C. The labeling was stopped with the addition of DMEM containing 5 mM methionine and, if not "chased," two washes of ice-cold PBS. To follow the subsequent changes in the labeled subunits, the cells were chased. Specifically, these cells were washed two more times with DMEM containing 5 mM methionine and incubated for various times in NB medium at 20 °C in the absence or presence of 5 mM dithiothreitol (DTT). After a [35S]methionine pulse chase, cultures were rinsed with ice-cold PBS, scraped, and pelleted by centrifugation at 5,000 × g for 3 min, and the pellets were resuspended in lysis buffer: 150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4, 0.02% NaN3, 2 mM phenylmethylsulfonyl fluoride, 2 mM N-ethylmaleimide plus 1% solubilizing agent. In some of the experiments, 1 mM MgATP replaced the 5 mM EDTA to reduce the amount of actin that nonspecifically precipitated with the subunits. To solubilize the labeled subunits, a Lubrol-phosphatidylcholine mixture composed of 1.83 mg/ml phosphatidylcholine and 1% Lubrol was used. Antibody-subunit complexes were precipitated with Protein G-Sepharose and electrophoresed on 7.5% SDS-polyacrylamide gels, fixed, enhanced for 30 min, dried on a gel dryer, and exposed to film at -70 °C with an intensifying screen. Autoradiographs were quantified by scanning densitometry using a flatbed scanner and analyzed with the Intelligent Quantifier software from BioImage.

125I-BuTx Binding

To measure cell surface 125I-BuTx binding, cultures were grown at 37 °C for 36 h in NB medium and then shifted to 20 °C for 48 h. Cultures were washed with PBS and incubated at room temperature in PBS containing 4 nM 125I-BuTx (140-170 cpm/fmol) for 2 h, which results in the saturation of binding. Cultures were washed again and solubilized, and the cell surface counts were determined by gamma -counting, or counts were immunoprecipitated with appropriate antibodies and counted. Total cell 125I-BuTx binding was measured after solubilization. In this case, cell lysates were incubated in 10 nM 125I-BuTx at 4 °C overnight to saturate binding at this lower temperature. The 125I-BuTx in the lysates was then immunoprecipitated with the indicated antibodies.

Sucrose Gradients

To distinguish subunit complexes on the basis of their size, solubilized subunits (labeled with [35S]methionine and/or bound by 125I-BuTx) were sedimented on sucrose gradients. For this procedure, lysates were layered on a 5-ml 5-20% linear sucrose gradient prepared in the appropriate lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm to omega 2t = 9.0 × 1011. ~17 fractions (300 µl) were collected from the top of the gradient, and the appropriate antibodies were then added to the fractions to be assayed.


RESULTS

Reduction of Disulfide Bonds Alters Subunit Assembly

When added extracellularly to cultured cells, DTT reaches the endoplasmic reticulum and prevents the formation of protein disulfide bonds without altering most other cellular functions (18). Cells stably expressing the four Torpedo AChR subunits were subjected to an [35S]methionine pulse-chase protocol in the absence or presence of 5 mM DTT (Fig. 2, A and B). Labeled subunits were immunoprecipitated with either a gamma  subunit-specific monoclonal antibody (mAb 168) or alpha  subunit, conformation-dependent mAb 14, to assay for subunit assembly and formation of the mAb 14 epitope. During a 30-min pulse of [35S]methionine, alpha beta gamma trimers formed as shown by the coprecipitation of predominantly alpha  and beta  subunits with the gamma  subunits (Fig. 2A). During the chase in the absence of DTT, progressively more delta  subunits are added to the trimers, followed by the addition of the second alpha  subunit as shown by the doubling in the amount of alpha  subunit relative to the other coprecipitated subunits. These two subunit additions are better resolved by the mAb 14 immunoprecipitations (Fig. 2, A and C). Since the mAb 14 epitope forms on alpha beta gamma trimers just prior to the addition of the delta  subunit (8), these immunoprecipitations show the complete time course of both the addition of the delta  and second alpha  subunits to the alpha beta gamma trimers.


