Phosphorylation and assembly of nicotinic acetylcholine receptor ...

THEJOURNALOF BIOLOGICAL CHEMISTRY 8 1987 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 262, No. 30, Issue of October 25, pp. 14640-14647,1987 Printed in U.S.A.

Phosphorylation and Assemblyof Nicotinic Acetylcholine Receptor Subunits in Cultured Chick Muscle Cells* (Received for publication, January 26, 1987, and in revised form, June 16, 1987)

Anthony F. Ross$, Mary Rapuanon,Jakob H. Schmidts, and Joav M. PrivesSll From the Departmentsof $Anatomical Sciencesand §Biochemistry, State University of New York at Stony Brook, Stony Brook, New York 11794

The assembly of the nicotinic acetylcholine receptor (AChR),an oligomeric cell surface protein, was studied in cultured muscle cells. To measure this process, the incorporation of metabolically labeled a-subunit into oligomeric AChR was monitored in pulse-chase experiments, either by the shift of this subunit from the unassembled (5 S) to the assembled (9 S) position in sucrose density gradients, or by its coprecipitation with antisera specific for the &subunit.We have found that AChR assembly is initiated 15-30 min after subunit biosynthesis and is completed within the next 60 min. The a-subunit is not overproduced, as all detectable pulse-labeled a-subunit can be chased into the oligomeric complex, suggesting that AChR assembly in this system is an efficient process. The rate of AChR assembly is decreased by metabolic inhibitors and by monensin, an ionophore that impairs the Golgi apparatus. We have observed that the y- and &subunits of AChR are phosphorylated in vivo. The &subunit is more highly phosphorylated in the unassembled than in the assembled state, indicating that its phosphorylation precedes assembly and that its dephosphorylation is concomitant with AChR assembly. These findings suggest that subunit assembly occurs in the Golgi apparatus and that phosphorylation/dephosphorylation mechanisms play a role in the control of AChR subunit assembly.

ometry to form the pentameric AChR ligand-gated ion channel that appears on the cell surface. According to several recent models of AChR structure (7-9), each subunit contributes one amphipathic transmembrane span to form the wall of the ion channel. Because the hydrophilic residues that are to line the aqueous pore must be excluded from thebilayer in subunits before assembly, markedintrasubunitconformational changeswould be expected toaccompany AChR assembly and aqueous channel formation (see Ref. 3). The molecular basis for dynamic events in subunit assembly, subunit-subunit recognition, conformational rearrangement, and channel formation, is not understood. The availability of electrogenic tissues of Torpedo sp. and Electrophorus electricus, rich sources of AChR, has contributed to the advanced stateof knowledge of AChR structural properties. However, the study of AChR expression requires a system inwhich AChRsynthesis is both rapid and accessible to measurement with metabolic labeling techniques. Differentiation of myogenic cellsin cultureismarked by rapid biosynthesis of AChR, allowing the measurement of AChR assembly by methods utilizingmetaboliclabeling (10-12). Recent studieshave utilizedthis approach to show that AChR assembly rate isdecreased by elevated serum levels in BC3H1 cells (11) andisincreasedinculturedrat myotubes by exposure to tetrodotoxin, an inhibitorof membrane electrical activity (12). Thus,subunit assemblycould be a point of regulation of AChR expression. Evidence has been obtained tosuggest a role for phosphorylation/dephosphorylation mechanisms in the regulation of The nicotinic acetylcholine receptor (AChR)’ is a pentacell surface ionic channel proteins (13-15), including AChR meric glycoprotein complex, comprisedof four different mem(13). Torpedo AChR has been shown to contain substantial brane-spanning subunits with the stoichiometryof cy2, pl, yl, amounts of phosphoamino acids (16) and to undergo phos61 (for reviews, see Refs. 1-4). In intact muscle, AChR is proteinkinases (17). concentrated on the postsynaptic surface at the neuromus- phorylation catalyzedbyendogenous Phosphorylation sites have been identified on all four subcular junction and mediates synaptic transmissionby transTorpedo AChR (17), and changes in the phosphorylunits of ducing the bindingof the neurotransmitteracetylcholine into ation state of AChR can be correlated with altered AChR a change in membrane permeability to cations(1-4). desensitization rate (18-21). In addition, recent studies indiThe four AChR subunits are synthesizedas separate polycate that AChR expressed in cultured muscle is phosphorylpeptides ( 5 ) and inserted cotranslationally into membranes ated in situ (22, 23). In our study, we haveanalyzed the of the rough endoplasmicreticulum (6). The subunits are subsequently assembled in aprecise stoichiometry and ge- relationship of AChR subunit assembly and phosphorylation incultured chick myogenic cells. The results provide an * This work was supported by grants from the American Heart outline of the assembly kinetics andsuggest a role for reversAssociation and the National Science Foundation. The costs of pub- ible subunit phosphorylation in theregulation of assembly. lication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertiseEXPERIMENTAL PROCEDURES ment” in accordance with 18 U.S.C. Section 1734 solely t o indicate MateriaZ~-[~~S]Methioninewas obtained from American Carp. this fact. 32Piwas purchased from ICN Radiochemicals. ’*’I-Bgt, ‘251-protein A, 7 Cellular and Developmental Biology Graduate Program. The abbreviations used are: AChR, nicotinic acetylcholinerecep- and EN3HANCE were from New England Nuclear. Protein A-Sephtor; Bgt, a-bungarotoxin;SDS-PAGE,sodium dodecyl sulfate-polya- arose was purchased from Pharmacia Biotechnology, Inc.Chemicals for polyacrylamide gel electrophoresis and Western blotting were crylamide gel electrophoresis; PBS, phosphate-buffered saline; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid;BSA, bo- obtained from Bio-Rad. Bgtwas isolated from Bungarus multicinctus venom, and cobra toxin was isolated from Naja naja siamensis venom vine serum albumin. 14640

