Neuron, Vol. 7, 659-666, October, 1991, Copyright 0 1991 by Cell Press

Acetylcholine Receptor Assembly Is Stimulated by Phosphorylation of Its y Subunit

William N. Green, Anthony F. Ross, and Toni Claudio Department of Cellular and Molecular Physiology Yale University School of Medicine New Haven, Connecticut 06510

Summary

Different combinations of Torpedo acetylcholine recep- tor (AChR) subunits stably expressed in mouse fibro- blasts were used to establish a role for phosphorylation in AChR biogenesis. When cell lines expressing fully functional AChR complexes (a&G) were labeled with 32P, only y and S subunits were phosphorylated. For- skolin, which causes a 2- to s-fold increase in AChR expression by stimulating subunit assembly, increased unassembled y phosphorylation, but had little effect on unassembled 6. The forskolin effect on subunit phos- phorylation was rapid, significantly preceding its effect on expression. The pivotal role of the y subunit was established by treating aby and a@ cell lines with for- skolin and observing increased expression of only af3y complexes. This effect was also observed in ay, but not a6 cells. We conclude that the CAMP-induced increase in expression of cell surface AChRs is due to phosphory- lation of unassembled y subunits, which leads to in- creased efficiency of assembly of all four subunits.

Introduction

Muscle-like nicotinic acetylcholine receptors (AChR) are composed of four different but homologous sub- unitsinthestoichiometrya&& Duringdevelopment of the neuromuscular junction, the number of AChR complexes increases dramatically at the junction, and there is a subunit switch whereby fetal 7 is replaced by adult y (referred to as a) (Witzemann, et al., 1989). The mature junctional AChRs have several properties that differ from those of embryonic muscle. Fetal and adult AChRs have different surface half-lives, mem- brane mobilities, channel open times, and channel conductances; a few differences in immunological epitopes and biochemical properties have also been reported (reviewed in Scheutze and Role, 1987; Sal- peter, 1987; Brehm and Henderson, 1988; Claudio, 1989). These developmental changes in the AChR may result from regulation at several levels: transcrip- tional, posttranscriptional, translational, and post- translational. Our interest has been to investigate pos- sible posttranslational control mechanisms of AChR expression (Green et al., 1991). In this paper, we fur- ther investigate the CAMP effect on AChR expression and specifically address the role of phosphorylation in the regulation of AChR expression.

Phosphorylation of cell surface receptors is a com- mon mechanism by which the cellular response to external signals (neurotransmitters, hormones, and

growth factors) is altered, usually affecting signai trans- duction through a change in receptor activity or sub- cellular distribution (Sibley et al., 1987; Huganir and Greengard, 1990). It is well established that nicotinic AChRs are phosphorylated, but the consequence of this phosphorylation is unclear. Previous work sug- gests several possible effects of AChR phosphoryla- tion. AChR phosphorylation may accelerate its rate of desensitization (the rapid shutdown of the channel after prolonged exposure to acetylcholine) (Huganir et al., 1986; Hopfield et al., 1988; Wagoner and Pallotta, 1988). A role for phosphorylation in AChR assembly was postulated when it was observed that unassem- bled 8 subunits from chick muscle were more highly phosphorylated than the assembled form (Ross et al., 1987). Data indicating that developmental changes in the AChR are induced by its phosphorylation have also been presented (Saitoh and Changeux, 1981; Qu et al., 1990). Using different combinations of Torpedo AChRsubunits stablyexpressed in mousefibroblasts, we address whether the observed CAMP stimulation of AChR expression is due to phosphorylation of AChR subunits. The Torpedo AChR is the best char- acterized of the nicotinic AChRs, with the greatest number of subunit-specific and conformation-specific antibodies available for its study. Combining the ad- vantages of the Torpedo AChR with an expression system in which the subunits and receptor can be readily manipulated in vivo has allowed us to show thatwhiletwoAChRsubunits,yandS,arephosphory- lated, phosphorylation of y alone stimulates assembly of all four subunits into the AChR complex.

Results

Only y and S Subunits Are Phosphorylated Iln Viva Fully assembled AChRs are pentamers composed of four subunits with the stoichiometry of a&6. When Torpedo AChR subunit cDNAs are stably integrated into the genome of mouse fibroblasts (All-?1 cells), only a single class of a$$ receptors is expressed on the ceil surface (Claudio et al., 1987; Hartman et al., 1990). Previously, we found that CAMP increased cell surface AChR expression through a posttranslational effect on AChR subunits prior to pentamer formation (Green et al., 1991) and that this posttranslationai event led to increased assembly efficiencies of the four subunits (Ross et al., 1991). If the increase in as- sembly is caused by phosphorylation of AChR sub- units, then it follows that the subunits are phos- phorylated before they assemble into pentamers. The phosphorylation of unassembled as well as fully as- sembled y and 8 subunits is demonstrated in Figure 1.