Fig. 2. Reduction of disulfide bonds blocks AChR subunit assembly. A and B, AChR subunit assembly in the absence or presence of 5 mM DTT. A mouse L cell line, stably expressing all four Torpedo AChR subunits (the alpha beta gamma delta cell line; Ref. 7), was pulse-labeled with [35S]methionine for 30 min at 37 °C and chased at 20 °C in the absence or presence of 5 mM DTT for the indicated times. Labeled subunits were immunoprecipitated with either the gamma  subunit-specific mAb 168 or the alpha  subunit, conformation-dependent mAb 14 (24). The band labeled as alpha ' has previously been shown to be different from the delta  subunit (8) (see also Fig. 5B), although it migrates just above the delta  subunit. Further evidence that this band is not the delta  subunit is displayed in lane 1 of Fig. 2A. Cells stably expressing only the Torpedo alpha , beta , and gamma  subunits were subjected to the same pulse-chase protocol as the alpha beta gamma delta cells and immunoprecipitated with mAb 168. The alpha ' band coprecipitates with the other subunits in the absence of any delta  subunit expression. C, time course of mAb 14 epitope formation. Displayed is the quantification of the mAb 14-precipitated alpha , beta , gamma , and delta  subunit bands analyzed by SDS-polyacrylamide gel electrophoresis and quantified from resultant fluorographs by scanning densitometry. Also displayed for comparison are the scanned values of the delta  subunits coprecipitating with the precipitated gamma  subunits. The percentage assembled values are the scanned values divided by the value for the beta  subunit at the 48-h chase time (100%) and are shown to emphasize the time course of the alpha  subunit doubling. D, the rate of gamma  subunit degradation in the absence or presence of DTT. Displayed are the scanned values for the gamma subunit bands chased in the absence or presence of DTT and precipitated by the gamma  subunit-specific mAb (Fig. 2, A and B). Also displayed are values for the beta  subunits that coprecipitate with the gamma  subunits in the absence of DTT (Fig. 2A). The data are plotted on a semilog scale. In the absence of DTT, the decay of the gamma  subunit signal is biphasic. The slowly decaying component corresponds to assembled gamma  subunits as shown by the similar rate of decay of the assembled beta  subunits. To estimate the rate of decay of unassembled gamma  subunits, the 48-h value was subtracted from the other values and plotted as the gamma  adjusted symbols. E, the presence of DTT blocks BuTx site formation. 125I-BuTx binding to the cell lysate of alpha beta gamma delta cells was measured for cells grown in the absence or presence of 5 mM DTT for different lengths of time. The 0 time point is the time at which the cultures were shifted from 37 to 20 °C to start subunit assembly. A single 10-cm culture was used for each time point.
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In the presence of DTT, the subunits clearly retain the ability to assemble into alpha beta gamma trimers and with approximately the same efficiency as occurs in the absence of DTT (Fig. 2B). This result is at odds with a recent study (19), which suggested that 5 mM DTT completely blocks AChR subunit assembly. There are several differences between this and the other study that explain the conflicting results. Probably the most important difference in terms of the ability to observe alpha beta gamma trimers was that their only assay for measuring the assembly of alpha  subunits with other subunits was an immunoprecipitation with delta  subunit-specific antibodies. Obviously, alpha beta gamma trimers could never be observed with this assay. Another difference was the protocol used to solubilize the AChR subunit complexes. We have shown previously that solubilization in 1% Triton X-100 causes the assembly intermediates to dissociate and that the subunit associations are stable when solubilized with a combination of Lubrol PX and phosphatidylcholine (8).

After alpha beta gamma trimers assemble in the presence of 5 mM DTT, the ensuing subunit associations, the addition of the delta  and second alpha  subunits, fail to occur, and the alpha beta gamma trimers that had assembled were degraded 48 h after the [35S]methionine pulse (Fig. 2B). gamma  subunits in the presence of DTT degrade at about the same rate as unassembled gamma  subunits in the absence of DTT (Fig. 2D). Subunit complexes degrade more rapidly in the presence of DTT because the complexes are no longer stabilized by events subsequent to trimer formation. Although there are almost as many alpha beta gamma trimers present with DTT at the times when delta  subunits normally assemble with alpha beta gamma trimers (Fig. 2, A and B; 3- and 6-h chase), the formation of alpha beta gamma delta tetramers and alpha 2beta gamma delta pentamers is inhibited in the presence of DTT. Furthermore, the BuTx binding site (Figs. 2E and 3A) and the mAb 14 epitope (Fig. 2B) do not form on the alpha beta gamma trimers in the presence of DTT. We conclude that the addition of DTT blocks subunit assembly after the association of the alpha , beta , and gamma  subunits and before the BuTx binding site and mAb 14 epitope form on the alpha beta gamma trimers. Since formation of the BuTx binding site and mAb 14 epitope precede the addition of the delta  subunit (8), the data suggest that a block of these folding events by DTT prevents subsequent subunit associations.


Fig. 3. The effect of DTT on formation of the BuTx site. A, cell surface 125I-BuTx binding. Cell surface 125I-BuTx binding was determined for the alpha 128/142beta gamma delta and alpha beta 128gamma delta cell lines. Also determined was cell surface 125I-BuTx binding for the alpha beta gamma delta cell line grown in the absence (alpha beta gamma delta cells) or presence (alpha beta gamma delta  + DTT) of 5 mM DTT. Nonspecific binding was estimated by adding 10 mM carbamylcholine (alpha beta gamma delta  + carb) during 125I-BuTx binding. Each bar is the mean and S.D. determined from measurements on 4-6, 6-cm plates. B, total cell 125I-BuTx binding. 125I-BuTx binding to the cell lysate of alpha beta gamma delta , alpha 128/142beta gamma delta , and alpha beta 128gamma delta cells was measured. 125I-BuTx-bound complexes were immunoprecipitated by the alpha  subunit-specific mAb 35 (Developmental Studies Hybridoma Bank) or the beta  subunit-specific mAb 148. For both the alpha beta gamma delta and alpha beta 128gamma delta cells, mAb 35 precipitated 1.5-2-fold more 125I-BuTx sites than mAb 148. Each bar is the mean and S.D. determined from measurements on 4-6 wells of a 6-well plate.
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Elimination of Cystine Loop Disulfide Bond on the alpha  Subunit Blocks Assembly

