skeletal muscle acetylcholine receptor

THE Jounw.~ 0~ Bm.mxc~~ CHEMISTRY

Vol. 253, No. 8, Issue of April 25, pp. 2882-2891, 1978 Printed in U.S.A.

Skeletal Muscle Acetylcholine Receptor PURIFICATION, CHARACTERIZATION, AND TURNOVER IN MUSCLE CELL CULTURES*

(Received for publication, June 29, 1977, and in revised form, August 24, 1977)

JOHN PAUL MERLIE,$ JEAN-PIERRE CHANGEUX, AND FRAN~OIS GROS

From the Dbpartement de Biologie Moldculaire, Institut Pasteur, 75015 Paris, France

Acetylcholine receptor has been purified from embryonic skeletal muscle cells grown and allowed to differentiate in tissue culture. The polypeptide composition of purified receptor has been determined by two-dimensional electrophoresis. The purest preparations are composed of a single M, = 41,000 class of polypeptide which exhibits some charge heterogeneity. By high resolution two-dimensional electrophoresis a spot corresponding to acetylcholine receptor was localized among total proteins of muscle membrane extracts. Synthesis of this component is shown to be developmentally regulated. Quantitative analysis of receptor synthesis and degradation has led to the conclusion that receptor is one of a class of proteins whose synthesis is tightly regulated during terminal steps of myogenesis.

The nicotinic ACh-receptor’ is the primary protein component of the postsynaptic membrane at the neuromuscular junction. When adult skeletal muscle fibers are surgically denervated they rapidly become supersensitive to iontophoret- ically applied ACh (1). The number of ACh-receptor sites determined by a-neurotoxin binding increases by more than 20-fold (2, 3). All of the new sites are localized in the extrasynaptic area of the muscle membrane and over its entire surface (4). The increase in the number of toxin binding sites following denervation can be prevented or reversed by chronic electrical stimulation of the muscle fiber (5). Since increase in the number of sites is accompanied by synthesis de nouo of receptor (6) and electrical stimulation has been shown not to increase the rate of receptor degradation (7), it is probable that denervation supersensitivity arises because of an increase in the rate of ACh-receptor synthesis. Experiments in which degradation of bound radioactive toxin has been used as a

* This research was supported by the Muscular Dystrophy Asso- ciations of America, Inc., the Fonds de Development de la Recherche Scientifiaue et Techniaue. the Centre National de la Recherche Scientifique, the Co&i&ariat a L’Emergie Atomique, and the Liaue Nationale Francaise Contre le Cancer. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelv to indicate this fact.

$ Supported by a postdoctoral fellowship frbm the Muscular Dys- trophy Associations of America, Inc. Present address, The Salk Institute for Biological Studies, P. 0. Box 1809, San Diego, Calif. 92112.

’ The abbreviations used are: ACh-receptor, acetylcholine receptor; SDS, sodium dodecyl sulfate; 12SI-B~T~, Y-labeled a-bungarotoxin; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

measure of receptor degradation have led to the conclusion that newly synthesized, extrasynaptic receptor is degraded more rapidly than the synaptic species present initially (8, 9). Embryonic muscle fibers are analogous to denervated fibers. The entire surface is initially sensitive to ACh and after innervation extrasynaptic sensitivity (10) and toxin binding sites disappear (9).

These observations on control of synthesis and degradation of ACh-receptor in response to denervation and innervation suggest that such processes play a critical role in synaptogen- esis. A model has been presented detailing possible biochemical mechanisms by which the acquisition of synaptic specific- ity may be coupled to regulation of synthesis and degradation

of receptor polypeptides (11). Embryonic skeletal muscle myoblasts have proven useful

for the study of ACh-receptor metabolism using radioactive a-neurotoxin as a probe (12-14). We have chosen to work with primary cultures of dissociated skeletal muscle myoblasts from fetal calf embryos because of the ease in obtaining large amounts of material, the reproducibility of tissue culture, and the possibility of doing quantitative precursor labeling experiments in the defined environment of a tissue culture system.

Skeletal muscle myoblasts grow and differentiate in tissue culture. A great deal is known about the synthesis and accumulation of specific muscle proteins and acquisition of muscle phenotype during differentiation in vitro (15, 16). The ACh-receptor is a member of that class of proteins whose activity is developmentally regulated during myogenesis. This phenomenon can be observed in vitro even in the absence of nerve cells. Innervation is the final step in the process of myogenesis and as such the regulatory effects of innervation on the expression of ACh-receptor synthesis must be superim- posed upon the developmental program. For these reasons we have attempted to define the molecular events which result in accumulation of ACh-receptor during myoblast differentiation.

In this paper we describe the direct precursor labeling of the AChR, its purification, and characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of its polypeptide components. By pulse-chase labeling methods we have demonstrated that synthesis of receptor polypeptides is tightly regulated during muscle differentiation.

EXPERIMENTAL PROCEDURE

Cell Culture and Labeling- Primary cultures of skeletal muscle myoblasts of fetal calf were prepared as described previously (17- 19). For routine culture a 3:l mixture of Dulbecco’s modified Eagles’

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Muscle ACh-Receptor 2883

medium and Medium 199 (Gibco, Grand Island, N. Y.1 containing 1% chick embryo extract (Eurobio, Paris) and 10% selected fetal calf serum (Gibco) was used. For labeling, the cells were washed and methionine-free Eagles’ minimal medium supplemented with either 5 or 20 FM L-methionine and up to 100 Z&i/lo-cm culture dish of L [35S]methionine (200 Cilmmol, CEA, France) in a final volume of 2 ml was added. For pulse experiments the labeling time was 4 h. For chase experiments radioactive medium was removed from the dish and the cells were washed twice with warm phosphate-buffered saline and returned to culture in myoblast-conditioned medium supplemented with 50 PM methionine. At the end of a pulse or a chase period culture dishes were washed with phosphate-buffered saline and immediately frozen at -80°C.