All-11 cells were labeled with 32P, and assembled cell surface, fully assembled internal, and unassembled subunits were isolated by sedimentation on 5%-20% sucrose gradients followed by sequential immuno-

NeLlrOn 660

Lane 1 2 3 4

-a .i:,:

5 6 12 3 456

C Assembled

Cell Intra- Surface cellular

Lane 1 2 3 4

Unassembled

Y 6

Expressed Al one Y 6

C F CF C F C F

5 6 7 8 9 IO 11 12

Figure 1. Phosphorylation of Unassem- bled and Assembled AChR Subunits Is Stimulated by Forskolin

(A and B) Only y and 8 subunits are phos- phorylated. Shown are the phosphoryla- tion patterns of Torpedo AChR subunits stably expressed in fibroblasts in the ab- sence of forskolin. L-pSV2-aBy&, (All-11 cells) were induced to express AChRs and then labeled with either [3*P]orthophos- phate (A) or [XS]methionine (B). intact cells were then surface-labeled with 1251-BuT~, solubilized, and run on 5%-20% sucrose gradients. The 95 peak (fractions 9-11) was pooled to isolate assembled AChRs, and the 6s peak (fractions 6-8) was pooled to isolate unassembled subunits (subunit monomers plus partially assembled sub- unit complexes). Cell surface AChRs were isolated by immunoprecipitating with anti-BuTx antiserum (lane 6), and then in- tracellular assembled AChRs were isolated by immunoprecipitating the immune-de- pleted supernatant with MAb 35 (lane 5). Unassembled subunits (lanes I-4) were iso- lated from the 6s peak after first removing any assembled AChRs with MAb 35 and then immunoprecipitating equal aliquots of immune-depleted supernatant with anti-a antiserum (lane I), anti-B antiserum (lane 2), anti-y MAb 168 (lane 3), and anti-8 antiserum (lane 4). (C) Stimulation of y and 8 subunit phos- phorylation by forskolin. All-11 cells were induced to express AChRs. in the presence or absence of 100 PM forskolin and then labeled with [32P]orthophosphate. Sucrose

gradients were run, and the different pools of subunits were isolated and run on SDS polyacrylamide gels. Shown are phosphorylated y and 8 subunits from control (lane C) and forskolin-treated (lane F) cells. Assembled cell surface AChRs (lanes 1 and 2), fully assembled intracellular AChRs (lanes 3 and 4), unassembled y subunits (lanes 5 and 6), and unassembled 8 subunits (lanes 7 and 8). For the gel lanes under “Expressed Alone,“y subunits from L-pSV2-y, cells (lanes 9 and IO) and 8 subunits from L-pSV2-i$ cells (lanes 11 and 12) are shown. For these cell lines, y and 8 subunits were immunoprecipitated from fraction 7 (-6s) from a 5%-20% linear sucrose gradient on which the 32P-labeled cell lysates were run.

precipitations with l&and-specific, subunit-specific, or conformation-specific antibodies. Intact cells were labeled with a-bungarotoxin (BuTx), atoxin that binds specificallytoAChRs. Solubilizedcellswerethen sedi- mented on sucrose gradients, and two different peak fractions were collected: fully assembled AChRs, which sediment as a sharp 9s peak (Claudio et al., 1987), and unassembled subunits (subunit monomers and partially assembled complexes), which have a broad profile centered at -6s (Paulson et al., 1991). Assembled cell surface AChRs were isolated and re- moved from the 9s fraction by immunoprecipitation with anti-BuTx antiserum (Figures IA and IB, lane 6). Assembled internalAChRswerethen isolated(Figures IA and IB, lane 5) with monoclonal antibody (MAb) 35,aMAbdirectedagainstthectsubunitthat preferen- tially binds assembled rather than unassembled a subunits (Ross et al., 1991). Unassembled subunits were isolated by pooling the 6s peak fractions and depleting any assembled AChR complexes by immu- noprecipitation with MAb 35. The immune-depleted supernatantwas then divided into four equal aliquots

and immunoprecipitated with four different subunit- specific antisera (in Figures IA and IB, a is shown in lane 1, p in lane 2, y in lane 3, 8 in lane 4). As shown in Figure IB, when dishes of All-11 ceils are labeled in parallel with [35S]methionine, all four subunits are clearly present in these cells. As shown in Figure IA, however, only the y and 8 subunits are phosphory- lated. This pattern of phosphorylation, which is identi- cal to that of in vitro studies using endogenous or purified protein kinase A (PKA) to phosphorylate na- tive Torpedo AChRs (Huganir and Greengard, 1983), suggests that PKA is the kinase phosphorylating AChR in fibroblasts.

