TE671 Cells Express an Abundance of a Partially Mature Acetylcholine Receptor (Y Subunit Which Has Characteristics of an Assembly Intermediate*

A partially mature form of the nicotinic acetylcholine receptor a subunit was found to be expressed in the human cell line TE671. We found that 40-50% of the a-bungarotoxin-binding sites in detergent extracts of these cells corresponds to this unassembled a sub- unit. These unassembled a subunits are not found in the surface membrane. The unassembled a subunits in extracts from TE671 cells appear, like mature recep- tors, to have a disulfide bond between Cys-192 and Cys-193 near the acetylcholine-binding site. The unassembled a subunit binds a-bungarotoxin with high affinity, but its dissociation constant is still 5-fold higher than the native assembled acetylcholine receptor. The cholinergic ligands d-tubocurarine and carbamylcholine have negligible affinity for the immature a subunit. Similarly, Xenopus oocytes injected with RNA transcripts for the TE671 a subunit express an a-bungarotoxin-binding component which is insensitive to carbamylcholine and has a sedimentation coef-ficient

A partially mature form of the nicotinic acetylcholine receptor a subunit was found to be expressed in the human cell line TE671.
We found that 40-50% of the a-bungarotoxin-binding sites in detergent extracts of these cells corresponds to this unassembled a subunit. These unassembled a subunits are not found in the surface membrane.
The unassembled a subunits in extracts from TE671 cells appear, like mature receptors, to have a disulfide bond between  near the acetylcholine-binding site. The unassembled a subunit binds a-bungarotoxin with high affinity, but its dissociation constant is still 5-fold higher than the native assembled acetylcholine receptor. The cholinergic ligands d-tubocurarine and carbamylcholine have negligible affinity for the immature a subunit.
Similarly, Xenopus oocytes injected with RNA transcripts for the TE671 a subunit express an a-bungarotoxin-binding component which is insensitive to carbamylcholine and has a sedimentation coefficient on sucrose gradients of 5.0 S. Oocytes injected with RNA for the Torpedo a subunit did not have abungarotoxin binding activity under similar conditions, suggesting a possible differential efficiency in the maturation of this a subunit. We examined the binding of monoclonal antibodies specific to the main immunogenic region and found that this epitope on the unassembled a subunit was formed, but was not in a fully mature conformation because although these antibodies bound, they bound with lower affinity than to native acetylcholine receptors. Antibodies in myasthenia gravis patient sera also bound to the unassembled a subunits, but with an average 14-fold lower titer. The nicotinic acetylcholine receptor (AChR)' found at the neuromuscular junction is an acetylcholine-activated cation channel composed of four structurally related subunits arranged as an @a$ pentamer (reviewed in Karlin et al., 1986;Lindstrom et al., 1988;Unwin, 1989). The extracellular surface of the LY subunit has the binding site for acetylcholine; other agonists and competitive antagonists, including a-bungarotoxin (~Bgt), are presumed to bind at the same site. The disulfide-linked cysteine 192 and 193 of the (Y subunit are near this binding site (Kao et al., 1984;Kao and Karlin, 1986). An additional site on the extracellular surface of the (Y subunit is the main immunogenic region (MIR). Amino acids within the sequence a68-76 contribute to the structure of the MIR (Bellone et al., 1989;Tzartos et al., 1990;Das and Lindstrom, 1989;Saedi et al., 1990). The majority of antibodies produced in animals immunized with intact AChR bind to this epitope . Antibodies to the MIR have also been implicated in the pathology of myasthenia gravis (MG) (Lind-Strom et al., 1988). About two-thirds of the anti-AChR antibodies in sera from MG patients bind to the MIR (Tzartos et al., 1982). Additionally, antibodies to the MIR can cause experimental autoimmune MG, and when added to cultured muscle cells can cause loss of AChRs (Tzartos et al., 1987;Tzartos and Starzinski-Powitz, 1986). The ability of the (Y subunit to bind acetylcholinomimetic ligands, ol-Bgt, and MIR-specific antibodies with high affinity is not an intrinsic property of the polypeptide chain. Cell-free translation systems produce full-length (Y subunit polypeptides which cannot bind (Y-Bgt (Anderson and Blobel, 1981;Sumikawa et al., 1981). Similarly, LY subunits of Torpedo AChR dissociated with sodium dodecyl sulfate and purified do not bind a-Bgt with high affinity (Haggarty and Froehner, 1981;Tzartos and Changeux, 1983). Merlie and co-workers (Merlie and Sebbane, 1981;Merlie and Lindstrom, 1983;Carlin et al., 1986)  or d-tubocurarine. Similar results were obtained when the mouse (Y subunit was expressed in quail fibroblasts in the absence of 0, y, or d subunits (Blount and Merlie, 1988). When mouse LY subunits were expressed in pairwise combinations with y or 6 subunits in fibroblasts, a fraction of the (Y subunits were associated with y or 6 subunits and could be differentiated from unassembled LY subunits by their ability to bind d-tubocurarine and carbamylcholine with high affinity (Blount and Merlie, 1989). The combination of o( and p subunits did not lead to efficient assembly and had binding properties indistinguishable from (Y subunits expressed alone. Similar results were obtained when combinations of Torpedo AChR subunits were expressed in Xenopus oocytes (Kurosaki et al., 1987;Sumikawa and Miledi, 1989).' Thus, the assembly of OL with y or 6 subunits is the next step in the maturation of the (Y subunit. Merlie and Lindstrom (1983) showed in BCSH-1 cells that the assembly of (Y with other subunits proceeds from 30 to 90 min after synthesis. The question remains as to what happens to the nascent (Y subunit which causes it to change from an "immature" form to a "mature" form that can bind a-Bgt and MIR antibodies and assemble with other subunits. A number of possibilities have been suggested, in the form of covalent or noncovalent modification of the CY subunit. These modifications include addition of carbohydrate, formation of disulfide bonds between amino acids , fatty acid acylation, and phosphorylation (Merlie, 1984). These covalent modifications may initiate or accompany other noncovalent conformational changes of the (Y subunit, which may take place with the help of "molecular chaperonins" such as immunoglobulin heavy-chain binding protein and heat-shock proteins (reviewed in Ellis and Hemmingsen, 1989;Pelham, 1988).