Acetylcholine Receptor Purification - The purification of receptor from [3sSlmethionine-labeled cells was done as described previously (20, 21). Erabutoxin b (22) was coupled to cyanogen bromide-activated Sepharose 4B* by the method of Ratner (23) except that the use of reducing agents was omitted. A ratio of 100 pg of toxin/ml of packed beads was found to be optimal. Higher concentrations of toxin seemed to result in greater difficulty in receptor elution.

Cells from which receptor was to be isolated were used fresh or stored for short periods at --80°C. After washing with phosphate- buffered saline, cells were scraped from tissue culture dishes with a rubber policeman in distilled water. The distilled water lysate was centrifuged at 100,000 x g for 30 min and the pellet was extracted 1 h at 4°C with 1% Triton X-100 containing buffer (0.025 M sodium phosphate, 0.18 M NaCl, pH 7.6). In this and subsequent steps buffer contained 1 rnM EDTA, 0.1 mM phenylmethanesulfonyl fluoride, and benxethonium chloride (24) to inhibit protease activity. After extrac- tion and recentrifugation at 100,000 x g for 30 min, the supernatants were filtered through a tight plug of glass wool and adsorbed to small quantities of toxinlagarose (0.1 to 0.2 ml) either at room temperature or at 4°C for periods of 3 to 8 h. Shorter times (3 hl were employed when the purity and integrity of the preparation were important, longer times (8 h) when quantitation was the goal. Adsorption and washing were done in batch in lo-ml test tubes. Washing with 1% Triton X-100, 0.05 M sodium phosphate, and 0.36 M NaCl, pH 7.6 was done by adding one lo-ml aliquot/O.l- to 0.2-ml sample of beads followed by centrifugation and aspiration of the supernatant. The process was repeated until no radioactivity could be detected in the wash, about 10 to 20 times or 1000 to 2000 column volumes.

Elution of receptor bound to toxin agarose was done with 0.1 M

decamethonium in batch at 4°C for 40 h. Affinity elution proved the most variable step in the purification (21). Eluted material was diluted lo-fold with 0.01 M Tris-Cl, 0.01 M NaCl, 0.1% Triton X-100, pH 7.6, and adsorbed to a 0.2-ml Whatman DE52cellulose column and eluted with 0.05 M sodium phosphate, 0.36 M NaCl, 0.1% Triton X-100, pH 7.6. The recovery of toxin binding sites was monitored at every step by a toxin binding assay using L2”I-B~T~ and Whatman DE81 filter paper discs as described (21).

Sucrose Gradient Analysis - Analysis of 35S-labeled receptor labeled with 3H-labeled Naju nigricollis a,-isotoxin was done on 10 to 40% exponential convex sucrose gradients (25) as described previously (20, 21).

Polyacrylamide Gel Electrophoresis - SDS-polyacrylamide gel electrophoresis was done as described by Laemmli (26), on gels of 10% acrylamide. Peptides were analyzed on 20% acrylamide gels as described (27). Except where indicated samples contained 9 M urea in addition to sample buffer and were placed in a boiling water bath for 1 min before application to the gel.

Two-dimensional electrophoresis was done almost exactly as described by G’Farrell(28). The pH gradient of the first dimension was established using pH 5 to 8 and 3.5 to 10 Ampholines in the proportion of 1.6:0.4%. The second dimension was 10% SDS-polyacrylamide gel (26). All samples for two-dimensional electrophoresis were made up to 9 M urea, 2% Ampholines, 2% NP-40, and 5% p- mercaptoethanol and frozen at - 80°C as soon as possible to minimize protein modification (28). Molecular weight estimates were done by comparison to standards. The apparent p1 was determined as described (28). Visualization of radioactive protein in gels was either by autoradiography with Kodak Kodirex x-ray film or by fluorography as described by Laskey and Mills (291. Scanning of autoradiograms was done using a scanning spectrophotometer with peak

* It has been suggested that coupling of toxin to CNBr-activated beads at high pH (9.0) results in improved desorption, J. Lindstrom, personal communication.

integrator. Adsorbance of the exposed film was a linear function of radioactivity with the conditions used.

Analysis ofpeptides Produced by Cleavage at Cysteine Residues- In order to examine the relatedness of the multiple species of 41,000 molecular weight observed in purified receptor, these were localized by autoradiography and cut from fixed and dried electrofocusing gels (see Fig, ID). Radioactive protein was eluted by incubation of gel slices with NH,HCOI buffer, pH 9.0, and 0.1% SDS. Eluates were lyophilixed and resuspended in small volumes and the cleavage products were prepared essentially as described by Jacobson et al (30). The samples were reduced for several hours with 10 mM dithiothreitol and dialyzed against 1% SDS, 1 mM dithiothreitolO.5 M NH,HCOI, pH 9.0. Then, samples were adjusted to 5 rnru DTNB and NaCN was added to 50 mM. Alter 24 h incubation at 37°C an excess of @mercaptoethanol was added and the samples were lyophilixed. After resuspension in distilled water they were analyzed on 20% acrylamide gels (27).