14641

Acetylcholine Receptor Subunit Phosphorylation and Assembly (Miami Serpentarium, Miami, FL). All other reagents were from Sigma. Preparation of Antisera-AChR was purified from denervated leg muscle of adult chickens by affinity chromatography, using cobra toxin conjugated to CNBr-activated Sepharose CI-4B, and fractionated, by SDS-polyacrylamide gel electrophoresis, into polypeptides of approximately 40, 47, 52, and 55 kDa, molecular mass. The 40-kDa band was identified as the a-subunit by its ability, after electrophoretic transfer onto nitrocellulose, to bind '251-Bgt.The remaining bands were designated as p-, 7 - , and &subunits. These designations will be used throughout this manuscript, although definitive identification clearly will have to await subunit sequence analysis. Slices containing the a- and &bands were cut from the gel and eluted, and their purity as single bands was established by re-electrophoresis on SDS-polyacrylamide gels. New Zealand White rabbits were given subcutaneous injections, repeated in 2-week intervals, of the appropriate antigen (10 pgof purified receptor or receptor subunit, and 30 pg, increasing to 120 pg, of Rgt). Titers, as determined by indirect immunoprecipitation of chick muscle receptor. IzsI-Bgtcomplexes, were: anti-a-subunit antiserum, 12.5nM; anti-hubunit antiserum, 3.3 nM; and anti-native . antibodies were chick muscleAChR antiserum, 5.5 p ~ Anti-Bgt prepared by affinity chromatography of anti-Bgt antiserum on BgtSepharose and diluted in PBS to a final concentration of 10 nM. All antisera were found to quantitatively immunoprecipitate "'I-Bgt. AChR complexes from embryonic chick muscle. The preparation and characteristics of the rat monoclonal antibody mAb35, specific for the main immunogenic region of the a-subunitof Electrophorus AChR but cross-reactive with other vertebrate AChR, have been described (24). mAb35 was isolated from the supernatant of hybridoma TIB 175 (American Type Culture Collection). Cell Culture-Primary cultures of skeletal muscle cells were prepared from breasts of 12-day-old chick embryos as described previously (25, 26). Cells were plated on collagen-coated culture dishes a t an initial density of 1.8 X lo6 cells/6-cm diameter culture dish or 1.2 X 10' cells/lO-cm diameter culture dish. The cultures were grown in Dulbecco's modified Eagle's medium supplemented with 10% horse serum and 2% embryo extract, at 37 "C in an atmosphere of 92% air, 8% CO,. After 2 days in culture, cells were fed with growth media containing 10 p~ cytosine arabinoside for 48 h to minimize fibroblast proliferation (27). Labeling and Immunoprecipitation-Cultures were labeled a t 37 "C with ["S]methionine (170 pCi/ml) in methionine-free Dulbecco's modified Eagle's medium supplemented with 10% complete medium for the specified time or with ["2P]orthophosphate ("Pi; 2 mCi/ml) in phosphate-free medium for 4 h. In both cases, the labeling medium included 10-* M "'I-Bgt to monitor cell surface AChR as described previously (28). Cultures were washed twice with buffer A (150 mM NaCI, 10 mM Tris-HCI, pH 7.4, 2 mM EGTA, 2 mM EDTA) supplemented with the protease inhibitors 5 mM benzamidine, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin, 1 pg/ml leupeptin, and 10 mM N-ethylmaleimide. In 32Pi-labeledcultures the phosphatase inhibitors 50 mM sodium fluoride, 40 mM sodium pyrophosphate, 20 mM potassium phosphate, 10 mM sodium molybdate, and 1 mM sodium orthovanadate were included. These washes and subsequent steps were carried out at4 "C. The cells were scraped in buffer A supplemented with 0.5% Triton X-100, extracted for 30 min, and theextracts clarified by centrifugation at 100,000 X g for 30 min. The supernatantswere incubated with 10-15 pl of appropriate antisera for 3 h at 4 "C and then incubated with protein A-Sepharose (15 pl/tube) for 1 h. For preabsorption controls, the antibody was incubated in 100-fold excess of purified antigen for 3 h at 4 "C immediately before incubation with the cell extract. The protein A-Sepharose beads were pelleted, washed 5 times with buffer A containing the enzyme inhibitors, and resuspended in 50 pl of SDS sample buffer. After incubation for 10 min a t room temperature, the beads were centrifuged, and the supernatantswere fractionated by SDS-PAGE on 10% gels according to Laemmli (29). The gels of ["'Sjmethionine-labeled extracts were stained with 0.1% Coomassie Brilliant Blue in 40% methanoland 10% acetic acid, destained, treated with EN:'HANCE for 1 h, dried, and exposed to Kodak XAR-5 x-ray film. For gels containing :'?Pi-labeledproteins, the staining solution consisted of 0.1% Coomassie Brilliant Blue in 50% trichloroacetic acid, and the EN'HANCE was omitted. Densitometry was performed on an EC scanner densitometer connected to an IRM XT computer through an A to D board and analyzed numerically. fmmunohlotting-Transfer of proteins from SDS-polyacrylamide

gels to nitrocellulose paper was carried out as described (30). AChR was purified by batchwise affinity chromatography on cobra toxin conjugated to Sepharose beads. A 100-pl volume of a 1:3 slurry of beads in buffer A was added to clarified supernatant prepared from 3-day muscle cultures as described above. After a 3-h incubation at 4 'C, the beads were collected by centrifugation, washed 3 timeswith buffer A, and incubated in SDS sample buffer for 10 min. Samples were fractionated by SDS-PAGE using a 10% gel concentration, and the proteins were transferred to nitrocellulose paper. Transfer was carried out for 2 h at 70 V a t 4 'C, and after quenching with 2% BSA in PBS, the nitrocellulose paper was incubated with the appropriate antibody diluted in 2% BSA/PBS for 6 h. The strips were washed 5 A (loficpm/ times with 2% BSA/PBS, and incubated with 1251-protein ml) in 2% BSA/PBS for 2 h a t room temperature. Where specified, the strips were incubated in '251-Bgt(lo-" M, loficpm/ml) after the quenching step. The paper was washed extensively in 2% BSA/PBS, dried thoroughly, and exposed to Kodak X-Omat film. Sucrose Gradient Fractionation-Cell extracts prepared as described above were layered on a 4.4-ml5-20% linear sucrose gradient, prepared in buffer A supplemented with protease inhibitors and, where applicable, phosphatase inhibitors. Gradients were centrifuged in a Beckman SW 55 rotor at 45,000 rpm for 8 h (w't = 6.5 X 10"). Twenty-drop (0.2 ml) fractions were collected, immunoprecipitated, and analyzed as described above. RESULTS