Up Regulation of Subunit Phosphorylation by Forskolin We have shown previously that forskolin, as well as other agents that increase intracellular CAMP levels (theophyllin, cholera toxin, and CAMP analogs), stimu- lates the cell surface expression of AChRs expressed in fibroblasts and muscle cells in the absence of any new protein synthesis (Green et al., 1991). To deter-

;6ybunit Phosphorylation Regulates AChR Assembly

mine whether the posttranslational event causing this increase by forskolin is phosphorylation, All-11 cells were labeled with 32P in the absence or presence of forskolin, and assembled and unassembled subunits were isolated as described above. As shown in Figure IC, “P labeling of the y and 6 subunits dramatically increased in the presence of forskolin. However, for- skolin also increased the number of y and 6 subunits in the pools (Green et al., 1991) so that the increase in phosphate per subunit is less than one would infer from the gels of Figure IC. The actual increase in sub- unit phosphorylation was determined, taking into ac- count the change in moles of subunit; these values are compiled in Table 1. Surprisingly, cells exposed to forskolin for 48 hr had little or no effect on the phosphorylation of unassembled 6 subunits. This re- sult suggests that prior to subunit assembly, phos- phorylation of the y subunit is the important conse- quence of treatment by forskolin and, furthermore, that the 6 subunit may have little to do with this event.

While significant effort was made to isolate unas- sembled subunits from assembled AChRs, it is still possible that heterologous subunit-subunit interac- tions occur among the unassembled subunits and, possibly, that the phosphorylation of y and/or 6 could be influenced by associations with other subunits. To test whether the presence of the other subunits is affecting y or S subunit phosphorylation, cell lines expressing y or S subunits alone were treated with 32P and processed as described above. As can be seen in FigurelC(lanes9-12),yandSsubunitsarephosphory- lated when expressed alone, demonstrating that they do not require interactions with other subunits to be phosphorylated. The effect of forskolin on subunit phosphorylation was virtually identical to that found in cells expressing all four subunits (Table I), with the phosphorylation of y subunits being stimulated to a .much larger degree than the 6 subunit phosphoryla- tion. These results again indicate that the primary tar- get for the forskolin effect on subunit phosphoryla- tion is unassembled y and that even though the 6 subunit is phosphorylated, it may not play a role in the observed up regulation of cell surface AChRs.

Table 1. The Effect of Forskolin on AChR Subunit Phosphorylation

Ratio: Forsk. to Control.

32P per subunitc

Surface AChRsa Intracellular AChRs’ Unassembled Subunitsa Subunits Expressed Aloneb

Y 2.1 * 0.7 3.3 f 1.3 2.8 rt 0.1 2.4 8 1.6 + 0.6 1.9 f 1.1 0.9 * 0.3 1.3

Values were obtained from cells exposed to forskolin for 48 hr and are displayed as ratios of forskolin to control. a Data were obtained from All-11 cells and plotted as mean + SD for 2 determinations. b Data were obtained from 3T3-pSV2-ys cells for the y subunits and from 3T3-pSV2-S2 cells for the 6 subunits. c 32P per subunit ratios were determined by dividing phosphate values, obtained from gel scans of 32P-labeled y and 8 subunits, by the change in subunit moles. For unassembled and individually expressed subunits, the change in moles was determined from gel scans of [35S]methionine-labeled y and 8 subunits. For the surface and intracellular pools of AChR, the change in moles was determined by 1251-BuT~ binding to [35S]methionine-labeled AChRs.