Materials- a-Bungarotoxin was a gift from Dr. A. Kato, College de France, erabutoxin b was a gift from Dr. N. Tamiya, Tokyo, and 3H-labeled N. nigricollis toxin was a gift from Dr. Andre Menez, CEA, France. a-Bungarotoxin was iodinated with iz61 exactly as described by Vogel et al. (311, and the mono and diiodo species isolated by chromatography on CM-Sephadex. The mono-iodo species was used for the experiments described here.

FIG. 1. Analysis of purified ACh-receptor by two-dimensional high resolution polyacrylamide gel electrophoresis. Aliquots of purified ACh-receptor labeled in culture with radioactive amino acids were analyzed by the two-dimensional electrophoretic system described by G’Farrell (28). First dimension electrofocusing (horizon- tal, left to right, basic to acidic) was done for 16 to 20 h. The second dimension (vertical, approximate molecular weight in units x 1O-3 is given) SDS-polyacrylamide gel electrophoresis was done on 1.2- mm slabs of 10% acrylamide as described by Laemmli (26). Gels were prepared for fluorography by the method of Laskey and Mills (291 and exposed to Royal X-Omat for the times indicated. A), W- labeled receptor eluted from toxinlagarose with 0.1 M decamethonium. Approximately 6,500 cpm were applied to the gel. The fluorogram was exposed for 244 h. B), 35S-labeled receptor eluted from the toxinlagarose column (previously eluted with decamethonium) with 9 M urea, 1% SDS, plus 5% p-mercaptoethanol. Approximately 20,000 cpm were applied to the gel. The fluorogram was exposed for 244 h. Cl, 14C-labeled receptor eluted with urea/SDSI@mercaptoethanol from a toximagarose column. Approximately 1000 cpm were applied to the gel. The fluorogram was exposed for 2.3 months. D), 35S-labeled receptor eluted with urea/SDSI/3-mercaptoethanol from a toxin/agarose column, the same as in B. These isoelectric focusing gels were dried and an autoradiogram was done. The indicated bands were cut from the dried gel and eluted for peptide mapping (see Fig. 3).


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2884 Muscle ACh-Receptor

RESULTS Our experience with toxinlagarose affinity chromatography

Purification is that the yield by specific choline@ ligand elution is generally quite low and variable. Those factors which we have

The method of cu-neurotoxin/agarose affinity chromatography used here is described in detail under “Experimental

found to give better results are the following: 1) a relatively low density of toxin sites (100 pg of toxin/ml of beads), 2)

Procedures.” It is essentially similar to that used and described by several other laboratories (32, 33). However, it was

saturation of binding capacity of toxinlagarose (i.e. receptor excess), 3) repeated washing with greater than 1000 column

necessary to adapt this procedure to a small scale so that volumes by centrifugation in batch, and 4) elution times of the receptor from one or a few culture dishes could be isolated in a order of 40 h at 4°C. rapid manner. Table I summarizes results of a typical purification of ACh-

TABLE I

Receptor purifzatim ‘*%BuTx binding’ Recovery of ‘251-B~T~ Sites [WlMethionine Specific activity*

Cell homogenate Triton extract Toxin column adsorbed Toxin column deca eluted Toxin column urea/SDS eluate

pm01 96 CPm pmOllllmOl 7.07 x 108 0.64

45 100 1.05 x 108 4.3 26 58

4.35 9.7 116.8%1’ 1.19 x 104 [15.5%1” 3,700 6.50 x lo4

o Two separate extracts were adsorbed to the toximagarose column. First an extract of “S-labeled cell protein containing 7.25 pmol

c Under the column labeled % recovery of ‘2sI-BuTx binding sites

of toxin binding sites was incubated with the column; 67% of toxin the bracketed value refers to recovery of sites bound to the column. Under the column labeled P?Slmethionine the bracketed value

binding activity was adsorbed. This was followed by an extract of cold carrier cells containing 38 pmol, of which 45% was adsorbed.

refers to the per cent of total PSlmethionine radioactivity eluted by

The binding data are a summation of these values. treatment with decamethonium and with denaturing agents which can be eluted with decamethonium alone. Since the YS-labeled

b Specific activity is expressed in picomoles of toxin binding per nanomole of [Yllmethionine. Since the number of molecules of YG

protein eluted with denaturing agents is shown by electrophoretic

labeled receptor can not be determined, these values are only a analysis to be identical to decamethonium eluted receptor, it is

relative indication of purification and can not be compared directly concluded that all receptor not normally recovered from the toxin/

to normally reported values of toxin binding activity per mg of agarose remains bound to the column and may be recovered by conditions which disrupt protein-protein interaction of the

protein. toxin * receptor complex.

FIG. 2. SDS-polyacrylamide gel analysis of receptor purified at room temperature and at 4°C. A Triton X-100 extract of YS-labeled

washed and eluted in an identical manner with urea/SDS/P-mercap-

cells was first adsorbed to toxinlagarose at 4°C for 3 h. Fifty per cent toethanol. The eluates were analyzed directly by SDS-polyacryl-

of the toxin-binding activity was adsorbed. The supernatant from amide gel electrophoresis on a 10% acrylamide gel. A reproduction

the first adsorption was reincubated with fresh toxin/agarose at of the autoradiogram and its spectrophotometric scan are shown. In

room temperature for 3 h. Fifty per cent of the remaining activity the top trace (4°C preparation) the major peak at 41,000 daltons and

was adsorbed. Both preparations of agarose-bound receptor were the peak at 46,000 are present in a ratio of 4~1. The peaks at the extreme right co-migrate with the dye front.