The addition of DTT in the above experiments prevents disulfide bond formation for all AChR subunits as well as for other proteins that might affect AChR subunit assembly. To test whether the cystine loop on AChR subunits is involved in the DTT block of assembly, we obtained mutations of the Torpedo AChR alpha  and beta subunits in which cysteines forming the cystine loop were replaced by serines (15). Of the four AChR subunits, only alpha  subunits contain an additional cystine formed between adjacent cysteines (cysteines 192 and 193) located at the ACh binding site (20) (Fig. 1B). Deletion mutations eliminating that cystine cause the loss of ACh binding but have no effect on BuTx binding, subunit assembly, or cell surface expression (12, 14). An alpha  subunit construct was created, alpha 128/142, where both cystine loop cysteines were replaced by serines to avoid the possibility that aberrant disulfides form between either cysteine 192 or 193 and the remaining cystine loop cysteine. For the beta  subunit mutation, beta 128, the formation of the cystine loop was eliminated by replacing only cysteine 128 with serine. Each of the two mutated subunits, along with the other three wild type subunits, was stably transfected into L fibroblasts. Cell lines were isolated that expressed each of the mutated alpha  and beta  subunits and the corresponding three wild type subunits (Figs. 4A, lane 1, and 5A, lane 1).


Fig. 4. Elimination of the cystine loop disulfide on the alpha  subunit. A, AChR subunit assembly in the alpha 128/142beta gamma delta cell line. alpha 128/142beta gamma delta cells were pulse-labeled with [35S]methionine and chased for the indicated times. Lane 1, labeled subunits were immunoprecipitated with a mixture (cocktail) of alpha , beta , gamma , and delta  subunit-specific antibodies, which consisted of alpha  and beta  subunit-specific polyclonal antiserum (39) and the gamma and delta  subunit-specific mAb 88b (American Tissue Culture Collection). Lanes 2-6, labeled subunits were immunoprecipitated with the alpha subunit-specific polyclonal antiserum, eluted from protein G-Sepharose with a 0.1 M glycine buffer (pH 2.5), and precipitated a second time with the alpha  subunit-specific polyclonal antiserum. Only significant amounts of the beta  and gamma  subunits coprecipitate with the alpha 128/142 subunit, and the efficiency of these interactions was reduced 2-3-fold relative to wild type subunit assembly (see Fig. 2A, lane 2, and Fig. 6C, lane 1). B, formation of the mAb 35 epitope during subunit assembly in the alpha 128/142beta gamma delta cell line. alpha 128/142beta gamma delta cells were pulse-labeled and chased for the same times as in Fig. 4A (lanes 2-6) except that labeled subunits were immunoprecipitated with mAb 35. C, the rate of alpha 128/142subunit degradation. Displayed are the scanned values for the alpha 128/142 subunit bands (Fig. 4A). The data are plotted on a semilog scale. The half-life was estimated to be 10.9 h for alpha 128/142 based on a least squares fit of an exponential function to the data, which is within the range of values found for the wild type subunits under the same conditions (8).
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Fig. 5.