Forskolin-Induced Subunit Phosphorylation Precedes AChR Up Regulation If an increase in unassembled subunit phosphoryla- tion up regulates AChR, then the increase in subunit phosphorylation would have to precede the increase in expression. This prediction was tested by compar- ing the time course of forskolin’s effect on surface expression with subunit phosphorylation (Figure 2). Because of the temperature-sensitive nature of Tor- pedo subunit assembly, we were able to devise a pro- tocol to synchronize assembly of subunits. Torpedo AChR subunits are synthesized at 37%, but no assem- bly occurs unless the temperature is lowered 40°C (Claudio et al., 1987; Paulson and Claudio, 1990). Once shifted to a permissive temperature, the polypeptides synthesized at 37OC are able to refold into an assem- bly-competent conformation (Ross et al., 1991). The protocol used here was to synthesize subunits at 37OC, shift cells to 20°C, and add forskolin at this time (time zero). As shown in Figure 2A, forskolin caused an -2-fold increase in both the rate of expression and the number of AChRs.

To compare the effects of forskolin on surface- expressed AChRs with newly synthesized unassem- bled subunits and to determine whether the phos- phorylation of unassembled subunits preceded the rise in surface AChRs, ceils were temperature-shifted and grown at 20°C for 48 hr, during which the cells were exposed to forskolin for different periods of time (Figures2B-2D). Forskolin had littleeffecton the num- ber of surface AChRs (Figure 2B) within 1 hr of treat- ment, whereas it had a maximum effect’on subunit phosphorylation within this time period (Figures 2C and 2D). After al hr exposure, unassembled y subunit phosphorylation increased 3.6-fold, whereas unas- sembled 6 subunit phosphorylation increased only 1.3-fold. These values are similar to those obtained after a 4%hr treatment with forskolin (Table 1). The results support the notion that phosphorylation of unassembled y is the important event leading to in- creased surface expression. The 2.6- to 3.6-fold in- creases in unassembled y phosphorylation are quite consistent with the 2- to 3-fold increases in surface

Time (h) Time (h) Time in Forskolin (h) Time in Forskolin (h)

O.ll 0 Tyme izFors:olin G) Time in Forskolin (h)

Figure2. Time Dependence of the For- skolin Effect on Subunit Phosphorylation

(A) The effect of forskolin on the t ime courseof surface AChRexpression at 20%. After the temperature was shifted from 37OC to 20°C (time zero), All-11 cells were treated without (open squares) or with (closed squares) 100 P M forskolin and as- sayed for surface AChRs at different times. Forskolin was added at t ime zero and was present during the entire 20°C incubation period. Values are given in picomoles, as determined by binding of 1251-B~T~ to the cell surface, and represent the mean of 2 determinations. (B)Timedependenceoftheforskolin effect on AChR cell surface expression. All-11 cells were temperatureshifted from 37OC to 20°C and then exposed to 100 P M for- skolin at 20°C for different lengths of time: 0, 1,3,24, or 48 hr. The number of ceil sur- face AChRs, monitored by 9-BuTx bind- ing, increased as a linear function of t ime exposed to forskolin.

(C) Time dependence of the forskolin effect on y subunit phosphorylation. All-11 cells, temperature-shifted and forskolin-treated as in (B), were labeled with 32P, and SDS gels were run of the assembled and unassembled subunits. Shown arevalues obtained from scans of phosphorylated y subunit bands from the gels of cell surface AChRs (open squares), intracellular AChRs (closed triangles), and unassembeld y subunits (open circles). The number of picomoles of subunit was determined by simultaneously labeling intracellular AChRs with P%]methionine and 1251-B~T~. From this dual labeling, the number of picomoles of y subunits (measured by the ‘251-BuT~ labeling) for a given [35S]methionine signal was obtained. Picomoles of [%]methionine-labeled unassembled y subunits could then be estimated. Assembled and unassembled y subunits were isolated as described in Figure 1. (D) The t ime dependence of the forskolin effect on 6 subunit phosphorylation. 6 subunit incorporation of 32P per picomole of subunit was determined as described in (C) for they subunit. Results qualitatively similar to those displayed in (B), (C), and (D) were obtained when the cells were temperature-shifted from 37OC to 26°C instead of 37OC to 20°C.

AChRs observed in All-11 and muscle cell lines after treatment with forskolin (Green et al., 1991). In addi- tion to this finding, unassembled subunits in general were found to be the most highly phosphorylated pool. The phosphorylation per subunit for unassem- bled y and 6 subunits was -2-fold greater than the phosphorylation of intracellular assembled y and 6 subunits and MO-fold greater than that of cell surface y and 6 subunits (Figures 2C and 2D). These results lend further support to the notion that forskolin stim- ulation of AChR expression is exerted through unas- sembled subunits.