Identification of AChR Subunits-Because the interpretation of the data from many of the experiments in this study relies on the ability of the subunit-specific antiserato distinguish between the CY- and &subunits, it was essential at the outset to establish the specificity of the antisera, particularly i n view of t h e high degree of sequence homology between AChR subunits (31-32). Fig. 1 is a Coomassie Brilliant Bluestained gel showing the AChR subunits purified from adult denervated chick muscle (lane2) which wereused as antigens, as detailed under "Experimental Procedures." For comparison, purified Torpedo AChR subunits are shown in Fig. 1, lane 1. T h e specificity of the antisera was established by immunoblotting extracts of cultured muscle cells with antisera made against the CY- and &subunits of AChR from denervated muscle of adult chicken. To obtain sufficient quantities of t h e AChR subunits for these immunoblotting experiments, AChR-enriched fractions were prepared by affinity chromatography of Triton X-100 extracts of cultured muscle cells on cobra toxin conjugated to Sepharose and elution in

1

2

3

4

5

6

66Y

FIG. 1. Purification of chick AChR and characterizationof antisera by immunoblotting. AChR from 7 b r p c ~ helectric organs and denervated muscle of adult chickens was purified by affinity chromatography on cobra toxin-Sepharose as described under "Experimental Procedures," and fractionated by SDS-PAGE. Lanes I and 2, Coomassie Brilliant Blue-stained gel of purified Torpedo AChR subunits (lane I ) and purified adult chicken muscle AChR (lane 2). Lanes 3-6, overlay of blots of partially purified AChR from cultured chick muscle with '"I-Bgt (lane 3), anti-a-subunit antiserum ( l o n e 4 , anti-undenatured chicken AChR antiserum (lane 5). and antichicken &subunit antiserum(lane 6). Bound immunoglobulins in lanes 4-6 are visualized by '"I-protein A autoradiography.

Acetylcholine Receptor Subunit Phosphorylation

14642

and Assembly

SDS sample buffer. The 40-kDa band was identified as thea- polypeptides by 1)preabsorption of the anti-Bgtwith purified subunit by overlaying the blot with '"I-Bgt (Fig. 1, lane 3 ) . Bgt (lane 2); 2) preabsorption of the mAb35 with purified As shown in Fig. 1, this same40-kDa band was recognized by Torpedo AChR (lane 4 ) ; 3) preabsorption of the anti-a-subunit the anti-a-antiserum (lane 4 ) , as well as by the anti-native antisera with purified chicken AChR (lane 6).The larger of AChR antiserum (lane 5) which, like most anti-native AChR these polypeptides, with a molecular mass of 55 kDa, coantisera, recognizes only the a-subunit (24). In contrast, the migrates witha peptide that is specifically recognized on anti-&antiserum did not react with the a-subunit butrecog- immunoblots of AChR-enriched muscle cell extracts by antinized a 55-kDa peptide (lane 6 ) . These results indicate that b-subunit antiserum (Fig. 1, lane 6) and is designated the 6the antisera recognize the appropriate subunits and are non- subunit. This designation is in agreement with a previously cross-reactive. published report which suggests that a 54-kDa peptide from Immunoprecipitation experiments were performed (Fig. 2) chicken muscle AChR ishomologous to Torpedo &subunit on to identify the AChR subunits inmetabolically labeled cells. the basis of proteolytic digest patterns (33, 34). The second As seen in Fig. 2A, the a-subunit is clearly discernible as a phosphopeptide present in these immunoprecipitations hasa 40-kDa band upon SDS-PAGE of Triton X-100 extracts of molecular mass of 50kDa,between that of the a- and 6[:"S]methionine-labeled cells that have been immunoprecipi- subunits in culturedmuscle and is tentatively designated the tated with antiserum against the &subunit (lane I), or in Bgt/ y-subunit. In contrast to the 6 - and putative y-subunits of [""Slmethionine-labeled cells that have been immunoprecipi- AChR, no phosphorylation of the a- or @-subunits was detated with anti-Bgt antibodies (lane 2). In contrast, the larger tected in our experiments. subunits were not clearly visible in these immunoprecipitates Our findingof AChR subunitswhich are difficult to resolve because of high levels of contaminating ["S]methionine-lain ["S]methionine-labeled preparations, yet are readily idenbeled proteins in this region of the gel. An alternative possi- tifiable upon"Pi labeling, is reminiscentof a similar situation bility, that these larger subunits were absent from the gels encountered in the immunoprecipitation of another surface due toproteolysis, was ruled out by a2Pilabeling experiments channel protein, the a-subunit of voltage-sensitive Na' chanand, in the case of the b-subunit, by resultsobtained by nels from cultured neurons (35,36). In thesestudies, the Na' immunoblotting (Fig. 1).When cultureswere labeled with '*Pi channel polypeptides from [RsS]methionine-labeledcells were and immunoprecipitated with several independent antibodies, not visible due to high backgrounds, but were clearly resolved two phosphoproteins were clearly visible in the 50-60-kDa in "Pi-labeled preparations (35, 36). region of the gels.As shown in Fig. 2B, these bands were Immunoprecipitation of AChR Subunits from Sucrose Graselectively immunoprecipitated with anti-&subunit antiserumdient Fractions-Metabolically labeled AChR oligomer has (lane 7);Bgt-anti-Bgt (lane I); mAb35, a monoclonal antibody been shown to sediment asa 9 S peak upon density gradient against the AChR (lane 3);and by anti-a-subunit antiserum fractionation, whereasunassembled subunits sedimentas a 5 (lane 5). The identity of these bands as AChR subunits was S peak (10). The analysisof density gradient fractionsoffers confirmed by the inhibition of immunoprecipitation of these a means to distinguish between the monomeric and oligomeric forms of AChR subunits and to measure the rate of assembly of metabolically labeled AChR subunits. As a first step, we A B monitored the fractionation pattern of cell surface AChR. 1 2 31 2 3 4 5 6 7 8 Intact muscle cellswere treated with1251-Bgt(lo-' M) to label - " AChR on the cell surface, extracted, layered on a 5420% sucrose density gradient, andcentrifuged (u2t= 6.5 x 10") as -116- 92 described under "Experimental Procedures." When the individual gradient fractions were immunoprecipitated with anti- 66 a-subunit antiserum, '2sI-Bgt.AChR complexes were seen to sediment asa 9 S peak (Fig. 3). T o monitor the fractionation pattern of metabolically labeled AChR, cultures were labeled with [3sS]~ethioninefor 10 h, extracted, fractionated on a sucrose gradient, and the fractions immunoprecipitated with anti-a-subunit antiserum. The immunoprecipitates were subjected to SDS-PAGE and fluorography, and the a-subunit was quantitated by densitometry. As seen in Fig. 3, with this extended labeling period the fractionationprofile of metabolically labeled a-subunitisidenticaltothat of assembled surface AChR, revealing that the major proportion of[:%] FIG. 2. Identification of metabolically labeled AChR subunits. Cultured muscle cells were labeled with either["%]methionine methionine-labeled a-subunit is in theassembled oligomer. To monitor the assembly of newly synthesized a-subunit ( A ) or "P ( R ) ,as described under "Experimental Procedures." The into oligomeric AChR, cultures were pulse-labeled with ["SI labeled cultures were extracted and immunoprecipitated withthe specified antibody, and the immunoprecipitations were fractionated methionine for 15 min and, after various chaseintervals, by SDS-PAGE. A, [JsS]methionine-labeledcultures were extracted fractionated on sucrose gradients as described above. The and immunoprecipitated with anti-&subunit antiserum ( l a n e I ) , Bgtanti-Bgt (lane 2), or preimmune serum ( l a n e 3). B,"Pi-labeled cul- fractionation profiles of pulse-labeled a-subunit after 15, 60, tures extracted and immunoprecipitated with Bo-anti-Bgt ( l a n e I ) ; and 180 min of chase are shown in Fig. 4, A-C. After a 15Bgt-anti-Bgt, using anti-Bgt which was preabsorbed with free Bgt min chase time, less than 25% of the labeled a-subunit is (lone 2); mAb35 (lane 3); mAb35 preabsorbed with purified Torpedo found in the 9 S peak. Subsequently, the a-subunit accumuAChR ( l a n e 4); anti-a-subunit antiserum ( l a n e 5); anti-a-subunit lates in the 9 S peak with increasing chasetimes. The extent antiserum preabsorbed with purified chicken AChR (lane 6).Lanes of a-subunit accumulation in the 9 S peak as a function of 7-8 are anti-&subunit antiserum immunoprecipitations of 3ZPi-labeled cell extract after fractionation of theextract on a sucrose chase time,reflecting the rateof AChR assembly, is shown in gradient. Lane 7, the peak AChR fraction (9 S);lane 8, a nonpeak Fig. 5. Two sequential phases are distinguis!lable: an initial fraction (15 S). lag phase that lasts approximately 15 min and a subsequent