Elimination of the cystine loop disulfide on the beta  subunit. A, AChR subunit assembly in the alpha beta 128gamma delta cell line. alpha beta 128gamma delta cells were pulse-labeled with [35S]methionine and chased for the indicated times. Labeled subunits were immunoprecipitated with a mixture (cocktail) of alpha , beta , gamma , and delta  subunit-specific antibodies (lane 1), the beta  subunit-specific mAb 148 (lanes 2-6), or mAb 14 (lane 7). As in Fig. 2, A and B, the band labeled as alpha ' coprecipitates with the other subunits. The band (~43 kDa) that migrates between the alpha  and beta  subunit bands is believed to be actin. B, the rate of beta 128 subunit degradation. Displayed are the scanned values for the beta 128 subunit bands and the alpha  subunits that coprecipitated with the beta 128 subunits in Fig. 5A. The data are plotted on a semilog scale. The half-life was estimated to be 15.4 h for beta 128 subunits based on a least squares fit of an exponential function to the data, which is within the range of values found for the wild type subunits under the same conditions. C, sedimentation of alpha beta 128gamma delta subunit complexes. alpha beta 128gamma delta and alpha beta gamma delta cells were bound with 125I-BuTx as in Fig. 3B and size-fractionated on a 5-20% linear sucrose gradient. Shown are 125I counts from the 125I-BuTx-bound intracellular complexes in fractions 4-14, which were immunoprecipitated with the beta  subunit-specific mAb 148. alpha beta gamma delta cell surface complexes were first bound with cold BuTx to block 125I-BuTx binding to the surface AChRs. Also displayed on the figure are the standards, alkaline phosphatase (5.4 S), catalase (11 S), and surface alpha 2beta gamma delta complexes (9 S; dashed line). The alpha beta 128gamma delta complexes peak at 8 S as estimated from a least squares linear regression fit to the S values of the standards. The shape of the intracellular alpha beta gamma delta profile can be duplicated by the sum of the intracellular alpha beta 128gamma delta profile reduced by 60% plus the 9 S peak cell-surface alpha 2beta gamma delta complexes. This indicates that intracellular alpha beta 128gamma delta complexes are similar in size and composition to intracellular alpha beta gamma delta complexes with the exception of the alpha 2beta gamma delta complexes in the 9 S peak. The broad profile observed for the intracellular AChR complexes relative to the cell surface alpha 2beta gamma delta 9 S peak has been seen in other studies both for the Torpedo subunits at reduced temperature (4, 8, 40) and the mouse subunits at 37 °C (3, 5, 6, 8). D, the effects of the alpha 128/142 and beta 128 subunits on subunit assembly are consistent with the model shown. The alpha 128/142 subunit and DTT block assembly after the formation of trimers but before the formation of the BuTx binding site and mAb 14 epitope. The beta 128 subunit blocks assembly after the addition of the delta  subunit but before the addition of the second alpha  subunit.


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125I-BuTx binding experiments were performed on the stably transfected cells to characterize the effect of the mutations on AChR expression (Fig. 3, A and B). No 125I-BuTx binding was detected on the cell surface of either the alpha 128/142beta gamma delta or the alpha beta 128gamma delta cell lines (Fig. 3A) or for the intracellular compartments of the alpha 128/142beta gamma delta cells (Fig. 3B). However, intracellular 125I-BuTx binding sites were expressed in the alpha beta 128gamma delta cells (Fig. 3B). The intracellular 125I-BuTx binding sites were immunoprecipitated using beta  subunit-specific antibodies, which demonstrates that these sites in the alpha beta 128gamma delta cells contained the mutated beta  subunits.

Since the alpha 128/142beta gamma delta cells failed to express any BuTx sites, we tested whether the mutated alpha  subunit assembled with other subunits. [35S]methionine pulse-chase experiments were performed on the alpha 128/142beta gamma delta cell line to characterize the assembly of the mutated subunit with the three wild type subunits. Displayed in Fig. 4A are the [35S]methionine-labeled subunits from the alpha 128/142beta gamma delta cells immunoprecipitated with alpha  subunit-specific polyclonal antibodies (lanes 2-6). As shown by the coprecipitation of the wild type beta  and gamma  subunits with the alpha 128/142 subunit, these two wild type subunits assemble with the mutated alpha  subunits in approximately a 1:1 ratio. No assembly was observed between delta  subunits and the mutated alpha  subunits. These results indicate that alpha 128/142beta gamma trimers assemble, but neither the delta  nor second alpha 128/142subunits subsequently assemble with the alpha 128/142beta gamma trimers. We were unable to precipitate any [35S]methionine-labeled subunits with conformation dependent mAb 14 (data not shown); thus, the mAb 14 epitope does not form on the alpha 128/142beta gamma complexes during assembly. Furthermore, BuTx binding sites failed to form on the alpha 128/142beta gamma complexes (Fig. 3, A and B). Subunit assembly in the alpha 128/142beta gamma delta cells is thus blocked at the same step as the block of assembly by DTT, i.e. after assembly of the alpha beta gamma trimers and before the BuTx binding site and mAb 14 epitope form.

Similar to the alpha beta gamma trimers assembled in the presence of DTT, the assembled alpha 128/142beta gamma complexes degrade more rapidly than alpha beta gamma complexes in the absence of DTT. The faster rate of degradation appears to occur because the complexes are no longer stabilized by events subsequent to trimer formation (Fig. 2D). As shown in Figs. 4C and 5B, the rate of alpha 128/142 and beta 128 subunit degradation is in the range found for the unassembled wild type subunits (8). Therefore, the failure to complete the assembly process is not caused by an increased rate of degradation of the mutated subunits.