Forskolin Stimulates Expression Only of AChR Complexes Containing y Subunits We have determined that forskolin stimulates phos- phorylation of unassembled 7 much more than 6, its phosphorylation is independent of 6, and increased AChR expression correlates with y phosphorylation. To test directly whether the forskolin effect on AChR expression acts through y and not S subunits, stable fibroblastcell I inesthatexpressedagyoraBSsubunits were established. Torpedo AChR complexes lacking a y or a S subunit are assembled into 9s a@r or apS oligomers, they are expressed on the cell surface, and they bind BuTx (Ross, Green, and Claudio, unpub- lished data). As shown in Figure 3, forskolin treatment of cells expressing a8r AChRs results in a 1.6-fold in- crease in surface AChRs (lane 3), whereas treatment

of ap6 cells results in only a l.l-fold increase (lane 4). the effect on aSy AChRs was nearly as large as that on a8ySAChRs (1.8-fold). We conclude that the stimu- lation of AChR by forskolin is independent of the 6 subunit.

We also established two other stable fibroblast cell lines to investigate the interactions between a and either y or 6. Torpedo ay and aS subunits form stable associations that bind BuTx, but the complexes are not transported to the cell surface (Ross, Green, and Claudio, unpublished data). We treated ay and a6 cell lineswith forskolin, solubilized thecells, labeled with 1251-BuT~, and immunoprecipitated each with anti-y or anti-8 antibodies, respectively. As shown in Figure 3 lane 5, anti-y antibody immunoprecipitated 1.9-fold more 1251-BuTx-ay in forskolin-treated cells than in control ay cells. In contrast, there was no difference between forskolin-treated and control with a6 cells (Figure 3, lane 7). These results demonstrate once again that forskolin stimulation of expression is inde- pendent of the 6 subunit and, furthermore, that it is independent of the f3 subunit as well. Analysis of a subunits not associated with y subunits (those re- maining in ay cells after immunoprecipitation with anti-y antibody) revealed that theytoo were not stimu- lated byforskolin treatment (Figure 3, lane 6), indicat- ing that forskolin has no direct effect on a subunits. The striking result is that the forskolin effect on in- creasing ay interactions is the sameas it ison stimulat-

;6ybunit Phosphorylation Regulates AChR Assembly

I II I Cell Surface

Subunit Complexes Intracellular Subunits

Figure 3. Effect of Forskolin on Cells Expressing aBy8, aBr, aB8, ay, or a8 Subunit Complexes The bars represent ratios of the number of cell surface AChRs (lanes I-4) or subunit-subunit interactions (lanes 5-8), forskolin to control, as determined by’*51-BuTx binding and are shown as the mean + SD. The effect of 100 PM forskolin on the number of cell surface AChRs is displayed for proper AChRs (aBy from All-11 cells; lane 2), aBr AChRs (from 3T3-pSVl-aByzO cells; lane 3), and aBS AChRs (from 3T3-pSV2-a& cells; lane 4). The effect of 100 uM forskolin on the number of intracellular cry complexes is displayed in lane 5 (3T3-pSV2-cry9 cells), and its effect on a8 complexes is displayed in lane 7 (3T3-pSV2-a& cells). ay and aS cell lines were first labeled with ‘*51-B~T~, and then the ay or a8 complexes were immunoprecipitated with either the anti-y MAb 168 (lane 5) or the anti-8 antiserum (lane 7X After immunodepletion of the complexes containing y or 8 subunits, the remaining 1Z51-B~T~ bound a subunits were immunoprecipi- tated with anti-a MAb 35 (lanes 6 and 8). The control bar in lane 1 represents untreated cells. All cell lines were induced to express AChR subunit complexes with our temperature-shift protocol: IO-cm plates of confluent cultures were supplemented with 20 mM sodium butyrate medium at 37OC for 24 hr, then shifted to 20% in fresh medium for 48 hr in the absenceor presenceof 100 PM forskolin.

ing expression of surface AChRs. Our results indicate that phosphorylation of the y subunit leads to in- creased associations between a and y and that this causes more efficient assembly of the subunits, which in turn leads to increased expression of surface AChRs.