3-

-

-

Acetylcholine Receptor Subunit Phosphorylation and Assembly

14643

1

" 3

1

B)

1

5

37 5

7

9

Y

l

I

11

13

91311

IS

17 19 21

15 17 19 21

" " " " "

11692

66 -

-

v)

2

5

10

15

--

45

31

-

FIG. 3. Comparison of the sucrose density gradient sedimentation profile of AChR detected by immunoprecipitation of '""IBgt-AChR complexes ( A ) and ["'Slmethionine-labeled AChR a-subunit ( B ) .Intact muscle cells were double-labeled with 170 pCi/ml [""Slmethionine for 10 h and "sI~Rgt(lo-" M) for the last hour, detergent extracted, and fractionated by sucrose density gradient centrifugation as described under "Experimental Procedures." The individual gradient fractionswere immunoprecipitated with antia-subunit antiserum. The "'1-Rgt.AChR in each fraction was quantified by y-spectroscopy; metabolically labeled a-subunit was identified by SDS-PAGE andfluorography and quantifiedby densitometry. Numbering of the lanes refers to the sucrose gradient fractions, with fraction I representing the topof the gradient. Thearrowhead on the sides of the gel shows the location of the a-subunit on the fluorograph. Migration of molecular mass standards is indicated onleft. Markers are catalase, 11 S (arrowhead);alkaline phosphatase, 5.4 S (arrow).

FIG.4. AChR oligomerization measured by the shift in sedimentation of immunoprecipitable a-subunit from 5 S to 9 S peaks in pulse-chase experiments. Muscle cultures were pulselabeled with 170 pCi/ml [""S]methionine for 15 min and chased for 15 min ( A ) , 60 min ( R ) ,or 180 min (C). Cultures were extracted, fractionated on sucrose density gradients, and the fractions immunoprecipitated with anti-a-subunit antisera. ["SS]Methionine-labeled a-subunit in each fraction (the lowermost band in each fluorograph) was quantified by densitomety (solid line in each panel). The sedimentation profile of 12'II-Bgt-labeledcell surface AChR (broken line) shows the position of oligomeric AChR in the sucrose gradients. Markers are catalase, 11 S (arrowhead);alkaline phosphatase, 5.4 S (arrow).

period of rapid assembly. During theperiod of rapid assembly, the time required for half of the a-subunit to reach the 9 S peak was 15-20 min, based on a semilogarithmic plot of the data (Fig. 5, inset). AChR assembly was also monitoredby measuring the physical association of a- and &subunits in pulse-chase experiments. After a 15-min pulse with [:"S]methionine followed by increasing chase times, cultures were extracted, and equal aliquots of the extract were immunoprecipitated with either anti-a- or anti-&antiserum, or with nonimmune serum. Because the anti-&antiserum does not cross-react with the asubunit, only the a-subunit physically associated with the bsubunit will be precipitated by it. An increase in a-subunit precipitated by anti-&antiserum with increasing chase time would reflect AChR assembly. After a 15-min pulse, the a-subunit precipitatedby anti-b-

antiserum comprised only approximately 10% of the total labeled a-subunit precipitated with anti-a-antiserum(Fig. 6). With increasing chase intervals, the proportion of labeled asubunit precipitated by anti-&antiserum increases (Fig. 6). As was the case with the sucrose gradient measurements (Fig. 5), the co-immunoprecipitation assay also indicates that the assembly process consists of two phases: an initial lag period followed by a periodof relatively rapid assembly. When these data were plotted on a semilogarithmic scale (Fig. 6, inset), the chase time required for one-halfthe a-subunit to associate with the &subunitwas estimated tobe approximately 30 min. There is no net decrease in the amount of labeled a-subunit during the chase intervals, and ofallthe a-subuniteventually asembles with the &subunit, demonstrating that the unassembled a-subunit is metabolically stable and its assembly into

5

10

Fraction No.


