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Skeletal Muscle Acetylcholine Receptor

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Text of 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.
(Received for publication, June 29, 1977, and in revised form, August 24, 1977)
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 electro- phoresis. The purest preparations are composed of a single M, = 41,000 class of polypeptide which exhibits some charge heterogeneity. By high resolution two-dimensional electro- phoresis a spot corresponding to acetylcholine receptor was localized among total proteins of muscle membrane ex- tracts. Synthesis of this component is shown to be develop- mentally regulated. Quantitative analysis of receptor syn- thesis and degradation has led to the conclusion that recep- tor 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 compo- nent 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 extrasy- naptic 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 recep- tor; SDS, sodium dodecyl sulfate; 12SI-B~T~, Y-labeled a-bungaro- toxin; 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 biochemi- cal 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 exper- iments 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 differentia- tion.
In this paper we describe the direct precursor labeling of the AChR, its purification, and characterization by sodium dode- cyl sulfate-polyacrylamide gel electrophoresis of its poly- peptide components. By pulse-chase labeling methods we have demonstrated that synthesis of receptor polypeptides is tightly regulated during muscle differentiation.
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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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-acti- vated 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 la- beled with 3H-labeled Naju nigricollis a,-isotoxin was done on 10 to 40% exponential convex sucrose gradients (25) as described previ- ously (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 de- scribed 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-poly- acrylamide 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 de- scribed (28). Visualization of radioactive protein in gels was either by autoradiography with Kodak Kodirex x-ray film or by fluorogra- phy as described by Laskey and Mills (291. Scanning of autoradi- ograms 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 pur- ified ACh-receptor labeled in culture with radioactive amino acids were analyzed by the two-dimensional electrophoretic system de- scribed 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 decametho- nium. Approximately 6,500 cpm were applied to the gel. The fluoro- gram 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/[email protected] anol 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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RESULTS Our experience with toxinlagarose affinity chromatography
Purification is that the yield by specific [email protected] ligand elution is generally quite low and variable. Those factors which we have
The method of cu-neurotoxin/agarose affinity chromatogra- phy 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 de- scribed 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-
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 col- umn. 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 incorpo- ration. 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-la- beled 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 elu- tion 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 com- position 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…