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receptor from fetal calf skeletal muscle cells labeled in tissue culture with [%S]methionine. A binding assay using cG51- bungarotoxin was employed to monitor recovery of receptor activity. [VlMethionine-labeled protein was monitored by scintillation counting of hot trichloroacetic acid-precipitable radioactivity. In this experiment toxin binding activity eluted from the toxinlagarose conjugate by decamethonium was 5000- fold purified relative to the crude cell homogenate. From this value it can be calculated that incorporation of [~Slmethionine into receptor accounts for at most 0.02% of the total incorporation. As will be shown in a later section this value varies depending upon the exact time of labeling.

Complete and quantitative recovery of receptor polypeptides may be obtained from the affinity column. When the toxin/ agarose conjugate previously eluted with decamethonium was further subjected to protein denaturants, 9 M urea, 1% SDS, plus 5% p-mercaptoethanol, a significant amount of 35S-labeled protein was recovered. Because of denaturation, this material could not be assayed for toxin binding activity, but it was shown to be identical to ACh-receptor polypeptides by electrophoretic analysis (Fig. 1 and below). Furthermore, the quantity of YS-labeled receptor recovered by denaturant elution corresponded exactly to that left unaccounted for by decamethonium elution (bracketed values, Table Il. In other words, all of the receptor initially adsorbed to the column remained bound throughout the washing procedure and could be eluted by urea/SDS/P-mercaptoethanol.

Characterization of Affinity Purified Receptor

Sucrose Gradient Analysis-The PSlmethionine-labeled protein recovered from the purification procedure has been identified and characterized as receptor protein by both im- munological and biochemical criteria (20, 21). Analysis by sucrose gradient centrifugation has shown that greater than 80% of the YS radioactivity eluted from toximagarose column co-migrates with the peak of toxin binding activity. Further- more, both toxin binding activity and 35S-labeled cross-reacted with antiserum prepared against purified ACh-receptor from Electrophorus electricus (see Ref. 21). By comparison with enzyme markers of known s value (34), the purified receptor. toxin complex was found to have a sedimentation coefficient of approximately 9.5 s, identical to that found for the complex in crude cell homogenates.

Electrophoretic Analysis-Analysis of the polypeptide composition of S5S-labeled purified receptor by high resolution two- dimensional gel electrophoresis (28) demonstrates its remark- able homogeneity. The fluorograms reproduced in Fig. 1, A and B, were deliberately over-exposed in order to identify the minor components. One of these at M, = 43,000 and apparent p1 6.25 has been identified by co-electrophoresis as actin. The faint streak at M, = 200,000 is myosin heavy chain. The spot of A4, = 46,000 and apparent p1 6.25 is so far unidentified. Although the fluorograms for these two dimensional gels were not quantitatively scanned, visual comparison indicated that the content of the M, = 46,000 species was much less than that found in later preparations such as those shown in Figs. 2, 5, and 8.

FIG. 3. lJ5S]Methionine-labeled cysteine peptides of ACh-receptor and actin. Proteins were subjected to specific chemical cleavage at the amino peptide bond of cysteine residues by a modification of the method of Jacobson et al. (30). The peptides were separated by SDS- polyacrylamide gel electrophoresis on 20% acrylamide gels. Radio- active peptides were visualized by fluorography (29). Lanes A to C are from the three isolated 41,000-dalton species eluted from an electrofocusing gel similar to that shown in Fig. ID. Lane D is from the total column eluate of purified ACh-receptor. Lane E is from purified actin from muscle cells in culture (provided by R. G. Whalen). The difference in relative intensity of the same peptides from one lane to the next is due to incomplete and variable cleavage of the different preparations. The highest molecular weight band in Lane C does not appear to be present in the other lanes. The reason for this is unknown, but the molecular weight of this band calculated from molecular weight standards is greater than 41,000.

This suggests some heterogeneity in the apparent molecular weight of this species, resulting in band spreading during the second dimension. These three species have indistinguishable apparent molecular weights on one-dimensional SDS-gel electrophoresis (Fig. 2) in which only a single symmetric band at M, = 41,000 is seen.

In order to be certain that the use of [35S]methionine did not result in our missing a methionine-free polypeptide associated with receptor or give a false idea of the relative abundance of the species which were detected, similar analyses were per- formed on receptor purified from cells labeled with a mixture of 15 PC-amino-acids. The fluorogram of the two-dimensional gel analysis of ‘VXabeled receptor is shown is Fig. 1C. These results confirm the findings from analysis of 35S-labeled receptor. Direct analysis by SDS-polyacrylamide gel, as shown in Fig. 2, demonstrates that two-dimensional analysis of purified receptor does not miss polypeptides that might be too basic or too acidic for the Ampholines (pH 5 to 8) employed in the first dimension of two-dimensional gels.