In addition to the block of assembly, the efficiency of assembly of the alpha 128/142beta gamma trimers was reduced 2-3-fold relative to wild type subunit assembly (see Fig. 2A, lane 2, and Fig. 6C, lane 1). A similar reduction in assembly efficiency was observed for subunit complexes containing the beta 128 subunit (Fig. 5A). The decrease in assembly efficiency could be caused by misfolding of some of the mutated subunits, which has been observed for other proteins where cysteines have been mutated (21). To examine whether alpha 128/142 subunits are misfolded, we tested whether the alpha 128/142 subunits are recognized by mAb 35. mAb 35 is a conformation-dependent antibody specific for the alpha  subunit (2). mAb 35 differs from mAb 14 in that its epitope forms on unassembled alpha  subunits, and it forms well before the mAb 14 epitope (8). As shown in Fig. 4B, the results obtained with the mAb 35 precipitation are similar to the results with alpha  subunit-specific polyclonal antibodies (Fig. 4A, lanes 2-6). mAb 35 recognizes a large percentage of unassembled alpha 128/142 subunits as well as the alpha 128/142beta gamma complexes, which indicates that the unassembled alpha 128/142 subunits are not grossly misfolded.


Fig. 6. Block of assembly correlates with a conformation change of the alpha  subunit cystine loop. A, the specificity of the mAb that specifically recognizes the alpha  subunit cystine loop (cystine loop mAb). Lanes 1 and 2, alpha beta gamma delta cells were pulse-labeled with [35S]methionine, chased 3 h in 5 mM DTT, and immunoprecipitated with alpha  subunit-specific polyclonal antiserum (lane 1) or the cystine loop mAb (lane 2). Lanes 3 and 4, alpha 128 cells were pulse-labeled with [35S]methionine and immunoprecipitated with alpha  subunit-specific polyclonal antiserum (lane 3) or the cystine loop mAb (mAb 259; lane 4). B, alpha beta gamma delta cells were pulse-labeled with [35S]methionine and chased for the indicated times. Labeled subunits were precipitated with alpha  subunit-specific polyclonal antiserum (lane 1) or the cystine loop mAb (lanes 3-8). In lanes 1 and 8, subunits were denatured with 1% SDS prior to the immunoprecipitation. The marked alpha ' band, which migrates just above the delta  subunit band, is different from the delta  subunit. It is specifically precipitated by alpha  subunit-specific antibodies even after coprecipitating subunits are dissociated by the SDS treatment (lane 1) and is expressed in cells, the alpha beta gamma cells, which lack the delta  subunit (lane 2). C, alpha beta gamma delta cells were pulse-labeled with [35S]methionine and chased for the indicated times. Labeled subunits were precipitated with alpha  subunit-specific polyclonal antiserum. D, alpha beta gamma delta cells were pulse-labeled with [35S]methionine and chased for the indicated times in the presence of 5 mM DTT. Labeled subunits were precipitated with alpha  subunit-specific polyclonal antiserum.
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Elimination of the Disulfide Bond on the beta  Subunit Blocks Assembly at a Later Step

The finding that the BuTx binding site forms on complexes that contain the beta 128 subunit (Fig. 3B) suggests that subunit assembly with the beta 128 subunit progresses to a later step than assembly with the alpha 128/142 subunit. The assembly of the beta 128 subunit with the wild type alpha , gamma , and delta  subunits was characterized using an [35S]methionine pulse-chase protocol with the alpha beta 128gamma delta cells as shown in Fig. 5A, lanes 1-7. In contrast to subunit assembly with the alpha  subunit mutation or after DTT treatment, assembly continued after the formation of alpha beta gamma trimers. Immunoprecipitation of the labeled subunits with beta  subunit-specific antibodies (lanes 2-6) coprecipitated the delta  subunit as well as the alpha  and gamma  subunits. The mAb 14 epitope forms on the assembled complexes as shown by precipitation of the [35S]methionine-labeled subunits by mAb 14 (Fig. 5A, lane 7), and as with the beta  subunit-specific antibodies, all four subunits are precipitated. The BuTx binding site also forms on these complexes as shown by the 125I-BuTx-bound complexes precipitated by beta  subunit-specific antibodies (Fig. 3B).

The assembly of alpha beta 128gamma delta complexes differs from the assembly of the four wild type subunits in that the second alpha  subunit is not added to the alpha beta 128gamma delta complexes. The pulse-chase experiments demonstrate that the amount of alpha  subunit coprecipitated with beta 128 subunits (Fig. 5B) is constant throughout the pulse-chase protocol, in contrast to the doubling observed with wild type subunit assembly. To further investigate the nature of the alpha beta 128gamma delta subunit complexes formed, their sedimentation on a linear sucrose gradient was determined (Fig. 5C). The alpha beta 128gamma delta complexes, bound with 125I-BuTx and immunoprecipitated with beta  subunit-specific antibodies as in Fig. 3B, migrated in a peak at a value of 8.3 S. The size of the alpha beta 128gamma delta complexes is consistent with tetramers (8), which sediment at ~8 S and are smaller than the cell-surface alpha 2beta gamma delta complexes, which sediment at 9 S.