Because of the role of AChRs in MG, another question which should also be addressed is whether these maturation processes also occur for AChRs in human muscle.
Recently, our laboratory showed that the human cell line TE671 expresses functional muscle-type AChRs, and we also obtained full-length cDNAs for the (Y and d subunits from these cells (Luther et al., 1989;Schoepfer et al., 1988). AChRs from these cells are practically important as an alternative to amputated leg muscle for diagnostic immunoassays for MG . We now report that TE671 cells also express an abundance of unassembled (Y subunits of the AChR, which makes this cell line a good model system for studying the maturation of the cy subunit.  (Smith, 1968) and were fitted with a linear regression line (Fig. 1). The S values for Torpedo AChR dimers and monomers were 11.5 S and 9.5 S as determined from this regression line. Similarly, the S values for TE671 AChRs and unassembled 01 subunits were 9.5 S and 5.0 S. Preparative sucrose gradients were prepared in the same way as the analytical gradients except that 1.4 ml of extract was layered on 37 ml of 520% sucrose gradients and centrifuged in a VTi 50 rotor (Beckman) at 50,000 rpm for 6 h at 4 "C. Fractions of 1 ml were collected from the bottom of the tube. Samples (25 ~1) from each fraction were then assaved for ""I-labeled a-Bat binding in the solidphase radioimmunoassay.
The peak fractionscontaining "'I-labeled a-Bgt binding activit.y were pooled. Immunoadsorption of Native TE671 AChR-MAb 111 (p subunit specific; Luther et al., 1989)   Standard proteins with known sedimentation coefficients were sedimented on individual 5-ml 5-20% sucrose gradients, as described under "Materials and Methods." The gradients were fractionated from the bottom of the tube and assayed for protein using a BCA protein assay (Pierce). The peak fraction (mean of duplicate gradients) for each protein is plotted uersus the known S value (fractions are labeled from the bottom of the gradient to the top). The standard proteins (m) include catalase (11.1 S), human immunoglobulin G (7.2 S), human transferrin (5.5 S), bovine hemoglobin (4.3 S), P-lactoglobulin (3.1 S), and soybean trypsin inhibitor (2.3 S). Torpedo AChR dimers and monomers prelabeled with "'Ilabeled a-Bgt run in each gradient (Cl) have the calculated sedimentation coefficients of 11.5 S and 9.5 S, respectively. The unassembled (Y subunits in TE671 extracts were found to have an S value of 5.0 0. or 1 PM a-Bgt. After an overnight incubation at 4 "C!, the incubation mixture was aspirated and the wells were washed 4 x with 0.2 ml of PBS, 0.5% Triton X-100. The i" I-labeled a-Bg-t bound was determined by counting the wells in a y-counter. Radioimmunoassay-Pooled gradient fractions were combined with iz51-labeled a-Bgt (0.1 nM when ligand competition was studied), mAb, or antiserum, and 3 ~1 of normal rat serum in a total volume of 0.5 ml. PBS, 0.5% Triton X-100 was used as a diluent when needed. After 15-18 h of incubation at 4 "C, goat anti-rat immunoglobulin (0.1 ml) was added. The samples were incubated an additional 60 min at 4 "C, then 1 ml of PBS, 0.5% Triton X-100 was added, immune complexes pelleted, washed twice with 1 ml of PBS, 0.5% Triton X-100, and counted in a y-counter. Nonspecific and background counts were determined in the absence of specific antibody or in the presence of 1 PM a-Bgt. In ligand competition studies, mAb 210 (2 ~1 of stock solution/tube) was used to immunoprecipitate the '?-labeled a-Bgt binding components.
Titers for mAbs were determined at dilutions of mAb stock solutions that precipitated 520% of the '251-labeled c~-Bgt binding sites in the assay. The titers are reported as the moles 'Z51-labeled a-Bgtbinding sites precipitated per liter mAb stock.