The remaining and by far major species all migrate at M, Receptor was purified at 4°C and the remaining receptor in 41,000 and apparent ~16.5 to 6.7. These consist of two distinct the supernatant was then adsorbed at room temperature so spots and a less well defined spot on the basic pH side of the that the effects of proteolysis could be evaluated. Fig. 2 shows major spot. This pattern is completely reproducible and char- that receptor purified at the two temperatures is essentially acteristic. When an autoradiogram of the electrofocusing (first similar. The increase in amount of actin (43,000) found in the dimension) gel was examined it was found that the ill defined sample at room temperature is partially understood. Since the spot derived from a relatively normal protein band (Fig. ID). extract was first adsorbed at 4°C and the remaining receptor


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A B

FIG. 4. The resolution of ACh-receptor in unfractionated cell extracts by high resolution two-dimensional electrophoresis. Y3- labeled, purified ACh-receptor having an electrophoretic pattern similar to that shown in Fig. L4 was mixed in increasing proportions with Y-labeled unfractionated cell extracts. By observing which

in the supernatant was then adsorbed at room temperature, this could simply be due to an enrichment for actin in the supernatant as receptor is adsorbed out. It is also possible that actin binds nonspecifically to agarose and that this binding is greater at room temperature.

Peptide Mapping - In order to further characterize the polypeptide species associated with the ACh-receptor the methionine containing peptides produced by chemical cleavage at cysteine residues (30) have been analyzed. This method of protein cleavage was chosen because it permitted the analysis of minute quantities of protein species without destroying the tracer-labeled methionine residues necessary for detection. Proteolytic digestion produces fragments of too small size for the amount of labeled material available. Cyanogen bromide cleavage results in the loss of methionyl sulfur and could not be used. Fig. 3 shows the SDS-gel analysis of the methionine- labeled cysteine peptides of muscle actin, purified ACh-receptor, and the three M, = 41,000 species recovered from an isoelectric focusing gel loaded with purified ACh-receptor as in Fig. lD. This comparison demonstrates that the three M, = 41,000 species seen in the two-dimensional gel analyses of purified receptor are very similar in primary structure and probably differ by only a single charge (28, 35). The similarity of the peptides of total purified ACh-receptor with those of the isolated 41,000-dalton species confirms the findings that these species are major constituents of purified material and that all major receptor polypeptides have been detected by our electrophoretic analysis.

In summary, a purification procedure has been established which results in the rapid and quantitative isolation of a protein with the characteristics of the ACh-receptor. This material has been shown to be of high purity and composed of a small number of polypeptide components of identical molecular weight and probably of similar primary structure. In the following paragraphs we demonstrate the use of purified receptor as a marker to localize the snot corresponding to ACh-receptor in high resolution two-dimensional electrophoresis of crude unfractionated 100,000 x g extracts of YMabeled cells. By these and quantitative affinity purification tech-

spot became intensified when such mixtures were subjected to two- dimensional electrophoresis and subsequent fluorography, the position or expected position of the major ACh-receptor polypeptide was determined in gel patterns of (A) extracts of differentiated cells and (B) extracts of undifferentiated cells.

TABLE II

Receptor &gradation from pulse-chase labeling

Data were obtained from the experiment described in Fig. 5. 93 radioactivity in total protein and in ACh-receptor at the indicated times after the pulse labeling of the cells and chase with nonradioactive methionine was determined. Values are expressed as per cent of zero time value.

Time of chase %-1&&d protein ~totsllolate)

“S-labe;kd-&Ch-RY ACh-Rb

h x lo-‘cpm 96 x10-3cpm % %

0 3.58 (100) 5.46 (100) 100 14.5 2.12 (76) 3.20 (59) 69 25.0 2.42 68) 2.10 (38) 41

38.5 1.80 (50) 1.80 (33) 42

a Values were obtained by considering total counts per min eluted by urea/SDS/p-mercaptoethanol from toxin/agarose columns.

b Total eluted radioactivity was analyzed by SDS-polyacrylamide gel electrophoresis. The values listed in this column were determined by calculation of the area of only the M, = 41,000 component from spectrophotometric scans of the gel autoradiogram. Compari- son of the data in the last two columns suggests that a good approximation of receptor degradation can be made from analysis of total column eluates and thus for most purposes further analysis by electrophoresis is unnecessary.

niques we demonstrate the developmental regulation of ACh- receptor synthesis in differentiating muscle cells.

Resolution of ACh-Receptor from Cell Lysates by Two- dimensional Electrophoresis - Purified [35Slmethionine labeled ACh-receptor was added to total Triton X-100 extracts of %S-labeled differentiated muscle cell membranes. Fluoro- grams of the two-dimensional electrophograms of such mixtures were examined for spot enhancement. The spot indicated by the arrow in Fig. 4A is the only one of several hundred identifiable spots which is enhanced in crude lysates when purified 35S-labeled receptor is added. Only the major species is seen in these experiments due to the intensity difference among the three and the fact that these fluorograms have not been overexposed. When the fluorogram of the gel of an extract made from cells labeled early in culture, before any


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ABCDE F G Ii I

FIG. 5. SDS-polyacrylamide gel analysis of receptor purified from cells pulse-chase-labeled with [“Wmethionine. Twenty lo-cm culture dishes were labeled for several h (from 135 to 139 h of culture) with [Wlmethionine (2.5 Cilmmol, specific activity). Cultures were washed and returned to myoblast-conditioned medium supplemented with 0.05 rnM methionine. At 0, 14.5, 25, and 38.5 (corresponding to Lanes F through I), five dishes were washed and immediately frozen at -80°C. Extracts were prepared and receptor purified from each set of dishes as described in the text. Adsorption of toxin binding activity to toxin agarose was greater than 80% in

morphological signs of differentiation are apparent, is examined no spot corresponding to receptor can be detected (Fig. 4B). This analysis provides semiqualitative information about the control of expression of the gene(s) for ACh-receptor activity during myogenesis. The quantitative aspects of receptor synthesis and degradation were studied by the use of the affinity purification procedure described above.