Discussion

We have shown previously that forskolin, as well as other agents that increase intracellular CAMP levels (theophyllin, cholera toxin, and CAMP analogs), causes an increase in the number of surface-expressed Tor- pedo AChRs in fibroblasts and endogenous rat AChRs in rat muscle cell lines. The increase in surface AChRs in both the fibroblast and the muscle cell lines occurs in the presence of protein synthesis inhibitors, dem- onstrating that a posttranslational mechanism of ac- tion causes the effect. We also determined that for- skolin and a CAMP analog increased the half-life of each of the four subunits prior to the assembly of a&yS AChR pentamers. The forskolin effect was ob-

Figure 4. Model Detailing How AChR Subunit Phosphorylation Regulates Subunit Assembly

When All-11 cells are not treated with forskolin, CAMP-depen- dent phosphorylation of y subunits is low, associations between a and y are low, and the efficiency of assembly of AChR com- piexes is low (denoted by a single AChR pentamer). In the pres- ence of forskolin, CAMP-dependent phosphorylation of y sub- units increases, associations between a and y subunits increase, and there is a 2- to 3-fold increase in the number of AChR com- plexes compared with untreated cells.

served when subunits were present in the same ceil, but not when each was expressed in a separate ceil line, indicating that heterologous subunit-subunit in- teractions were also required (Green et al., 1991). In the present study, we investigated whether CAMP stimulation of AChR expression is caused by a cAMP- dependent phosphorylation of the AChR. The results from the previous study would suggest that if phos- phorylation of the AChR were important, then it would probably be occurring on one or more of the unassembled subunits.

Wefirstdetermined thatonlyyandSsubunitswere phosphorylated and that they were phosphorylated in three pools of subunits: assembted cell surface AChRs,fuIlyassembled internalAChRs,and unassem- bled subunits. Forskolin treatment of cells caused an increase in phosphorylation of both assembled and unassembled y subunits, but did not increase the phosphorylation of the unassembled S subunits. The increases in unassembled y subunit phosphoryla- tion correlated well with the increases in surface- expressed AChRs. By using cell lines expressing af3y and a@ AChRs, we were able to show that of the two phosphorylated subunits, the y subunit alone ac- counts for the increase in surface-expressed AChR subunit complexes. Studies using ay and a6 cell lines demonstrated that forskolin stimulated ay interac- tions and that neither S nor S was required for up regulation of the AChR by forskolin. A model consis- tent with all of these data is presented in Figure 4. We believe that phosphorylation of the y subunit leads to increased associations between a and y subunits.

Neuron 664

More efficient assembly of the subunits results from increased a-y associations, which lead to increased expression of surface AChRs. If this model is correct, then the association of a and y must occur early in the assembly of the AChR and may, in fact, be the event initiating subunit assembly, as suggested previously (Claudio, 1989).

We have shown previously that Torpedo subunits assemble in the endoplasmic reticulum (ER) of fibro- blasts and that transport through the Golgi to the plasma membrane is rapid (Ross et al., 1991). Since unassembled AChR subunits are phosphorylated, phosphorylation must therefore occur in the ER. It is likely that PKA is the kinase responsible for the phosphorylation of y, since phosphorylation of y is stimulated by forskolin. Proteins are often only tran- siently phosphorylated, and the degree to which they are phosphorylated depends on local kinase and phosphataseconcentrations. Since unassembled sub- units were phosphorylated more than assembled sub- units, the activity of PKA may be relatively high in the ER compared with other cellular compartments. It has been shown that PKA is present in the ER of canine pancreas cells (Nigam and Blobel, 1989), but the func- tion of PKA phosphorylation at this site is unknown. Our results indicate that PKA in the ER functions to regulate AChRassembly. It is possible that PKA serves asimilarfunctionforotheroligomericproteinsassem- bled in this compartment. PKA could be part of a sig- nal transduction mechanism in the ER whereby changes in intracellular CAMP could regulate protein assembly and possibly their release from the ER.

One curious finding is that although the 8 subunit contains a PKA phosphorylation consensus sequence that is phosphorylated in vitro (Yee and Huganir, 1987), forskolin fails to stimulate unassembled S sub- unit phosphorylation. One possibility is that this site is already occupied, since we have shown that both y and 6 can be phosphorylated without the addition of forskolin (Figure IA). It is also possible that unas- sembled S is a poorer substrate for PKA than y. If the latter is true, then what accounts for the observed phosphorylation of 8 in the absence of forskolin? PKA consensus sequences are not the only potential phos- phorylation sitesonyorLThe8subunitcontainsone to two PKC sites (Safran et al., 1987; Schroeder et al., 1990), both yand 8 contain tyrosine kinase phosphory- lation sites (Huganir et al., 1984; Schroeder et al., 1990), and it is possible to phosphorylate the fragment of y that contains the PKA sitewith PKC (Safran et al., 1990). We have shown in this study that phosphorylation of unassembled y leads to increased assembly efficiency of AChR subunits. This leaves at least six other phos- phorylation sites on y and 8 alone; these sites could serveas sites of regulation or modulation of theAChR.