14644

I

I

I

I

15

30

45

60

CHASE TIME (min)

FIG. 5. Kinetics of oligomerization of a-subunit as monitored by shift of a-subunit from 5 S into 9 S peaks. Cultures were pulse-labeledwith170pCi/ml[35S]methioninefor 15 min, chasedfor the indicatedtime, and the sucrosedensity gradient fractionation profile of the pulse-labeled a-subunit obtained by immunoprecipitating the fractions with anti-&-subunitantiserum followedby SDS-PAGE. Quantification oflabeled a-subunit in the AChRoligomerwasperformedby calculating the area in the 9 S peak. Values on the ordinate represent the percentage of the total labeled a-subunit which is in the 9 S peak after the specified chase interval. Inset, data plotted on logarithmic scale show two distinct phases, an initial lag followed by a period of rapid oligomerization.

/* 01

0

oligomeric AChR is a highly efficient process in embryonic chick muscle cells. Effects of Metabolic Inhibitors on AChR Assembly-The intracellular transportof newly synthesized proteins fromthe rough endoplasmic reticulum to the Golgi is an energy-requiring process (37).To study the relationshipof AChR assembly to this translocation process, we have monitored the effects on assembly of metabolic inhibitors which act at different steps along this translocation pathway. Cultures were pulselabeled with [35S]methionine, incubated in chase media either for 60 min, and the shift with or without metabolic inhibitors of immunoprecipitable a-subunit from the 5 S to the 9 S peak was measured. The results of these experiments are shown in Fig. 7. After this chase interval, approximately90% of the asubunit was in the 9 S peak in control cultures(Fig. 7A). The protein synthesis inhibitor cycloheximide had no effect on assembly (Fig. 7 B ) under conditions in which incorporation of [35S]methionine into the trichloroacetic acid-precipitable fraction was inhibited by greater than 90% (not shown). The metabolic inhibitors 2,4-dinitrophenol and potassium cyanide have been reported to inhibit the transfer of proteins from the rough endoplasmic reticulum to the Golgi apparatus (37). In the presence of 0.5 mM 2,4-dinitrophenol or 50 mM potassium cyanide, the shift of a-subunit into the 9 S peak was significantly inhibited, reaching64 and 77% of control levels, respectively (Fig. 7, C and D). At the same concentrations, both agents inhibited incorporationof assembled AChR into the cell surface by 70-80%, as monitored by the binding of lZ5I-Bgtto intact cells (not shown). Furthermore, AChR assembly was found to be temperature-dependent: incubation of cultures at 4 "C inhibited AChR assembly by 80% (Fig. 7F). These results suggest that metabolic energy is required

/ / I

15

'

30

1

60

I

I

I20

CHASE TIME (mi")

FIG. 6. Assembly of a- and &subunits measured by immunoprecipitation of labeled a-subunit using anti-&subunit antiserum in pulse-chase experiments. Cultures at 48 h postplating were pulse-labeled with 170 rCi/ml [35S]methionine for15 min, and, after the specified chaseinterval, the detergent extracts were divided into two equal fractions and immunoprecipitated witheither anti-asubunit or anti-&subunit antisera. The immunoprecipitates were fractionated by SDS-PAGE and the a-subunit in each sample quantified by densitometry of the fluorograph (given on the ordinate in relative units). Open circles, a-subunit immunoprecipitated by antia-subunit antiserum. Closed circles, a-subunit immunoprecipitated by anti-&subunit antiserum. Inset, A logarithmic plot of the data shows that two phasescanbedistinguished, an initial lagphase followed by a period of rapid oligomerization. Values onthe ordinate are expressed as the percentage of total labeled a-subunit which is immunoprecipitated by the anti-&antisera.

Frortion N ~ ~ m t ~ e f

FIG. 7. Inhibition of a-subunit oligomerization by metabolic inhibitors. Cultures were pulse-labeled with [35S]methionine for15 min and chasedfor 60 minwithmediacontaining the specified inhibitor or treatment. Cell extracts were fractionated on a sucrose density gradient, the fractions immunoprecipitated withanti-a-subunit antiserum, and the immunoprecipitablea-subunit in each fraction quantified by densitometry of the fluorograph (solid line) as described in the legend to Fig. 2. The sedimentation profile of lz5IBgt-labeledcell surface AChR showsthe position of oligomeric AChR in the gradients (broken line). A , control cultures; B , 1 rg/ml cycloheximide; C, 0.5 mM 2,4-dinitrophenol;D, 50 mM potassium cyanide; E, 50 PM monensin; F , cultures were incubated at 4 "C during the chase period.