MB;i'A Ak&lati& of a-Bgt-binding
Sites-The affinity reagent [4-(N-maleimido)benzyl]trimethyl ammonium iodide (MBTA) was synthesized by the method of Karlin et al. (1971). Pooled gradient fractions of native AChR or unassembled (Y subunits were adsorbed onto mAb 210~coated microtiter wells by overnight incubation at 4 "C. The wells were washed twice with PBS, 0.5% Triton X-100. Dithiothreitol (DTT) at 1 mM in 5 mM Tris, 100 mM NaCI, 0.5% Triton X-100, pH 8.0, was added to a set of wells and incubated for 30 min at room temperature. Control wells were incubated in buffer only. All wells were washed twice and then incubated for 30 min with 1 /IM MBTA in PBS, 0.5% Triton X-100 or with buffer only (control wells). The wells were washed twice and 1 nM iz51-labeled a-Bgt in PBS, 0.5% Triton X-100 was added. After an overnight incubation, the wells were washed three times with PBS. 0.5% Triton X-100 and counted. Maximum binding was determined from wells which were incubated with only the buffer during the reduction and alkylation steps. Nonspecific binding was determined from wells which were not coated with mAb 210.
In control exueriments, the abilitv of mAb 210 to bind AChRs in this assay was unaffectedby the reduction or alkylation treatments.

Expression of TE671 (Y Subunits in Xenopus
Oocytes-Full-length cDNA clones of Torpedo AChR subunits cloned under the control of SP6 promoter were generously provided by Dr. T. Claudio (Yale University). A full-length cDNA insert of TE671 a subunit isolated previously (Schoepfer et al., 1988) was subcloned into the BglII site of the plasmid vector pSP64T (Krieg and Melton, 1984) under the control of SP6 promoter, and the insertion verified by DNA sequencing. Plasmids were linearized by digestion with XbaI and used for in vitro transcription (Krieg and Melton, 1984). Oocytes were prepared from Xenopus lueuis (Colman, 1984) and injected with 1.5 ng of each subunit RNA synthesized in vitro. Oocytes were incubated at 19 "C for 2 days before analysis. Subunit expression was assessed by homogenizing the oocytes in extraction buffer, incubating the lysate at 4 "C for 30 min, centrifuging the lysate in a microfuge for 30 min at 4 "C, and sedimenting the resulting supernatant on a 5-ml 5-20% sucrose gradient and analyzing the fractions for '251-labeled a-Bgt, as described above.

Antigenic
Modulation of TE671 Surface AChRs-Several control experiments were conducted to show that iZ51-labeled a-Bgt binding sites were lost from the cell surface after mAb 210 treatment. TE671 cells were grown on 6-well plates (Costar) in the medium described above until confluent. Various concentrations of mAb 210, mAb 60, or PBS (also the diluent for the mAbs) in a volume of 10 ~1 was added to the TE671 cells which had 1 ml of medium. The cell cultures were incubated for 24 h at 37 "C. '251-Laheled a-Bgt binding to the cell surface was assayed after washing the cells twice with 2 ml of serumfree medium. '*'I-Labeled a-Bgt (10 nM) was added in serum-free medium, and the cells incubated for 60 min at room temperature. After the incubation the medium was aspirated and the cell layer washed three times with media. The cell layer was solubilized with 0.5 N NaOH (0.5 ml) and counted in a y-counter. Nonspecific binding was determined in the presence of 1 pM a-Bgt or 1 mM carbamylcholine chloride. All binding measurements were determined from 3-6 identically treated wells.
Extracts of cells treated with mAb 210 or PBS were also examined by velocity sedimentation. For these experiments, TE671 cells were grown to confluence in T-175 cm2 flasks. mAb 210 (2 nM) or PBS (200 ,ul) was added to flasks which had 100 ml of medium. The flasks of cells were incubated for 24 h at 37 "C. Extracts of the cells were made and analyzed by velocity sedimentation. The fractionated ma-iZ5 terial was assayed for I-labeled a-Bgt in the solid-phase radioimmunoassay. The cells from three separate flasks for each treatment (PBS or mAb 210) were examined independently.

Detergent extracts of TE671 cells when analyzed by velocity sedimentation
in sucrose gradients shows the presence of two cu-Bgt-binding components with very different molecular weights (Fig. 2). The larger component (9.5 S) cosediments with the monomer of Torpedo AChRs (9.5 S) and is probably the TE671 AChR monomer as characterized by Luther et al. (1989). The smaller component sediments with a size of 5.0 S. The 5.0 S component could go unnoticed if these analyses were conducted using '251-labeled cr-Bgt labeled extracts to determine the AChR, because excess free iz51-labeled cr-Bgt might not be resolved from the 5.0 S component. The size of this component and the ability to bind 1251-labeled cr-Bgt is similar to the unassembled LY subunit described by Merlie-Lindstrom (1983). Although not completely resolved from the 9.5 S component, the 5.0 S component consistently represents 40-50% of the total binding sites for cr-Bgt in extracts of the TE671 cells.
Consistent with the assumption that the 9.5 S component is the intact TE671 AChR is the fact that the binding of 1251labeled cu-Bgt is inhibited by the acetylcholinomimetic agonist carbamylcholine (Fig. 2). However, the binding of '251-labeled or-Bgt to the 5.0 S component is not inhibited by carbamylcholine, even at the high concentration of 10 mM. Cold a-Bgt in excess (1 pM) completely inhibited this binding, indicating that this is not just nonspecific.
More direct evidence that the 5.0 S component is composed of unassembled (Y subunits comes from an adsorption experiment using a specific mAb against the /3 subunit. Extracts were passed through an immunoadsorbent Zetaffinity-10 column that has mAb 111 (specific for the /3 subunit of TE671 AChR, Luther et al., 1989; with an epitope that maps within amino acids 370-410 of Torpedo fi subunits; Ratnam et al., 1986b) coupled to it, and then the unbound material was analyzed by velocity sedimentation.
ol-Bgt binding in the fractionated material was assayed in the presence and absence of carbamylcholine.