The effects of the two subunit mutations and DTT on subunit assembly are summarized in Table I and are consistent with the model displayed in Fig. 5D. Based on the results of both the pulse-chase experiment and the sedimentation of the mature alpha beta 128gamma delta complexes, the end product of subunit assembly in the alpha beta 128gamma delta cells is an alpha beta 128gamma delta tetramer. Thus, the beta 128 subunit, like the alpha 128/142 subunit, causes a block in subunit assembly. However, the beta 128 subunit blocks assembly at a later step, after the formation of alpha beta gamma delta tetramers and before the addition of the second alpha  subunit. The failure of alpha beta 128gamma delta complexes to fully assemble into pentamers provides an explanation for why alpha beta 128gamma delta complexes are not transported to the cell surface (Fig. 3A). Since alpha beta 128gamma delta complexes never fully assemble, most likely they are retained and degraded in the endoplasmic reticulum (22).

Table I. Characteristics of assembly intermediates isolated in different cell lines and under different conditions


 alpha beta gamma trimers  alpha beta gamma delta tetramers  alpha 2beta gamma delta pentamers Cell surface BuTx binding Total cell BuTx binding mAb 14 epitope

 alpha beta gamma delta cells Yes Yes Yes Yes Yes Yes
 alpha beta gamma delta cells with DTT Yes No No No No No
 alpha 128/142beta gamma delta cells Yes No No No No No
 alpha beta 128gamma delta cells Yes Yes No No Yes Yes
Cystine loop-specific mAb complexes Yes No No No No No

The alpha  Subunit Cystine Loop Changes Conformation during Assembly

To address when the alpha  subunit cystine loop forms during assembly, we made use of a mAb (mAb 259) that selectively recognizes the alpha  subunit only when the cystine loop is intact (23). This specificity of the mAb is demonstrated in Fig. 6A, where the mAb failed to recognize either the reduced alpha  subunit or the mutated alpha  subunit with the cystine loop eliminated. In Fig. 6, B and C, the alpha beta gamma delta cells were pulse-labeled with [35S]methionine and chased to test at what point in assembly the alpha  subunit is recognized by the cystine loop mAb. Immediately following the half-hour [35S]methionine pulse, the cystine loop mAb precipitated about the same amount of labeled alpha  subunit as our alpha  subunit-specific polyclonal antibodies (Fig. 6B, compare lanes 1 and 3). The alpha  subunit cystine loop, thus, must form shortly after the subunit is synthesized. Since the events blocked by DTT and by the alpha  subunit mutation occur several hours after the synthesis of the alpha  subunit, the block occurs after the formation of the cystine loop.

The ability of the cystine loop mAb to precipitate alpha  subunits diminished with time. The loss of the epitope occurs during subunit assembly as shown by the difference in the subunits precipitated by the cystine loop mAb compared with the subunits precipitated with the alpha  subunit-specific polyclonal antibodies (Fig. 6, B, lanes 3-7, and C, lanes 1-5). During the chase, the amount of alpha  subunit precipitated by the cystine loop mAb is progressively reduced. By the last chase time, the cystine loop mAb precipitated very little alpha  subunit (Fig. 6B, lane 7) and also was unable to precipitate any significant amount of cell surface AChRs (Fig. 7A). The mAb epitope, although inaccessible to the mAb, is still present on the alpha  subunit as demonstrated by the cystine loop mAb precipitating as much alpha  subunit as the alpha  subunit-specific antibodies after the subunits were denatured with SDS (Fig. 6, B and C, compare lanes 8 and 5). The loss of the epitope thus results from a conformational change that buries the epitope.


Fig. 7. The BuTx binding site does not form on subunit complexes recognized by the cystine loop mAb. A and B, the mAb that specifically recognizes the alpha  subunit cystine loop does not precipitate 125I-BuTx-bound complexes containing the gamma  subunit and mAb 14 epitope. Both cell surface (A) and total cell (B) 125I-BuTx binding were performed on the alpha beta gamma delta cells. The cystine loop mAb (mAb 259) precipitated less than the cell surface counts measured in the presence of carbamylcholine (alpha beta gamma delta cells + carb), while mAb 35 precipitated 60% of the total number of cell surface counts (alpha beta gamma delta cells). The cystine loop mAb precipitated a significant number of total cell 125I-BuTx binding sites, about 12% of the sites precipitated by mAb 14 and the gamma  subunit-specific mAb, mAb 168. However, the number of sites precipitated by the cystine loop mAb was not reduced if the precipitation was performed after the precipitation by mAb 14 or the gamma  subunit-specific mAb. The data indicate that the intracellular 125I-BuTx binding sites precipitated by the cystine loop mAb do not contain the gamma  subunit or the mAb 14 epitope. These BuTx sites appear to be a small population of the unassembled alpha  subunits. Each bar is the mean and S.D. determined from measurements on 4-6 wells of a 6-well plate (A) or 4-6 6-cm plates (B).
[View Larger Version of this Image (74K GIF file)]

The Conformational Change Occurs before Formation of the BuTx Binding Site and the Addition of the delta  Subunit