14645

A for theassembly of AChR subunits. Theionophore monensin 1 2 3 4 5 6 7 8 9 1 0 1 11 2 1 3 1 4 inhibits intracellular transport of newly synthesized protein by acting on the Golgi complex (38). As shown in Fig. 7E, monensin (50 FM) markedly inhibits the shift of a-subunit 66 into the 9 S peak, indicating that functional integrityof the 6Golgi apparatus is necessary for AChR assembly. YPhosphorylation of the Assembled and Unassembled AChR 45 Subunits-AChR subunit assembly in cultured chick muscle cells does not occur immediately upon subunit synthesis, but A B apparently requires an interval of 15-30 min between synthe1 2 3 4 5 6 7 8 9 1 0 1 1 12 1 3 1 4 sis and the onset of assembly (Figs. 5 and 6), in agreement with recent findings in othercell types (10, 12). Thisdelay is consistentwiththe idea that assembly isdependenton changes in the covalent structureof subunits. The well documented phosphorylation of AChR subunits is Torpedo (13, 16-18,39-41) and our current observation that the AChR in cultured chick muscle is phosphorylated in situ have led us to A investigate therole that phosphorylation plays in theregulaC tion of AChR assembly. 1 2 3 4 5 6 7 8 9 10 11 1 2 1 3 1 4 To compare the phosphorylation states of assembled and unassembled subunits, we fractionated extractsof "Pi-labeled cultures on sucrose gradientsandimmunoprecipitatedthe fractions with anti-AChR antiserum. As can be seen in Fig. 8, immunoprecipitation with anti-&antiserum revealed that the assembled (9 S) &subunit is phosphorylated in situ. In addition, a prominent phosphoproteinwhich co-migrates with A the &subunit in SDS-PAGE is found in the5 S region of the FIG.9. Immunoprecipitation of unassembled and assembled sucrose gradient, suggesting that unassembled &subunit is also phosphorylated. T o eliminate the possibility that this 5 AChR subunits and quantification of b-subunit by immunoSucrose gradient fractions of 32Pi-labeled cellswere immuS phosphoprotein represents a contaminant that co-migrates blotting. noprecipitated with anti-&subunit antiserum ( A ) or anti-n-subunit with the b-subunit, we immunoprecipitated the "Pi-labeled antiserum ( B ) . The immunoprecipitation profile shows that the ygradientfractionswithanti-a-antiserum.Theanti-a-antiand &subunits canbe immunoprecipitated from assembled oligomers serum precipitated phosphorylated b-subunitfrom the 9 S by both antisera, but unassembled &subunit is immunoprecipitated region only; no similar band was seen in the 5 S region (Fig. only by anti-&subunit antisera. C , sucrose gradient fractions of unlabeled cell extracts were immunoprecipitated with anti-b-subunit 9B). Furthermore, the partial chymotrypticdigest pattern of antisera, thegel transferred to nitrocellulose paper and overlaid with the 5 S phosphopeptide very closely resembles that of the 9 S anti-&subunit antiserum followed by lZsII-proteinA. This method of b-subunit (notshown).The 5 S phosphoproteinmigrates quantifying the d-subunit in the various regions of the sucrose p a identically to the &subunitin SDS-PAGE, it isrecognized by dient shows that b-subunit in the 9 S peak is much more abundant the anti d-subunit antiserum but not by the anti-a-subunit than &subunit in the 5 S peak. Markers are catalase, 11 S (arrowantiserum, and the proteolytic digest pattern is similar to the head);alkaline phosphatase, 5.4 S (arrow).

4

k

4

&subunit isolated from oligomeric AChR, strongly suggesting that this phosphoprotein is an unassembled form of the 6subunit. As shown in Fig. 9, the putative y-subunit is as intensely phosphorylated as the &subunit. The phosphorylated y-sub66 unitisevident onlyin immunoprecipitates of assembled d AChR (Fig. 9, A and B ) , consistent with the idea that the yV subunit is physically associated with both the a- and the 645 subunits andis not recognized directly by the antiseradirected 36 against a-or 6-subunits. 29 T o compare the relative extent of phosphorylation of unassembled and assembled &subunits, quantitative immunoblotting experiments were performed to estimate the relative amounts of assembled and unassembled b-subunit present in the cell extracts. In these experiments, the sucrose gradient 4 1, fractions were immunoprecipitatedwithanti-&antiserum, FIG. 8. Phosphorylation of assembled and unassembled 6- transferred to nitrocellulose paper and overlaid with anti-dsubunits. At 48 h after plating, cultureswere labeled with 2 mCi/ml subunit antiserum and "'I-protein A. As shown in Fig. 9C, '"P, for 4 h, rinsed and extracted in bufferA supplemented with phosphatase inhibitors. The cell extract was fractionated by sucrose the 9 S peak of &subunit isclearly evident in these conditions density gradient centrifugation and the fractions immunoprecipitatedwhereas the5 S peak is below the resolution of the technique. with anti-&subunit antiserum. Immunoprecipitated material was sep- This finding is consistent with the interpretation that the arated by SDS-PAGE and the driedgel exposed to x-ray film for 72 relative amount of the unassembled &subunit is much lower h. Markers are catalase, 11 S (arrowhead);alkaline phosphatase, 5.4 S (arrow). TheintactAChR oligomer, as monitored by IY5II-Bgt than that of the assembled &subunit. An equivalent amount of net phosphorylation is seen in the assembled and unassemlabeling of cell surface AChR, sedimented in a peak in fractions 6-8 (not shown). bled peaks of &subunit under these conditions, indicating 1

2

3 4 5 6 7 8 91011121314

14646


that the relative amount of phosphorylation of the unassembled peptide greatly exceeds that of the assembled peptide. In additional experiments, cells were labeled with 32Pifor 24 h between day 1 and day 2 after plating (not shown). Under these conditions, all AChR present was synthesized in the presence of 32Pi.The resultsobtained with this extended labeling period were not detectably different from those obtained with 4-h labeling periods (Fig. 8), obviating the possibility that the higher apparent labeling of unassembled 6 subunit reflects a difference in radioactive ATP pools between the Golgi and thecell surface.