Phosphorylation of y may not be required for as- semblyof the AChR, but it could serve as an important mechanism for regulating its assembly. Torpedo AChR subunits assemble with efficiencies of 20%- 37% in fibroblasts, and mouse AChR subunits assem- blewiththesesameefficiencies in muscleand infibro-

blasts (Ross et al., 1991). Since muscle AChR is not assembled with an efficiency of lOO%, it is plausible that efficiency of assemblywould be a site for regulat- ing AChR expression. Thus, although this study was performed on Torpedo AChR, an analogous situation may exist in mammalian AChR. It is very intriguing that it is regulation of the Torpedo y subunit that causes stimulation of AChR expression. As mentioned earlier, fetal y is replaced by adult y (E) with develop- ment. This developmental switch occurs as y mRNA levels drop to undetectable levels with a concurrent increase in E mRNA levels (Witzemann et al., 1989). In vitro studies of Torpedo y subunit have mapped the PKA phosphorylation site to Ser-353 (Yee and Huganir, 1987). There is a homologous PKA phosphorylation site on the mammalian E subunit, but not on mamma- lian y. Thus one could envision a mechanism whereby regulation of AChR CAMP-dependent phosphoryla- tion could aid in regulating the level of expression of both adult and embryonic AChRs. If CAMP levels were stimulated as y subunit production decreased and E

subunit production increased, a, 8, and 8 subunits would preferentially assemble with the phosphory- lated E subunit rather than the nonphosphorylated y subunit. Such a mechanism would amplify the y to E switch during muscle development.

Experimental Procedures

Cell lines Cell lines expressing Torpedo AChR subunit cDNAs were estab- lished either by cotransfecting a thymidine kinase gene or a neomycin resistance gene with Torpedo subunit cDNAs engi- neered into pSVL vectors into mouse fibroblast Ltk- or NIH 3T3 cells, respectively (Claudia et al., 1987,1989). The cell lines used in this study include L-pSV2-aBy&, or All-11 cells (expressing all four AChR subunits), L-pSVL-y5 (expressing y alone), L-pSV2-82 (expressing S alone), 3T3-pSV2-a&, (expressing aBy), 3T3-pSV2- aBSIJ (expressing aBS), 3T3-pSV2-ay, (expressing cry), and 3T3- pSV2-a& (expressing a@. Transfected L-cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) plus 10% calf serum and Ix HAT (15 pm/ml hypoxanthine, 1 ug/ml amino- pterin, 5 ug/ml thymidine). Transfected NIH 3T3 cells were main- tained in DMEM pluslO% calfserumand 0.6mg/ml C418(CIBCO Laboratories). All cell types were maintained in incubators at 37”C, 5% CO,. To induce expression of subunits and assembled complexes, IO-cm dishes of confluent cultures were supple- mented with 20 mM sodium butyrate (NB medium) at 37’C for 24 hr, then shifted to 20°C in fresh NB medium for 48-72 hr (temperature shift protocol) in the absence or presence of 100 uM forskolin (Calbiochem).

‘251-BuT~ Binding Cell surface AChR levels were determined by ‘“I-BuTx (specific activity 140-170 cpm/fmol; ICN) binding as previously described (Green et al., 1991). Briefly, cultures were incubated in 4 nM ‘WBuTxin PBSforL hratroom temperature,afterwhichcuItures were washed three times with PBS, scraped from the plate in lysis buffer (150 mM NaCI, 5 mM EDTA, 50 mM Tris[pH 7.5],0.03% NaNI, 1% Triton X-100), and counted. Nonspecific binding was determined by measuring ‘251-BuTx binding in the presence of the competitive inhibitor carbamylcholine (1 mM; Sigma). Total cellular or intracellular lZSI-B~T~ binding was measured by incu- bating cell extracts for 24 hr at 4°C in 10 nM ‘**I-BUTX followed by immunoprecipitation with various subunit-specific antisera (see below). ‘WBuTx binding to [35S]methionine-labeled AChRs was also measured. A comparison of ‘“I-BuTx binding to [?S]methionine-labeled and unlabeled AChRs showed that the

;6Sgubunit Phosphorylation Regulates AChR Assembly

error caused by the additional [‘SSlmethionine counts was less than 5%.