As shown in Fig. 3, the 9.5 S component can be completely adsorbed from the extract, leaving behind the 5.0 S component which is unable to bind carbamylcholine. A small peak observed at a position slightly smaller than the size of the 9.5 S component after mAb 111 adsorption also does not bind carbamylcholine and may represent aggregates of (Y as seen in oocytes injected with TE671 cy RNA (described below).
This analysis indicates that only the 9.5 S component is associated with the /3 subunit. To further test whether the 5.0 S component was associated withy or 6 subunits, we separated the 9.5 S and 5.0 S components of TE671 extracts by velocity sedimentation on preparative sucrose gradients. The peak fractions of cu-Bgt binding activity were pooled and analyzed for associated subunits by immunoprecipitation using subunit-specific mAbs and antisera. Fig. 4 shows that mAbs to 01 and fl subunits and antisera to y and 6 subunits can readily immunoprecipitate the 9.5 S component. The quantitative difference in the ability of the mAbs and antisera to immunoprecipitate the TE671 AChR probably reflects differences in affinity or titer, not the absence or presence of subunits in the AChR. In contrast, only the a-specific mAb (mAb 210) effectively precipitates the 5.0 S component. The small effects of the antisera to y and 6 (12-22% of maximum) may indicate that a small percentage of the binding sites are composed of dimers of (Y with y or 6. When pairs of Torpedo AChR subunits are expressed in Xenopus oocytes and then analyzed, it is found that complexes of (Y and y or (Y and b subunits, but not N and p subunits, efficiently form, sediment at 6.1 S, and bind a-Bgt.' Contamination of the 5.0 S with 9.5 S material can be ruled out since mAb 111 (anti-p) did not precipitate any 5.0 S component, but was almost as efficient as mAb 210 (anti-a) in precipitating the 9.5 S component. In addition, we  Oocytes were injected with 1.5 ng of in uitro synthesized RNA for the Torpedo (Y subunit (panel A), the TE671 cy subunit (panel B), or TE671 01 subunit in combination with Torpedo 8, y, and 6 subunits (panel C). After incubating for 2 days at 19 "C, the oocytes were solubilized with Triton X-100 and analyzed by velocity sedimentation centrifugation in sucrose gradients. The fractionated gradients were assayed for '251-labeled oc-Bgt binding activity in the absence (H) and presence (0) of 10 mM carbamylcholine in a solidphase binding assay. Additionally, extracts of oocytes expressing Torpedo (Y subunits were sedimented and analyzed for binding of lZ51labeled mAb 142, an oc-specific mAb (Pun&A,0). The values reported are means of duplicate gradients.
To further confirm that the 5.0 S component was unassembled (Y subunit, we expressed the (Y subunit of Torpedo and TE671 AChRs in Xenopus oocytes and analyzed the expressed proteins by velocity sedimentation and lZ51-labeled LU-Bgt binding. The Torpedo (Y subunit, when expressed in oocytes and analyzed under these conditions, did not show any evidence of an cY-Bgt-binding component (Fig. 5A). However, the Torpedo (Y subunit protein was synthesized and could be detected in the gradient fraction by the binding of lZ51-labeled mAb 142 (Torpedo (Y subunit specific, epitope 01360-366; Ratnam et al., 1986a)3 in a similar solid-phase assay. In addition, a Western blot of the oocyte extract showed the presence of a protein with the electrophoretic mobility of an (Y subunit that was labeled with mAb 142 (data not shown). In contrast, ,' M. Das and J. Lindstrom, unpublished data. when proteins from Triton X-100 extracts of oocytes injected with TE671 01 subunit RNA were sedimented on sucrose gradients, a 5.0 S peak of '251-labeled a-Bgt binding activity was found (Fig. 5B). In addition, a smaller amount of an 8.5 S component was found. Binding of '251-labeled cw-Bgt to neither the 5.0 S nor the 8.5 S component was inhibited by 10 mM carbamylcholine. The 8.5 S component in these oocyte extracts could be oligomers of the (Y subunit or (Y subunits associated with accessory proteins from the oocytes. A similar peak of a-Bgt binding activity was also found when TE671 extracts were depleted of native AChR and analyzed by velocity sedimentation (Fig. 3). These results suggest that, in Xenopus oocytes, Torpedo (Y subunits do not efficiently mature in conformation from nascent chains to synthetic intermediates able to bind ol-Bgt, but that human a subunits do efficiently make this conformation change. Paulson and Claudio (1990) have shown that when expressed in mammalian cells, Torpedo (Y subunits fail to efficiently make a conformation change needed for assembly with other subunits, except at temperatures below 37 "C. This temperature-sensitive conformation change may be the maturation step from nascent chains to assembly intermediates which is efficiently achieved by human LY subunits. The TE671 (Y subunit that is expressed in oocytes can assemble with the p, y, and 6 subunits of the Torpedo AChR and forms a 9.5 S species the size of native Torpedo AChRs which is able to bind carbamylcholine (Fig. 5C). The smaller (6.1 S) peak of OI-Bgt binding activity corresponds in size and carbamylcholine affinity to an ay or ~8 pair.' Even though we were unable to show significant amounts of an (Y subunit with affinity for a-Bgt typical of the putative assembly intermediate when Torpedo (Y subunit RNA is injected alone (Fig. 5A), 9.5 S AChRs can be assembled when the Torpedo (Y, 6, y, and 6 subunits are co-injected into oocytes (Saedi et al., 1990). However, the total amount of 1251-labeied a-Bgt binding activity in extracts and on the oocyte surface is 5-lo-fold greater (90 fmol/oocyte in extracts and 11 fmol/ oocyte on the surface) when human (Y subunits replace the Torpedo LY subunits. The greater efficiency of assembly of hybrid AChRs probably results from the greater efficiency with which human (Y subunits mature to the conformation of an assembly intermediate as compared to Torpedo LY subunits.