reflect an assembly process in which sufficient subunit oligomerization to produce the S value shift occurs before the final stoichiometry is attained. Consistent with this idea is the observation that the 9 S peak is broad during intermediate chase times as compared with longer chase intervals (Fig. 4, B versus C), possibly reflecting the transient presence of incompletely assembled oligomers. The assembly process is efficient; all of thea-subunit synthesized duringthe chase period subsequently accumulates in the 9 S peak and is immunoprecipitated by the anti-6antiserum. This is in apparent contrast to the cases of rat myotubes or BC3H-1 cells, where assembly of the a-subunit DISCUSSION has been reported to be significantly less efficient (10, 12). In this study, we have examined the process of AChR Further, there is no net loss of immunoprecipitable pulsesubunit assembly in cultured chick muscle cells. Several im- labeled a-subunit during either the preassembly or assembly munological criteria were utilized to identify AChR subunits periods, demonstrating that newly synthesized a-subunit is metabolically stable. In this respect, AChR assembly in culfrom extracts of metabolically labeled cells. The a-subunit from [35S]methionine-labeledcultures was identified as a 40- tured chick muscle resembles assembly in rat myotubes (12) kDa protein on SDS-PAGE by 1) specific immunoprecipita- but is in apparent contrast to what has been observed in tion of intact AChR protein by a number of independent anti BC3H-1 cells (lo), where the a-subunit is overproduced and AChR antisera including anti-&-subunit antiserum, anti-6- metabolically labile. Previous studies have evidenced a delay between AChR subunit antiserum, and anti intact AChR antiserum; 2) immunoprecipitation by an affinity-purified antibody against subunit biosynthesisand oligomer assembly. In BC3H-1cells, Bgt, a highly specific AChR ligand; 3) co-migration on SDS- subunit assembly begins approximately 30 min after subunit PAGE with adult chicken muscle AChR a-subunit; 4) co- biosynthesis. During this preassembly period, the a-subunit undergoes conformational changes detected by the acquisition migration on SDS-PAGE with a band recognized by '"I-Bgt in Western blotting experiments; 5) co-migration on sucrose of the ability to bind Bgt and a monoclonal antibody that density gradientswith intact '251-Bgt.AChRcomplexes at the reognizes a conformational epitope not present on the initial 9 S peak, under labeling conditions in which all of the labeled translation product (10). In addition, experiments using electron microscopic autoradiography and '*'I-Bgt binding have a-subunit would be expected to be in the assembled form. Two phosphopeptides with molecular masses of 50 and 55 indicated that newly synthesized AChR peptides accumulate kDa in SDS-PAGE were identified as AChR subunits by: I) in the Golgi complex within 10 min of synthesis (42). These Direct recognition of the 55-kDa bandby anti-&-subunit anti- results suggest that both subunittranslocation into the Golgi serum, indirect immunoprecipitation of the 50-kDa band by apparatus andconformational modifications of the a-subunit anti-&subunit antiserum, and indirect immunoprecipitation occur during the preassembly period. Our results illustrate that AChR assembly is impaired by of the 50- and 55-kDa bands by anti-a-subunit antiserum. The immunoprecipitation of these bands was specifically pre- metabolic inhibitors and incubation of cells at low temperavented by preabsorption of the antisera with purified chick ture. These findings may indicate that metabolic energy is AChR. 2) Immunoprecipitation of these bands by the mono- required for conformational changes or post-translational clonal anti AChR antibody mAb35 and thespecific inhibition modifications of AChR subunits which are essential for asof immunoprecipitation by preabsorption of the antibody with sembly. Alternatively, because these treatments inhibit intrapurified Torpedo AChR. 3) Immunoprecipitation of these cellular translocation of secretory and membrane proteins bands by anti-Bgt antibody that is eliminated by preabsorp- (37), theinhibition of assembly may reflect a block in transit tion of the antibody with purified Bgt. 4) Co-migration of of AChR subunits to an intracellularcompartment where these bands in sucrose density gradients in the 9 S peak with assembly can occur. Corroborating this possibility is our find'"I--Bgtf AChR complexes. The &subunit is immunoprecipi- ing that monensin, an ionophore that has been widely used tated by anti-&subunit antiserum from both the 5 S (unas- as a blocker of Golgi function (38), inhibits AChR assembly. sembled) and 9 S (assembled) peaks. The y-subunit is indi- The time course of AChR assembly, characterized by an initial rectly immunoprecipitated by anti-&subunit antiserumfrom lag period, and the susceptibility of the assembly process to the 9 S peak only. Both y - and &subunits are indirectly metabolic inhibitors, low temperature, andmonensin are conimmunoprecipitated by anti-a-subunit antiserum in the 9 S sistent with the Golgi apparatus asbeing the siteof assembly in chick muscle. However, our findings do not preclude the peak only. We have used pulse-chase experiments and immunoprecip- possibility that some AChR assembly takes place in therough itation to measure AChR assembly by two criteria: the shift endoplasmic reticulum immediately before entering the Golgi, of a-subunit from a 5 S to a 9 S form in sucrose gradients, as has recently been suggested for BC3H-1 cells (43). A plausible mechanism for the regulation of AChR assembly and the coprecipitation of a-subunit during immunoprecipitation with anti-&subunit antiserum. Results of both AChR could involve post-translational modification of the newly assembly assays are in agreement on several features of the synthesized AChR subunits. In this context, we have studied assembly process. After an initial delay of approximately 15 the role of phosphorylation/dephosphorylationof AChR submin, AChR assembly occurs rapidly, with a half-time of 15- units in intact muscle cells. Phosphorylation of AChR is well 35 min after onset (Figs. 5 and 6). Comparison of the time documented in preparations from Torpedo and Electrophorus courses of assembly by the two methods suggests that oligo- (13) and has recently been reported in cultured rat (22) and merization of the a-subunit, as monitored by the shift into chick myotubes (23). The potential sites of phosphorylation the 9 S peak, occurs earlier than association of the a- and 6- on each subunit of Torpedo AChR are located intracellularly subunits, as monitored by co-immunoprecipitation of the a- (17), adjacent to a putative membrane-spanning region that subunit with anti-&subunit antiserum. This difference may may form the ion channel portion of the receptor, according

Acetylcholine Receptor Subunit Phosphorylation and Assembly to proposed models of AChR structure (7,8). These potential phosphorylation sites are present in the homologous regions of chick y- and d-subunits (32). In addition, several recent reports suggest that AChR functional properties are altered under conditions that promote phosphorylation (18-21). If all the subunits contribute segments to the channel wall, as is currently believed, then some of the putative phosphorylation sites will be in proximity to regions of interaction between subunits, andchanges in phosphorylation state may influence subunit-subunit interactions. The use of muscle cell cultures allows investigation of the role of phosphorylation/dephosphorylation in the regulation of AChR expression. In thepresent study,we have found that the AChR is phosphorylated in intact muscle cells on two of its peptides, tentatively identified as the y- and &subunits. Our resultsindicate that the net phosphorylation of the unassembled d-subunit is higher than that of the assembled &subunit. This finding suggests that phosphorylation occurs shortly after translation (within 30 min postsynthesis), and that dephosphorylation of this subunit occurs at about the time of assembly. An alternative interpretation, the existence of a large pool of unassembled, stable phosphorylated 6 subunit, is negated by our finding that the assembled pool of b-subunit is significantly larger than the unassembled pool, as shown by immunoblotting of sucrose gradients (Fig. 9). What role might phosphorylation of the unassembled 6subunit play in the expression of functional AChR? Our evidence supports the plausibility of a phosphorylation/dephosphorylation mechanism in the regulation of subunit assembly, and suggests two experimentally testable alternative pathways. First, dephosphorylation of the d-subunit may precede, and be a necessary precondition for, subunit assembly. &subunit phosphorylation/dephosphorylation could constitute a mechanism that prevents aberrant or premature assembly of subunits by controlling the point at which b-subunit becomes competent for assembly. Alternatively, &subunit dephosphorylation may befound to occur after subunitassembly. This could contribute to a mechanism by which correctly assembled oligomers are stabilized, while incorrectly assembled oligomers arenot dephosphorylated and may either disassemble or be removed from the translocation pathway. The identification of the phosphorylation site(s) onunassembled and assembled d-subunits, as well asa more precise temporal correlation between dephosphorylation and assembly could yield insight into the mechanism of subunit assembly. Acknowledgment-The expert technical assistance of Martha Hayes and Catherine Adee is gratefully acknowledged.