Quantitative Measurement of Receptor Synthesis and Degra- dation during Myoblast Development

Receptor Degradation-The steady state level of any protein is defined by the rate constants of its synthesis and degradation. In eukaryotes regulation of either or both of these processes leading to alterations in steady state levels of specific proteins is known (36). ACh-receptor degradation and synthesis has been measured directly in order to elucidate the mechanism by which this protein is accumulated in differentiating skeletal muscle.

Receptor degradation in differentiated cultures has been measured by pulse-chase labeling techniques (21). In this previous publication the first order decay curve for 35S-labeled receptor was obtained by purification of receptor from cells labeled in culture and chased with nonradioactive methionine for various times afterward. After purification, each of the column eluates was freed of decamethonium by DE52 cbro-

each case. All conjugates were washed and eluted with urea, SDS, and P-mercaptoethanol identically. Eluates were applied directly to a 10% SDS-polyacrylamide gel. The gel was dried and an autoradiogram prepared. Contamination with high molecular weight (150,000 to 200,000) material resulted from less extensive washing of affinity bound receptor. Each lane of the gel was scanned with a scanning spectrophotometer and the area under each peak integrated for quantitation. Lanes A to E represent a pulse experiment to measure rates of synthesis and are described in Fig. 8.

matography and analyzed by incubation with 3H-labeled (Y- toxin and sucrose gradient centrifugation. Each purification was corrected to 100% recovery of toxin binding sites present in the initial extract in order to arrive at the amount of 35S- labeled receptor present in the initial extract. The half-times for decay by this method were approximately 17 h for receptor and 48 h for total protein (21). The half-time for the decay of receptor-bound ‘251-B~T~ was also 17 h. It was concluded that toxin decay was a valid measure of receptor degradation.

In order to be certain that using corrected values for the calculation of receptor content did not lead to error in the half- time determination, we repeated the receptor degradation experiments using a simplified procedure. Bather than eluting the affinity columns with decamethonium and assessing the yield of each purification, the columns were quantitatively eluted with urea/SDS, plus P-mercaptoethanol. As described above this procedure was found to yield all of the receptor initially bound to toxinlagarose. In the experiment described in Fig. 5 and Table II, toxin binding activity in all extracts was adsorbed to greater than 80% by prolonged incubation with the conjugate at room temperature. The urea/SDS//3- mercaptoethanol eluate from each column was analyzed by SDS-polyacrylamide gel electrophoresis. In Fig. 5 is shown the autoradiogram and scanner traces of this gel. The radioactivity in the M, = 41,000 molecular weight component was determined by scanning of the autoradiogram. The half-time


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Muscle A.Ch-Receptor 2888

90 J ,o

FIG. 6. Receptor degradation during differentiation. Cells were labeled with ‘251-BuT~ (approximately 20 Ci/mmol, specific activity, at 5 x 10m9 M for 4 h) at three different times during the culture period. Unbound toxin was washed out and the cells were returned to culture in myoblast-conditioned medium. At the indicated times the amount of cell-associated toxin was determined. For each point two or five 6-cm culture dishes were pooled. Values are recorded as counts per min per 6-cm dish on the ordinate and hours after plating on the abscissa.

FIG. 7. Synthesis and accumulation of ACh-receptor (AC/L-R). ACh-receptor accumulation during myoblast differentiation was measured by the DEAE-filter assay using ‘251-B~T~ (10 Cilmmol). Values recorded are total counts per min per lo-cm culture dish. The accumulation of total protein was determined by incorporation of [YSlmethionine added at 0 h of culture. The rate of synthesis of total protein was determined by pulse labeling for 4-h periods with [35Slmethionine (10 Ci/mmol, 5 pM) and trichloroacetic acid precipitation of the unfractionated cell extract. The rate of synthesis of ACh-receptor was determined by affinity purification of labeled ACh-receptor from cells pulse-labeled for 4 h. Adsorption was greater than 80% in each purification. Elution was done with urea/ SDS/P-mercaptoethanol. Values represent total counts per min in the eluate per lo-cm culture dish. Since the eluates are almost certainly contaminated with nonreceptor polypeptides, these values represent only an estimate (see Fig. 8).

for receptor degradation by this method was between 18 and 22 h, while that for total protein was 40 h.

In order to account for the greater than loo-fold increase in steady state receptor sites which occurs during in vitro differentiation either the rate of receptor degradation must be

FIG. 8. SDS-polyacrylamide gel analysis of receptor purified from cells pulse-labeled with PSlmethionine. Several lo-cm culture dishes (20 for the earliest time point and five each thereafter) were pulse-labeled with IYSlmethionine (2.5 Ci/mmol, 20 PM) for 4 h at 44, 92, 139, 164, and 212 h of culture, corresponding to Lanes A to E of Fig. 5. ACh-receptor was purified by affinity chromatography from each set of cultures. Adsorption was greater than 80%; elution was done with urea/SDS/P-mercaptoethanol. Eluates were applied directly to a 10% polyacrylamide gel. The dried gel was exposed for autoradiography, the autoradiogram was scanned, and the area under each peak was integrated for quantitation. As in Fig. 5 the orientation of the gel is right to left, early to late times. The orientation of the scans is top to bottom, early to late.

slower than in undifferentiated cultures or the rate of its synthesis must be more rapid. Fig. 6 shows an experiment to detect a difference in the rate of receptor degradation from the earliest time at which such an experiment is possible using toxin of the specific activity available (20 Cilmmol) until late in the process of differentiation when maximal toxin binding activities are attained. (See Fig. 7 for the time course of accumulation of toxin binding activity.) No difference in the rate constant for receptor degradation could be observed during this period.