Binding
Site Characteristics of the Unassembled N Subunits--Because of the abundance of the unassembled (Y subunits (5.0 S component) in extracts of TE671 cells, we were able to isolate them from the native TE671 AChR (9.5 S component) and characterize the binding of a-B& d-tubocurarine, and carbamylcholine. For analysis of Y-labeled (Y-Bgt, pooled fractions of native AChR and unassembled a were incubated overnight with various concentrations of '251-labeled mu-Bgt and immunoprecipitated with an excess of mAb 210 (which binds to the MIR). The data from this experiment are shown in a Scatchard plot in Fig. 6. The binding of lZ51labeled (Y-Bgt to both native AChR and unassembled (Y is concentration dependent, saturable, and of high affinity. For native AChR solubilized in Triton X-100 and labeled with mAb 210, 'Y-labeled cu-Bgt bound to a single class of sites with a dissociation constant (I&) of 1.3 X lo-" M. '251-Labeled a-B@ similarly bound to unassembled (Y subunits with a slightly lower affinity having a Kd of 6.2 X 10-l' M. These values agree with a kinetically derived Kd of 5.6 X lo-" M for '251-labeled c~-Bgt binding to membrane fragments of TE671 cells (Lukas, 1986) and a Kd of 2.6 X lo-" M for binding of ~Bgt to TE671 AChRs on intact cells (Sine, 1988).
The ability of d-tubocurarine and carbamylcholine to inhibit the binding of lz51-labeled cu-Bgt was also studied in Peak fractions for native TE671 AChR and unassembled 01 subunits from preparative sucrose gradients were pooled and assayed for binding of '251-labeled cu-Bgt (0.01-5 nM) in a radioimmunoassay.
Samples, in a total volume of 0.5 ml, were incubated overnight at 4 "C with '251-labeled ~Bgt and mAb 210 (5 ~1 detail. Various concentrations of d-tubocurarine or carbamylcholine were incubated with detergent-solubilized native TE671 AChR and unassembled cx in the presence of 0.1 nM lz51-labeled a-Bgt. MAb 210 was included in the incubation mixture to precipitate the AChR. d-Tubocurarine and carbamylcholine displace iz51-labeled a-Bgt from native TE671 AChRs with moderate to high affinity with inhibition constants (Ki) of 2.4 X 10-s M and 9.5 X 10m6 M, respectively (Fig.  7A). The inhibition of 'Y-labeled cu-Bgt binding to unassembled (Y by these ligands is more complex. Approximately 25% of the a-Bgt-binding sites have high affinity for d-tubocurarine and carbamylcholine, whereas the majority of the oc-Bgtbinding sites have negligible affinity (Fig. 7B). The K, values for the high-affinity site are 3.8 X 10-s M for d-tubocurarine and 2.5 X lO+j M for carbamylcholine, values comparable to the K; values for native TE671 AChR. The K, values for the low-affinity site are greater than lo-' M. Because reanalysis of the unassembled (Y fraction did not show evidence of contamination with native AChR, it is possible that the carbamylcholine-inhibitable a-Bgt binding is due to 01-6 or o(y dimers, as described by Blount and Merlie (1989), Kurosaki et al. (1987), and Sumikawa and Mieldi (1989).' Fig. 4 suggests that (~7 and a6 pairs could collectively account for 25% of the cr-Bgt binding in the unassembled (Y fraction. The formation of disulfide bonds is one of the post-translational modifications that may be involved in the maturation of the (Y subunit (Merlie, 1984). The disulfide between cysteines 192 and 193 is presumed to be in or near the AChbinding site and can be irreversibly labeled with the affinity reagent MBTA only after reduction of the disulfide Kao et al., 1984). The presence of the disulfide between  was tested using MBTA on the unassembled LY subunits isolated from sucrose gradients after velocity sedimentation.