labeling and lmmunoprecipitations Plates (10 cm) of confluent cells were used to label cells with either [95S]methionine or p*PJorthophosphate. To label cells with [35S]methionine, cells were incubated for 24 hr at 37OC in NB medium. Cultures were washed once with methionine-free DMEM (J. R. Scientific) and then incubated in fresh methionine- free DMEM for 15 min. Cells were shifted to 20°C for48 hr in NB medium composed of methionine-free DMEM, containing 75- 125 uCi/ml [35S]methionine (4 ml per dish; Trans 35S; ICN), and supplemented with 20% NB medium. To label cells with [32P]or- thophosphate, cells were incubated for 24 hr at 37°C in NB me- dium, shifted to 20°C for 30-33 hr in NB medium, and then labeled with [32P]orthophosphate for 15-18 hr at 20°C. Prior to labelingcultureswerewashed oncewith phosphate-freeDMEM u. R. Scientific) and incubated for 15 min in fresh phosphate-free DMEM. Cultures were labeled in NB medium composed of phosphate-free DMEM, containing 300-600 uCi/ml [32P]ortho- phosphate (2 ml per dish; ICN).

After labeling, medium was removed and cells washed three times with PBS (this and all subsequent steps were carried out at 4OC), scraped from the plate, and pelleted by centrifugation at 5000x g for 3 min. The pellets were resuspended in 200-300 ~1 of lysis buffer supplemented with protease inhibitors, phenyl- methylsulfonyl fluoride (2 mM) and n-ethylmaleimide (2 mM), and phosphatase inhibitors, sodium fluoride (50 mM) and pyro- phosphate (5 mM). Cells were extracted for 60-120 min and clari- fied for 10 min at 10,000x g, and the supernatants were loaded onto sucrose gradients (see below). Sucrose gradient fractions were incubated overnight with anti-BuTx antisera (Claudia, un- published data), MAb 35 (American Type Culture Collection), Torpedo subunit-specific antisera (anti-a, -8, and -8 polyclonal antiserum [Claudio and Raftery, 1977]), or MAb 168 (Gullick and Lindstrom, 1983; Wan and Lindstrom, 1985). Protein A-Sepha- rose (Sigma) or anti-rat agarose (Sigma) was added, the mixture was rocked for 3-5 hr, and the resin was washed four times with lysis buffer. lmmunoprecipitates were electrophoresed on 7.5% SDS polyacrylamide gels, fixed, treated for 30 min with Fluoro- hance (Research Products International), dried on a gel dryer, and exposed to Kodak XAR film at -70°C with an intensifying screen for 4-8 days.

Autoradiographs of the labeled subunits were quantified by scanning densitometry using a BioimageNisage system (Milli- pore). Initially, the linearityofour scanning method waschecked by making a serial dilution of Torpedo AChR and performing a Western blot of different amounts of AChR run on an SDS polyacrylamide gel as previously described (Paulson and Clau- dio, 1990). By scanning the autoradiographs, we established a linear range of intensities of the AChR subunit bands that ex- ceeded 2 orders of magnitude. Care was taken to ensure that the intensity of the bands scanned in this studywaswithin this linear range. The linearity of our scanning procedure was checked in eachexperiment byrunningdifferentamounts (overa2-to3-fold range) of [35S]methionine-labeled AChRs on the gels. The ob- served changes in both the [“Plorthophosphate and p5S]me- thionine-labeled bands due to the forskolin stimulation were generally within this range. The linear range of the band intensity was further tested by exposing each gel to film for different lengths of time and scanning these multiple exposures.

Sucrose Gradient Centrifugation Cell lysates prepared as described above were layered onto a 5.0 ml, 5%-20% linear sucrose gradient prepared in lysis buffer. Gradients were centrifuged in a Beckman SW 50.1 rotor at 40,000 rpm for&t = 9.0 x IO”. Sixteen fractions (310 ~1) were collected from the top of the gradient, immunoprecipitated, and analyzed as described above.

Acknowledgments

We thank Dr. S. Tzartos (Hel!enic Pasteur Institute, Athens, Greece) for generously providing us with MAb 168. This work

was supported by an OssermanlMcClure research feilowship from the Myasthenia Gravis Foundation (W. N. C.) and National Institutes of Health grants NS21714 and HL38156 (T. C., W. N. C., and A. F. R.).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Sec- tion 1734 solely to indicate this fact.

Received June 5, 1991; revised ]uly 19, 1991.

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