Receptor Synthesis - Since the rate of receptor degradation remains unchanged, it is expected that there should be a significant increase in the rate of receptor synthesis to account for the observed accumulation. Figs. 7 and 8 show the results of two experiments in which the rate of receptor synthesis was measured directly by pulse labeling. The time course of receptor and total protein accumulation are presented in Fig. 7A. The rate in counts per min per lo-cm culture dish per 4 h for both total protein and receptor are presented in Fig. 7B. From this experiment only an approximation of receptor synthesis is obtained because only total counts eluted from toxin/agarose are recorded. Since none of the preparations are pure receptor and since the earlier time points are likely to be less pure than later ones, these results are probably slightly skewed. When in a second experiment eluates were analyzed by SDS-gel electrophoresis (Fig. 5) and the rate of synthesis of only the M, = 41,000 species was tabulated, it was found that


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Muscle ACh-Receptor

TABLE III

2889

Rate of synthesis of receptor from pulse labeling

HOUW Nuclei pex6kke x Fused nucleic Protein/plated 35S-labeled protein (total/plate)e

35S-labeled ACh-R/ plate’

Relative rate ACh-R synthesis’

% of total w

44 3.4 0.5 0.58 92 10 16 1.48

139 13 32 1.97 164 13 30 2.76 212 14 34 2.31

Ii Hours after plating of culture when the 4-h pulse was started (see legends to Figs. 5 and 7).

L(’ In order to determine increase in cell number and degree of morphological differentiation parallel culture plates were fixed with methanol and stained with Giemsa. Two thousand total nuclei were counted for cells at each time. Data is expressed as total nuclei per lo-cm culture plate and the per cent of total nuclei which were found in multinucleate myotubes.

” Total [%lmethionine incorporated into protein during the 4-h pulse was determined by trichloroacetic acid precipitation,

d Total cell protein was determined, using bovine serum albumin as a standard (37).

the rate of receptor synthesis increased more than 60-fold (Table III, column F). When the increase in rate of total protein synthesis is taken into account (Table III, column E), the differential rate of receptor synthesis increases 17-fold (Table III, column G). The meager rates of synthesis detected in early cultures can be more than adequately accounted for by the small number of differentiated cells (detected as multinucleate fibers) (Table III, column C) present, or by the observation that occasional mononucleate bipolar cells present ear!y in culture exhibit low levels of ACh sensitivity (39).

DISCUSSION

In this report we describe the characterization of the polypeptide composition of mammalian muscle ACh-receptor. The major polypeptide species of M, = 41,000 occurs in at least three isoelectric forms. We have been unable to determine the origin of this charge heterogeneity and the relatively low abundance of ACh-receptor in muscle cells and the possibility of charge modification during purification make such analyses difficult. No obvious precursor product relationship among these three forms has been observed in pulse-chase experiments in uiuo.3 However, such relationships might exist and be difficult to resolve, as has been shown for the spot polymor- phism of the VP-l, major capsid protein of SV40 (39). Differ- ences in the carbohydrate portion of the receptor (40) or degree of methylation or phosphorylation (41, 42) might account for the heterogeneity observed (see Refs. 28 and 35). It has been suggested that receptor modification (6), in particular phosphorylation (41, 42), might play a role in one or more of several physiological events involved in ion permeability and synapse formation. Experiments involving ACh-receptor phosphorylation which are limited to in vitro analysis may make an evaluation of the physiological significance of such a modification difficult. The use of muscle cell cultures might allow such evaluations to be made.

The fact that some purifications have resulted in preparations almost completely lacking the higher molecular weight (43,000 and 46,000) polypeptides suggests that these may not be receptor subunits. The spot of M, = 43,000 is almost

R J. P. Merlie, unpublished observation.

cpm x IO-6 CPm % of total protein synthe-

sis

5.35 18.6 0.00035 18.5 199 0.00108 22.4 975 0.00435 20.6 1190 0.00580 19.5 464 0.00238

‘Rate of incorporation of [“%]methionine into receptor was deter-

g Relative rate of ACh-receptor expressed as a per cent of total protein synthesis.

mined by affinity purification of labeled extracts. Elution was done with urea/SDS/P-mercaptoethanol. Extracts were analyzed by SDS- polyacrylamide gel electrophoresis and the autoradiograms of such gels were scanned spectrophotometrically and the content of the M, = 41,000 species was determined (see Figs. 5 and 8). A value in counts per min for this species was derived from the measure of % counts per min in the eluted sample and the fraction of the total film density of the autoradiogram which was found under the 41,000 peak.

certainly contaminating actin. The spot of 46,000 is unidentified; its possible identity as a receptor component is not excluded. We have previously reported the occurrence of significant amounts of polypeptides of M, = 50,000 and 75,000 in receptor purified from the same cells (20). These higher molecular weight species may have been impurities since a greater proportion of the V-labeled protein of current preparations co-sediments with toxin binding activity (see Refs. 20 and 21).