Native AChR, also isolated from sucrose gradients, was used as a positive control. As seen in Table I, MBTA had no effect on the binding of Y-labeled 01-Bgt to either native AChR or unassembled (Y unless the samples were initially reduced with dithiothreitol, suggesting that a disulfide bond between  is present in unassembled cx subunits from TE671 cells. lo-'0 10-9 10-e 10-7 106 10-s 10." 10-3 10-Z IO-1  MIR Conformation of Unassembled a--We were also interested in characterizing the MIR site on the unassembled 01 subunits. Binding of anti-MIR mAbs was inferred from immunoprecipitation of lz51-labeled a-Bgt native TE671 AChR and unassembled a subunits. Two mAbs which are directed to the MIR were titrated against an equivalent number of o(-Bgt-binding sites of either native TE671 AChR or unassem-mAb 210 (to MIR, less conformation and unassembled LY subunits, as well as the ability of one antibody molecule to precipitate two o( subunits/cu-Bgt-binding sites in native AChRs and only one a-Bgt-binding site in unassembled (Y subunits. In contrast to the 5-fold difference in the titers for mAb 210, we found a 20-fold difference in titers for mAb 35 binding to native TE671 AChR and unassembled (Y subunits. This difference in titers for mAb 35 probably reflects a significant difference in affinity for binding, which suggests that the unassembled (Y subunit has a MIR that has an immature conformation. Furthermore, this conformation of the MIR is probably closer to the native AChR than denatured (Y subunits because mAb 35 can bind, although with low affinity. MAb 35, when added in sufficient quantity, can precipitate all of the cY-Bgt-binding sites of the unassembled a subunit, which means that the difference in titers is because of a difference in affinity and not because it binds to only a fraction of the (Y subunits present. AChR or unassembled LY subunits. Samples (O.l-pmol binding sites) with "' I-labeled a-Bgt (2 nM) and mAb were incubated overnight at 4 "C in a total volume of 0.5 ml. The maximum precipitated was determined using mAb 210 (5 ~1 of stock solution). The apparent titer is reported with units of moles of lz51-labeled o(-Bgt bound per liter of serum. bled a! subunits isolated from sucrose gradients. MAb 210, which was raised against mammalian AChR, binds to native TE671 AChR (Luther et al., 1989), denatured TE671 (Y subunit on Western blots,4 and to the synthetic LY subunit peptide sequence a68-76, which forms part or all of the human MIR epitope (Tzartos et al., 1990;Das and Lindstrom, 1989). MAb 35 raised against Electrophorus AChR binds to the MIR on TE671 AChR, but is highly conformation dependent, does not bind nascent BC3H-1 (Y subunits prior to a conformational maturation (Merlie and Lindstrom, 1983), does not bind to denatured a subunit on Western blots unless very high concentrations are used,4 and does not bind to any peptides corresponding to a MIR epitope (Tzartos et al., 1990;Das and Lindstrom, 1989). MAb 35 binds to the same basic region as does mAb 210, since in vitro mutagenesis of Torpedo Asn a68 to Asp or Asp a71 to Lys inhibits the binding of both mAb 210 and mAb 35 to Torpedo AChRs expressed in oocytes (Saedi et al., 1990).
Immunoprecipitation of native TE671 AChR and unassembled (Y subunits by mAb 210 gives titers of 5.4 and 1.1 pM, respectively (Fig. 8). Because the assay contained equal concentrations of cu-Bgt-binding sites for the native AChR and unassembled a subunits, the differences in the titers may reflect a difference in the affinity for mAb binding to native Reaction with MG Patient Autoantibodies-Because the MIR has been proposed as a major epitope for the binding of immunoglobulins in MG patient sera (Tzartos et al., 1982;Lindstrom et al., 1988), and the unassembled (Y subunits have a near native conformation of the MIR in the absence of other subunits, we had the unique opportunity to test MG patient sera for the contribution of a-specific antibodies to the total complement of anti-AChR antibodies. The titers of 45 different MG patients' sera with a wide range of titers were determined for native TE671 AChR and unassembled (Y subunits (Fig. 9). Of the 45 sera tested, only one did not have a titer for either the native or unassembled (Y subunits. Two sera had moderate titers for native AChR (18.7 and 19.9 nM), but did not immunoprecipitate the unassembled cy subunits. There was a strong linear correlation between the titers for native AchR and unassembled a subunits (r = 0.92; p 5 0.0001); the slope of the regression line was 13.6 (regression of native on unassembled LY). The high degree of correlation, but a slope very much greater than 1, may indicate that antibodies to other subunits make a significant contribution to the titer for native AChR; or it may indicate that anti-MIR antibodies make a major contribution to the titer, but bind with lower affinity to the unassembled (Y subunits, as was seen with mAb 35. Since there is a close correlation between titers 'Ooo t for 24 h and were then assayed for '251-labeled n-Bgt binding. The '251-labeled cu-Bgt bound was determined for three wells, and the mean -I-standard deviation for each treatment is expressed as a percentage of the control treatment (PBS). MAb 60 does not bind to TE671 AChRs and was also used as a negative control.
B, TE671 cells exposed to 2 nM mAb 210 (W) or PBS (Cl) for 24 h were extracted with Triton X-100 and the extract analyzed by velocity sedimentation and "Wabeled cr-Bgt binding.
against native AChR and unassembled a over a wide range of titers, and since the titers against unassembled cy are uniformly lower to about the same degree as the conformationdependent mAb 35, it seems most likely that in most MG patients, conformation-dependent autoantibodies to the MIR make up a large fraction of the anti-AChR titer.