REFERENCES 1. Karlin, A. (1980) in The Cell Surface and NeuronalFunction (Cotman, C. W., Poste, G., and Nicolson, G. L., eds) pp. 191260, Elsevier/North Holland, New York 2. Changeux, J.-P., Devillers-Thiery, A., and Chemouilli, P. (1984) Science 225, 1335-1345 3. Stroud, R. M., and Finer-Moore, J. (1985) Annu. Reu. Cell Biol. 1,317-351 4. Salpeter, M. M., and Loring, R. H. (1985) Prog.Neurobiol. ( N . Y.) 25, 297-325

14647

5. Anderson, D., and Blobel, G. (1981) Proc. Natl. Acad. Sci.U. S. A . 78,5598-5602 6. Anderson, D. J., Walter, P., and Blobel, G. (1982) J. Cell Biol. 9 3 , 501--506 7. GUY,H. R. (1984) Biophys. J. 45,249-261 8. Finer-Moore, J., and Stroud, R. M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 155-159 9. Ratnam, M.,Le Nguyen, D., Rivier, J., Sargent, P. B., and Lindstrom, J. M. (1986) Biochemistry 2 5 , 2633-2643 10. Merlie, J. P., and Lindstrom, J. M. (1983) Cell 34, 747-757 11. Olson, E. N., Glaser, L., Merlie, J. P., Sebbane, R., and Lindstrom, J. M. (1983) J. Bwl. Chem. 258,13946-13953 12. Carlin, B. E., Lawrence, J. C., Jr., Lindstrom, J. M., and Merlie, J. P. (1986) J . Biol. Chem. 261,5180-5186 13. Browning, M. D., Huganir, R., and Greengard, P. (1985) J. Neurochem. 4 5 , l l - 2 3 14. Rane, S. G., and Dunlap, K. (1985) Proc. Natl. Acad. Sci.U. S. A . 83,184-188 15. Madison, 0.V., Malenka, R. C., and Nicoll, R. A. (1986) Nature 32 1,695-698 16. Vandlen, R.L., Wu, W. C.-S., Eisenach, J. C., and Raftery, M. A. (1979) Biochemistry 1 8 , 1845-1854 17. Huganir, R. L., Miles, K., and Greengard, P. (1984) Proc. Natl. Acad. Sci. U. S. A . 81,6968-6972 18. Huaanir. R. L.. Delcour. A.H.. Greeneard, P., and Hess, G. P. " ( i986) Nature 32 1 , 774-776 ' 19. Eusebi, F., Molinaro, M., and Zani, B.M. (1985) J. Cell Bbl. 100,1339-1342 20. Albuquerque, E. X., Deshpande, S.S., Aracava, Y., Alkondon, M., and Daly, J. W. (1986) Febs. Lett. 199, 113-120 21. Middleton, P., Jaramillo, F., and Schuetze, S. M. (1986) Proc. Natl. Acad. Sci. U. S. A . 83,4967-4971 22. Anthony, D. T., Rubin, L. L., Miles, K., and Huganir, R. L. (1986) SOC. Neurosci. Abstr.12, 148 23. Rapuano, M., Ross, A., and Prives, J. M. (1986) J. Cell Biol. 103, 448a 24. Tzartos, S. J., Rand, D. E., Einarson, B. L., and Lindstrom, J. M. (1981) J.Biol. Chem. 256,8635-8645 25. O'Neill, M., and Stockdale, F. E. (1972) J. Cell Biol. 5 2 , 52-65 26. Bar-Sagi, D., and Prives, J. M. (1985) J. Biol. Chem. 260,47404744 27. Fischbach, G. D., and Cohen, S. A. (1973) Deo. Biol. 31,147-162 28. Prives, J. M., Fulton, A. B., Penman, S., Daniels, M. P., and Christian, C. N. (1982) J. Cell Biol. 92, 231-236 29. Laemmli, U. K. (1970) Nature 227,680-685 30. Burnette, N. W. (1981) Anal. Biochem. 112, 195-203 31. Noda, M., Takahashi, H., Tanabe, T., Toyosato, M., Kikyotani, S., Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T., and Numa, S. (1983) Nature 302,528-532 32. Nef, P., Mauron, A., Stalder, R.,Alliod,C., and Ballivet, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 8 1 , 7975-7979 33. Sumikawa, K., Mehraban, F., Dolly, J. O., and Barnard, E. A. (1982) Eur. J. Biochem. 1 2 6 , 465-472 34. Sumikawa, K., Barnard, E. A., and Dolly, J. 0.(1982) Eur. J. Biochem. 126,473-479 35. Schmidt, J. W., Rossie, S., and Catterall, W. A. (1985) Proc. Natl. Acad. Sci. U. S. A . 82,4847-4851 36. Schmidt, J. W., and Catterall, W. A. (1986) Cell 46,437-445 37. Palade, G. (1975) Science 189, 347-358 38. Tartakoff, A., and Vassalli, P. (1978) J. Cell Bwl. 79,694-707 39. Saitoh, T., and Changeux, J.-P. (1981) Proc.Natl.Acad.Sci. U. S . A . 78,4430-4434 40. Davis, C. G., Gordon, A. S., and Diamond, I. (1982) Proc. Natl. Acad. Sci. U. S. A . 79, 3666-3670 41. Huganir, R. L., and Greengard, D. (1983) Proc. Natl. Acad. Sci. U. S. A . 80, 1130-1134 42. Fambrough, D. M., and Devreotes, P. N. (1978) J. Cell Biol. 76, 237-244 43. Smith, M. M., Lindstrom, J. M., and Merlie, J. P. (1987) J . Biol. Chem. 262,4367-4376 ~~