It is not understood why our preparations of muscle receptor have a polypeptide composition somewhat different from those ofE. electricus, Torpedo californica electric organ, or a mouse muscle cell line (40, 43-45). It has been reported that the receptor from electric eel contains two or three components of approximately M, = 40,000, 47,000, and 53,000 (39, 42), and that of Torpedo contains four components of M, = 39,000, 48,000, 58,000, and 64,000 (43, 44, 46). Recently, Sobel et al. (47) have found that the polypeptide composition of highly purified receptor from Torpedo marmorutu varies from one purification step to the next, and from one preparation to another. The preparations with the highest specific activities are richer in the M, = 40,000 polypeptide. Only the M, = 40,000 polypeptide of both eel and Torpedo receptors is labeled with the affinity reagent 4-(N-maleimido)benzyltri([“H]methyl ammonium) (MBTA) (42).

Patrick et al. (48) have reported the purification of ACh- receptor from E. electricus having no polypeptides of molecular weight less than 48,000. Treatment of this receptor preparation with trypsin resulted in the production of smaller molecular species (42,000 and 44,000) with no loss of toxin binding. These authors have suggested that receptor is particularly sensitive to rather specific and limited proteolysis. This may be the case for muscle receptor as well. I f so, such proteolysis must also be very rapid or must occur while the protein is still in the membrane, or both, since a spot corresponding to the M, = 41,000 subunit is found by two-dimensional electrophoresis of unfractionated membrane extracts.

Differences in the polypeptide composition from ACh-receptors derived from different tissues or from different species in different laboratories could be explained by one or more of several possibilities: 1) it is possible that species or organ


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differences exist for ACh-receptor derived from varied sources; 2) it is possible that differences in purification procedures might result in retention of nonreceptor protein or removal of loosely associated subunits; 3) as indicated above proteolysis during purification may be an important factor in determining the final polypeptide composition.

At present it is impossible to distinguish among these possibilities and to describe a definitive polypeptide pattern for the ACh-receptor. It is however certain that the 40,000- dalton species is a true receptor component or a derivative of such a component. Whatever the subunit composition, it seems valid to base qualitative and quantitative measures of receptor turnover on those species which we have found in highly purified muscle ACh-receptor.

The qualitative demonstration by two-dimensional electrophoresis that the spot corresponding to the ACh-receptor is developmentally regulated is supported by quantitative meas- urements of receptor synthesis and degradation. Our studies have shown that the large increase in accumulated ACh- receptor during myogenesis in tissue culture is due exclusively to an increase in receptor synthesis. Since receptor synthesis increases 17 times more than global protein synthesis this cannot be explained simply by cell growth. Expression of ACh- receptor, like that of a-actin, several myosin light chains and tropomyosins (16, 19, 49) is developmentally controlled during muscle differentiation.

The steady state level of any protein is determined by both its rate of synthesis and its rate of degradation. In cultured myoblasts the steady state value of receptor sites can be measured in two ways. From toxin binding experiments we know that each lo-cm culture dish contains from 0.5 to 1 pmol of toxin-binding sites. From our determination of receptor synthesis and degradation a steady state value of toxin binding sites can be arrived at by the simple relation [RI = k&,! where h, and kcl are rate constants for receptor synthesis and degradation respectively. A value for K, of 0.030 pmol/h/lO-cm dish was calculated from the maximal rate of synthesis observed in the experiment described in Table III. The following assumptions were made: 1) that there is a content in methionine of 3.4 mol%, derived from data for E. electricus receptor (39), 2) that there is one toxin-binding site per 40,000 daltons of receptor protein, the apparent mass of the major species, and 3) that the specific activity of Che [“SS]methionine is unaffected by cellular pools or compartmentalization. A value for K,) of 0.04 h-’ was calculated from the measured half- life from the expression tlR = ln2/h, using the value oft,, = 17 h, an average of several determinations. Then, [RI equals 0.75 pmol of toxin binding sites/lo-cm dish, a value which agrees remarkably well with that determined by toxin binding. Agreement of these values indicates that most receptor sites appearing in cultured cells do so as a result of synthesis de nouo and not as a result of conversion of some preformed cell component. It cannot be excluded that receptor polypeptides pass through a relatively small rapidly turning over pool of precursor material.

Receptor protein constitutes only a small fraction of total cellular protein. Less than 0.01% of total protein synthesis is due to receptor synthesis. This represents a synthetic rate of approximately 5 molecules of receptorlminlmyotube nucleus. For comparison Emerson and Beckner (48) have reported a value of 30,000 molecules/min/nucleus for the myosin heavy chain (M, = 200,000) in cultures of quail myoblasts. Five molecules per min is the approximate production rate of a single active polyribosome of the size one would expect for the

receptor subunit. This value establishes definite limits for the way in which we might think about the mechanism of regulation of receptor synthesis and for the ways in which we approach the study of this problem.

Acknowledgments-We would like to thank Drs. Kato, Menez, and Tamiya for their gifts of a-toxins. J. P. M. is specially grateful to Robert Whalen for his interest and consistently constructive discussions during the course of this work, as well as his help with the identification of actin and myosin in two dimensional gels. We thank Kathy Verlander for help with the preparation of the manuscript.

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J P Merlie, J P Changeux and F Grosturnover in muscle cell cultures.

Skeletal muscle acetylcholine receptor. Purification, characterization, and

1978, 253:2882-2891.J. Biol. Chem.

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