Failure of Surface Expression of Unassembled 01 Subunits-
To test if the unassembled (Y subunits were on the cell surface, we analyzed the surface lZ51-labeled a-Bgt binding and velocity sedimentation of extracts after exposure of the cells to externally applied mAb 210. MAb 210 and other MIR antibodies cause antigenic modulation of surface AChRs (Conti-Tronconi et al., 1981;Tzartos et al., 1985;Sophianos and Tzartos, 1989), and thereby a loss of surface '251-labeled a-Bgt-binding sites. TE671 cells treated with mAb 210 (0.2-10 nM) for 24 h show a concentration-dependent reduction in surface 1251labeled a-Bgt binding which reaches a maximum of -80% (Fig. 1OA). An antibody which does not bind TE671 AChRs (mAb 60) has no effect. When extracts of TE671 cells similarly treated with mAb 210 are analyzed by velocity sedimentation, there is a 32% decrease in the amount of native AChR present, but no decrease in the amount of the unassembled (Y subunit component (Fig. 10B). DISCUSSION Several recent papers from our laboratory have shown that the human cell line TE671 expresses muscle-type AChRs Whiting et al., 1987;Schoepfer et al., 1988), which have been characterized by molecular genetic, biochemical, immunological, and electrophysiological studies (Luther et al., 1989). In addition, Sine (1988) has characterized the AChRs of TE671 cells pharmacologically and electrophysiologically, and their interaction with MG patient sera has been studied by Lang et al. (1988) and Walker et al. (1988). The fact that this cell line, originally identified as a medulloblastoma (McAllister et al., 1977), expressed a muscletype AChR was at first perplexing, but a recent report has suggested that this cell line was misidentified and may indeed be a rhabdomyosarcoma, allaying the confusion (Stratton et al., 1989). Nevertheless, TE671 cell line has been suggested as a source of human AChR for diagnostic radioimmunoassays for detection of anti-AChR antibodies in MG Luther et al., 1989) and has been used to detect acetylcholine binding site-blocking antibodies in MG patients' sera (Pachner, 1989) and to show that MG patient sera can modulate surface AChRs (Sophianos and Tzartos, 1989). However, Walker et al. (1988) indicated that TE671 cells had a heterogeneous population of a-Bgt-binding sites. They showed that mAbs to different regions of the AChR immunoprecipitated different fractions of the total '251-labeled a-Bgt binding activity and that a similar fraction of these binding sites were insensitive to d-tubocurarine. They suspected that the TE671 cell might also have an immature form of the AChR. Our findings in this report have confirmed this, and we have found that the immature form is unassembled cy subunits which have the characteristics of an assembly intermediate.
The abundance of the unassembled a subunits in the TE671 cells has allowed us to do extensive biochemical and pharmacological characterization. Unlike the BC3H-1 cell line (Merlie and Lindstrom, 1983) and cultured rat (Carlin et al., 1986) and chick myotubes (Ross et al., 1987), the TE671 cells produce such an abundance of the unassembled (Y subunit that we did not have to resort to metabolic labeling to show the presence of this component in extracts of the cells. For example, Carlin et al. (1986) found that 10% or less of the total '251-labeled a-Bgt binding activity was accounted for by a component which sedimented in the 5.0 S region in sucrose gradients; we found that the TE671 cells typically have 40-50% of the lz51-labeled a-Bgt-binding sites in the 5.0 S region and sometimes even more binding sites than in the 9.5 S fractions (see Fig. 3). Only mAbs specific for the a subunit were able to immunoprecipitate this 5.0 S component, confirming its identity as unassembled cy subunits. Further confirmation that this 5.0 S component was unassembled a subunits came from expression of the TE671 a subunit in Xenopus oocytes in the absence of other subunits. Extracts of oocytes injected with cy subunit RNA had an identical 5.0 S a-Bgt-binding component which bound to an a-specific mAb (mAb 210) and was insensitive to the agonist carbamylcholine.
In mouse BC3H-1 cells, the acetylcholine-binding site on the a: subunit matures during the 15-30 min after the subunit is synthesized (Merlie and Lindstron, 1983). It is at this time that the (Y subunit acquires the ability to bind a-Bgt with high affinity. We found that the unassembled LY subunit in TE671 cells binds a-Bgt with relatively high affinity (& = 0.62 nM), but still slightly lower affinity than does the fully mature a subunit in the assembled AChR (& = 0.13 nM). In contrast, the BCSH-1 cr subunit, when expressed in fibroblasts, binds a-Bgt with higher affinity than the native AChR (Blount and Merlie, 1989). This may result from species differences, or methodological differences because they studied the binding in crude membranes using low ionic strength buffers, whereas we studied the binding in Triton X-100 extracts using buffers with higher ionic strength. The dissociation constants we obtained (native, 0.13 nM; unassembled LY, 0.62 nM) are similar to those reported for a-Bgt binding to membrane fragments of TE671 cells (& = 0.56 nM; Lukas, 1986) and binding to the TE671 surface AChRs (& = 0.26 nM; Sine, 1988).
The binding of a-Bgt to the unassembled (Y subunit indicates that the acetylcholine-binding site has matured, but further maturation steps are still needed. This is especially true when the binding of small ligands to the unassembled (Y subunits is studied. We found that the majority of the a-Bgtbinding sites were not able to bind either d-tubocurarine or carbamylcholine. Native AChRs bound these ligands with high affinity. Similar results were found in rat myotubes (Carlin, 1986) and for (Y subunits of BC3H-1 AChRs expressed in fibroblasts (Blount and Merlie, 1988) and Torpedo AChR (Y subunits expressed in Xenopus oocytes (Kurosaki et al., 1987). We know from expression of (~7 and a6 subunit pairs in fibroblasts (Blount and Merlie, 1989) and oocytes (Kurosaki et al., 1987;Sumikawa and Miledi, 1989)' that the high affinity binding of d-tubocurarine and carbamylcholine is acquired when the (Y subunit is associated with either the y or the 6 subunits. Approximately 25% of the cu-Bgt-binding sites of the TE671 unassembled (Y subunits were displaced by d-tubocurarine and carbamylcholine with high affinity. These binding sites may be on cuy or (~6 pairs, which were not adequately resolved from the (Y subunit monomer on preparative sucrose gradients.