The only AChR subunit complexes that appear to be recognized by the cystine loop mAb are alpha beta gamma trimers. This is most clearly observed at the times when the delta  subunit is maximally assembled with the other subunits, i.e. 24-48 h after assembly begins (Fig. 2, A and C). At these times, only beta  and gamma  subunits coprecipitate with the alpha  subunits recognized by the cystine loop mAb (Fig. 6B, lanes 6 and 7), although complexes containing delta  as well as the other three subunits are present when all alpha  subunits are precipitated (Fig. 6C, lanes 4 and 5) or when the subunits are precipitated by the gamma -specific mAb or mAb 14 (Fig. 2A). At earlier times in the pulse-chase experiments of Fig. 6, B and C, the alpha ' band, which is specifically recognized by the cystine loop mAb and alpha  subunit-specific antibodies, may be obscuring the presence of any delta  subunit. The DTT block of assembly was used to address whether delta  subunits are present at the earlier times during assembly. As shown in Fig. 2B, the addition of 5 mM DTT blocks assembly so that only alpha beta gamma trimers assemble. In Fig. 6D, subunit complexes were precipitated with the alpha -specific antibodies after cells were treated with 5 mM DTT. There are no significant differences between the results in Fig. 6B (lanes 4-7) and those of Fig. 6D. From this we conclude that the complexes recognized by the cystine loop mAb are identical to the alpha beta gamma trimers precipitated by the alpha -specific antibodies in Fig. 6D.

To further characterize the subunit complexes recognized by the cystine loop mAb, we tested whether the BuTx binding site and the mAb 14 epitope form on these complexes. The cystine loop mAb precipitated only about 12% of the intracellular 125I-BuTx binding sites precipitated by either mAb 14 or the gamma  subunit-specific mAb alone (Fig. 7B). To ascertain whether these 125I-BuTx binding sites are on complexes containing gamma  subunits and the mAb 14 epitope, 125I-BuTx binding sites were first precipitated with either mAb 14 or the gamma  subunit-specific mAb before using the cystine loop mAb. The number of sites recognized by the cystine loop mAb was not significantly reduced if samples were first depleted of complexes containing gamma  subunits or the mAb 14 epitope (Fig. 7B). Therefore, the gamma  subunit-containing complexes recognized by the cystine loop mAb do not contain the BuTx binding site or the mAb 14 epitope. Since formation of the BuTx binding site and the mAb 14 epitope precedes assembly of alpha beta gamma delta tetramers (8), the complexes recognized by the cystine loop mAb appear to be alpha beta gamma trimers. The data further indicate that the cystine loop conformational change occurs prior to the formation of the BuTx binding site and the mAb 14 epitope.

The point during subunit assembly when the cystine loop conformational change takes place is evident in the pulse-chase experiment of Fig. 6B. The loss of the cystine loop epitope during assembly, shown by decreasing amounts of alpha , beta , and gamma  subunits that are precipitated by the cystine loop mAb, begins at the 3-h time point and is almost completed by the 24-h time point (Fig. 6B, lanes 4-6). This is the time period during which the BuTx site and mAb 14 epitope form and are quickly followed by the addition of delta  subunits to the trimers (Fig. 2, A and C; see also Ref. 8). The similarity in the kinetics of the cystine loop conformational change and these assembly events together with the block of these events by alpha 128/142 and DTT demonstrate a strong correlation between the alpha  subunit cystine loop conformational change and the assembly events. As summarized in Table I, there is a close identity between the subunit complexes precipitated by the cystine loop mAb and those assembled in the presence of DTT and with the alpha  subunit mutation. Altogether the data indicate that after the alpha  subunit cystine loop forms, it undergoes a conformational change, which is essential for the BuTx site and mAb 14 epitope to form and for the delta  subunit to associate with the alpha beta gamma trimer.


DISCUSSION

The strong conservation of the cystine loop among the different ionotropic neurotransmitter receptors and throughout evolution suggests that this structure plays a vital role with respect to the receptors. In this paper, we demonstrate that the cystine loop is essential for different events that take place during subunit assembly. Events occurring during the assembly of the AChR can be broadly classified as either subunit folding or oligomerization. Previously, we observed that AChR subunits continued to fold after associations with other subunits (8). The data suggested a link between subunit folding and subsequent subunit associations. We proposed that the folding events, BuTx binding site and mAb 14 epitope formation, are part of the process to create a recognition site for the incoming delta  subunit. In support of this hypothesis, we find that eliminating the alpha  subunit cystine loop blocks these folding events and also blocks the subsequent addition of the delta  subunit to the alpha beta gamma trimer. As shown in the model in Fig. 8, we suggest that a subunit recognition site for the delta  subunit is created concurrently with the alpha  subunit cystine loop conformational change. Because elimination of the beta  subunit cystine loop blocks the addition of the second alpha  subunit to the alpha beta gamma delta tetramer, we further propose that the recognition site for the second alpha  subunit is created in parallel with a change in the conformation of the beta  subunit cystine loop.