When we expressed the TE671 (Y subunit in Xenopus oocytes, we found that the LY subunits had the expected size, by velocity sedimentation, and bound cu-Bgt, but not carbamylcholine. Oocytes injected with Torpedo (Y subunit RNA and analyzed in the same way had no Lu-Bgt-binding component, but had o( subunit protein as detected by the binding of an (Y subunit-specific mAb (mAb 142). Other groups have shown that oocytes injected with Torpedo LY subunit RNA do express (Y subunits which bind cy-Bgt (Kurosaki et al., 1987;Sumikawa and Miledi, 1989), however, the binding is sufficiently low and variable that we may not detect it under our assay conditions. Although differences in the amounts of (Y subunit synthesized or differences in protein stability may explain the differences between Torpedo and human (Y expression in oocytes, these data might also indicate that the TE671 (Y subunit forms a mature conformation competent to bind (Y-Bgt, with greater efficiency than does the Torpedo (Y subunit. This may also explain the 5-to lo-fold difference in cr-Bgt binding observed when the TE671 (Y subunit is substituted for the Torpedo (Y subunit and co-expressed in oocytes with the Torpedo @, y, and 6 subunit RNA. The human TE671 LY subunit may be unique in this ability to efficiently form a more mature conformation. A low efficiency of formation of the mature conformation was also found for the mouse (Y subunit. Only 30% of the (Y subunit synthesized in BC3H-1 cells acquires the ability to bind (Y-Bgt (Merlie and Lindstrom, 1983). Similarly, the proportion of (Y subunit synthesized that binds a-Bgt in fibroblasts transfected with mouse LY subunit DNA is approximately 20% (Blount and Merlie, 1988). The differences in the efficiency with which the nascent (Y chain matures in conformation to an assembly intermediate may be due, in part, to the ability to form a disultide bridge between the cysteines at positions 192 and 193 near the acetylcholine-binding site. We found that the unassembled (Y subunit in detergent extracts from the TE671 cells had this disulfide intact. The binding of (Y-Bgt to the DTT-reduced unassembled (Y subunits was considerably decreased; thus if these cysteines were in the reduced state within TE671 cells, there would be a significant fraction of (Y subunit which does not bind a-Bgt, as seen in BC3H-1 cells and rat myotubes, as well as myoblasts expressing Torpedo or fibroblasts expressing mouse (Y subunits (Paulson and Claudio, 1990;Blount and Merlie, 1988). Conformation of the LY subunit is important for binding of mu-Bgt; although the intermediate conformation of unassembled human (Y subunits has relatively higher affinity for oc-Bgt than do Torpedo (Y subunits expressed in Xenopus, synthetic (Y subunit peptides from human LY have much lower affinity for mu-Bgt than do synthetic Torpedo (Y subunit peptides (Neumann et al., 1986;Wilson and Lentz, 1988), and fully assembled human AChR has lower affinity for cy-Bgt than does fully assembled Torpedo or mouse AChR (Sine, 1988).
The MIR is another extracellular region which is thought to change conformation as the LY subunit undergoes maturation. We found that the MIR of the TE671 unassembled (Y subunit bound MIR-specific mAbs, even the highly conformation-dependent mAb 35. Again, as we found for the acetylcholine-binding site, the MIR was still in an immature conformation. The maturation of the MIR has not been extensively studied, but there is evidence that the maturation of the MIR is coincident with the maturation of the acetylcholine-binding site (Merlie and Lindstrom, 1983). Further conformational changes in the MIR also take place as the Torpedo (Y subunit assembles with & y, or 6 subunits.' Because the unassembled (Y subunits from TE671 cells had a near native conformation of the MIR, we were able to test MG patient sera for the titer of LY subunit-specific antibodies. We found that there was a 14-fold difference in the titers for native AChR versus unassembled a and that this difference may be accounted for, in part, by the 2-fold difference in the number of cY-Bgt-binding sites in the native AChR and LY subunit, but probably also by a lower binding affinity of anti-MIR antibodies to the unassembled (Y subunits. The presence of the unassembled o( subunits in extracts of these cells, however, should not prohibit the use of this cell line as a source of human AChR for testing patient sera for autoantibodies to AChRs. The question arises: why are so many unassembled (Y subunits found in TE671 cells? These cells produce many AChRs compared to BC3H-1 cells and especially vast amounts as compared to normally innervated muscle, thus there may be unusually high levels of synthetic intermediates or the human unassembled QL subunit is quite stable, at least compared to Torpedo LY subunit, and accumulates to a high steady-state level. The large amounts of unassembled (Y subunits, small amounts of (~7 and CUC? pairs, and no detected c@ pairs suggests that the amount of 01 subunits is not rate-limiting for assembly of AChRs in TE671 cells. In transfected fibroblasts, Claudio et al. (1990) found that Torpedo @ subunits have the shortest half-life of the subunits and suggested that /3 subunits may be rate-limiting in assembly of AChRs. Our data are consistent with this suggestion.