Fig. 8. Cystine loop "switch" model. Based on our results, we propose the cystine loop "switch" model, where the formation of the subunit recognition sites for the delta  subunit and second alpha  subunit depend on the conformation of the alpha  and beta  subunit cystine loops as diagrammed. The subunit recognition sites are created by a series of folding events. The folding events that create the delta  subunit recognition site include the formation of the BuTx site and the mAb 14 epitope and the change in conformation of the alpha  subunit cystine loop. The change in conformation of the alpha  subunit cystine loop is essential and occurs early in this chain of events. The cystine loop is modeled as a switch. In the cystine loop "up position," delta  subunit recognition site formation and the rest of assembly are blocked. In the cystine loop "down position," subunit recognition site formation and assembly continue. A similar role for the beta  subunit cystine loop is proposed for the formation of the second alpha  subunit recognition site.
[View Larger Version of this Image (34K GIF file)]

Formation of the different subunit recognition sites is likely to involve large rearrangements of the assembly intermediates. Such a rearrangement occurs on the alpha  subunit. Three folding events, 1) the cystine loop conformational change, 2) the formation of the BuTx binding site, and 3) the formation of the mAb 14 epitope, occur at about the same time and precede the addition of delta  subunits to alpha beta gamma trimers. The three events occur on three separate regions that span the length of the N-terminal domain of the alpha  subunit. The mAb 14 epitope overlaps or is very near to the main immunogenic region (24) at amino acids 67-76 (25, 26), while the BuTx binding site is at the other end of this domain in the region of amino acids 185-196 (27, 28). Regions of the gamma  subunits are also involved in these events, since the presence of this subunit is necessary for the mAb 14 epitope to form and greatly enhances formation of the BuTx binding site (8). Since the alpha  subunit cystine loop conformational change is part of a large rearrangement of the alpha  subunit, it is possible that the cystine loop itself does not directly associate with the delta  subunit during the assembly of the alpha beta gamma delta tetramer. Instead, other regions, involved in subsequent folding events and distant from the cystine loop, could associate with the delta  subunit.

A feature of our model (Fig. 8) is that AChR subunit assembly is controlled by the ordered formation of subunit recognition sites. These events are rate-limiting, as shown by the kinetics of the delta  and second alpha  subunit additions, and as such provide checkpoints during assembly where the fidelity of the assembly process can be tested. Each subunit recognition site would form only if assembly had proceeded properly up to that point. Any misfolded or misassembled intermediates would be prevented from participating in later stages of assembly, and these improperly assembled subunits would be selectively retained and targeted for degradation by the endoplasmic reticulum "quality control" mechanisms (29). This paradigm, where the formation of subunit recognition sites is rate-limiting and contributes to quality control mechanisms during oligomer assembly, is likely to be found in the assembly of other ionotropic neurotransmitter receptors and ion channels (1) and may also apply in the assembly of other complex oligomeric proteins. Key to the formation of subunit recognition sites appears to be the cystine loop conformational change. As shown in Fig. 8, we envision the role of the cystine loop as a switch in subunit assembly. In the "up position," the cystine loop blocks subunit recognition site formation and assembly. In the "down position," subunit recognition site formation and assembly continue. In such a role, the cystine loop conformational change is an essential part of the assembly process, which allows the subunits themselves to guide proper subunit folding and maintains the correctly ordered pathway by which subunits oligomerize.


FOOTNOTES

*   This work was supported by National Science Foundation Grant IBN-9319656, National Institutes of Health Grant P01 NS33826-01A2, a Council for Tobacco Research Scholar Award, and an award from the Brain Research Foundation (to W. N. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: Dept. of Pharmacological and Physiological Sciences, University of Chicago, 947 E. 58th St., Chicago, IL 60637. Tel.: 773-702-1763; Fax: 773-702-3774.
1   The abbreviations used are: AChR, acetylcholine receptor; BuTx, alpha -bungarotoxin; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DTT, dithiothreitol.

ACKNOWLEDGEMENTS

We are most grateful to Dr. Katumi Sumikawa for Torpedo alpha  and beta  subunit mutations alpha 128, alpha 142, and beta 128; to Drs. S. Tzartos, J. Lindstrom, and T. Claudio for antibodies, in particular Dr. J. Lindstrom for mAb 259; to Dr. T. Claudio for the Torpedo alpha , beta , gamma , and delta  subunit cDNAs, the alpha beta gamma delta cell line, and pDOJ vector; and to Dr. D. Fambrough for the vector pSVDF4. mAb 35 developed by J. Lindstrom was obtained from the Developmental Studies Bank (Johns Hopkins University and University of Iowa). We also thank Alison Eertmoed, Arjun Natesan, Justin Chen, and Suleiman Salman for help with some of the subunit constructs and Dr. A. Fluet for discussions at the early stages of this work.


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