Cell. Vol. 15, 1287-1300,

December

1978,

Copyright

0 1978 by MIT

Metabolism of Acetylcholine Receptor in Chick Embryo Muscle Cells: Effects of RSV and PMA Ruth Miskin, Thomas G. Easton,’ Alfred Maelicket and E. Reich Laboratory of Chemical Biology The Rockefeller University New York, New York 10021

Summary We have investigated some aspects of the metabolism of the integral membrane protein acetylcholine receptor (AChR) in normal and transformed cultures of chick embryo muscle cells. Turnover of AChR in control muscle cell cultures was compared with turnover in cultures infected and transformed by a temperature-sensitive mutant of Rous sarcoma virus (RSV) and with cultures treated with the tumor promoter phorbol myristate acetate (PMA). The parameters of AChR metabolism were estimated using ‘251-a-bungarotoxin as a stoichiometric high affinity ligand for the AChR. We found that both RSV transformation and PMA increased the rate of degradation and decreased the rate of synthesis of AChR. The consequent reduction in steady state receptor levels suggests that oncogenic transformation and tumor promoter significantly alter the metabolism of cell surface membranes. We also observed that parameters of AChR metabolism in control cultures changed systematically in a pattern which depended upon the age of the culture as well as the use of embryo extract or fetal bovine serum as medium supplements. The muscle cell system allows quantitative measurement of an integral membrane protein and its metabolism, and may serve as a more general model for alterations in membrane and surface receptor metabolism associated with the transformed state. Introduction Although there is a large body of evidence to support the widely held view that cell surface changes accompany oncogenic transformation, the nature of the relationship between membrane alterations and the malignant phenotype remains obscure. Attempts to rationalize some aspects of malignant cell behavior in terms of particular membrane functions are beset with many difficulties. These include the small number of clearly identified integral membrane functions, only a minority of which have been characterized in molecular * Present address: Department of Anatomy and Cell Biology, SUNY-Downstate Medical Center, Brooklyn, New York 11203. t Present address: Max-Planck-lnstitut fijr ErnBhrungsphysiologie, Dortmund, West Germany.

terms; the low concentrations of the molecules of interest; and the shortcomings in sensitivity, specificity or convenience of available assay methods, all of which combine to hamper and discourage quantitative experimentation. A characteristic abnormality of neoplastic cells potentially related to membrane changes is their response to hormones: in comparison with their normal tissue counterparts, tumors may be hyperresponsive to growth-promoting hormones or refractory to growth-inhibiting hormones or both, and since many of the important controlling hormones are polypeptides believed to interact with membrane receptors, the study of such receptors in systems undergoing transformation is of interest. With this in mind, we have investigated the metabolism of a specific membrane macromolecule analogous to hormone receptors-the nicotinic acetylcholine receptor (AChR) in chick myogenic cells exposed either to an oncogenic virus or to a tumor-promoting agent. The study of AChR metabolism in cultures of differentiating skeletal muscle is of special interest for several reasons. First, the AChR is well characterized both pharmacologically and biochemically (for review, see Cohen and Changeux, 1975; Maelicke, Fulpius and Reich, 1977), and it can be measured accurately as moles of receptor binding sites by use of the specific high affinity ligand lz51a-bungarotoxin (cr-BuTx) (for review, see Lee, 1972). This neurotoxin dissociates extremely slowly from its complexes with AChR and is a useful specific label since its binding is essentially irreversible on the time scale of metabolic or physiological experiments lasting several days. The work of Fambrough and his colleagues on AChR metabolism in skeletal muscle culture demonstrates the utility of cu-BuTx as an assay for rigorous determination of both the steady state level of plasma membrane AChR and rates of receptor biosynthesis and degradation (Hat-tzell and Fambrough, 1973; Devreotes and Fambrough, 1975,1976). Second, as an integral membrane protein mediating transmembrane signaling and local ion permeability, the AChR may serve as a model for other membrane receptors or integral membrane proteins, and may also reflect more general aspects of surface membrane metabolism. Third, changes in the amount and distribution of AChR in reinnervated (Miledi, 1960) or maturing skeletal muscle (Diamond and Miledi, 1962) are of considerable interest as examples of tissue-tissue interaction and timed programming in developmental processes, and in this regard, skeletal muscle culture provides an appropriate model that is more accessible to experimental manipulation than any other presently available.

Cdl 1288

Fourth, when suitably infected by avian sarcoma viruses, cultured skeletal muscle cells show characteristic changes after expression of the transforming viral gene (Easton and Reich, 1972; Fiszman and Fuchs, 1975; Holtzer et al., 1975; Miskin, Easton and Reich, 1978); and since several properties of other transformed cells are associated with surface changes (such as cell shape, distribution of intramembranous particles, lectin agglutinability), it is of interest to explore the response of a specific measurable and identifiable membrane structure to the transforming action of an oncogenie virus. With the preceding considerations in mind, we report here further study of muscle cell transformation by an RNA tumor virus and, for the first time, a quantitative determination in molar terms of the effects of transformation on the steady state concentration, synthesis and degradation of an integral membrane protein, the AChR. We have also investigated the effects of phorbol myristate acetate (PMA), a potent tumor promoter in mouse skin (Hecker, 1968), on the metabolism of AChR. Exposure to PMA has been shown to mimic several aspects of the phenotype of cells transformed by tumor viruses, including altered morphology (Sivak and Van Duuren, 1967; Diamond et al., 1974), increased rate of glucose uptake (Driedger and Blumberg, 1977), increased synthesis and secretion of plasminogen activator (Wigler and Weinstein, 1976; Miskin et al., 1978; Wilson and Reich, 1979), and decreased fibronectin production (Blumberg, Driedger and Rossow, 1976). As a necessary background for assessing the effects of viral transformation and PMA, we have also measured synthesis and half-life of AChR in normal muscle cell cultures. We have observed that both synthesis and degradation rates of AChR decrease systematically with the age of cultures and may also be manipulated by changes in the composition of the culture medium.

well above the rate-saturating concentration. The time course of binding also showed saturation; at 37C, binding was nearly complete by 30 min and showed no perceptible increase between 45 and 60 min. Identical time courses were found for control, PMA-treated and RSV-transformed cultures. Binding of ‘251-~-B~T~ was competed effectively (>95%) by unlabeled cu-BuTx (2 x 1O-6 M for 1 hr) or by dimethyltubocurarine (lo-’ M for 15 min). AChR-‘*+cu-BuTx complexes solubilized from cultures with 1% Triton X-100 and analyzed by sedimentation on sucrose gradients showed a single peak of radioactivity consistent with previous reports (Devreotes and Fambrough, 1975, 1976). Finally, most of the initially cell-bound radioactivity subsequently released into the medium was of low molecular weight, predominantly iodotyrosine (Devreotes and Fambrough, 1975). Estimation of the half-life of AChR-1251-(U-BuTx complex in muscle cells depends upon the determination, as a function of time, of the fraction of initially bound radioactivity which remains cell-associated. Half-life is estimated by graphical representation of the logarithm of the fraction of cellassociated receptor-toxin complex versus time, and corroborated by a least-squares fit of this linear relation. Devreotes and Fambrough (1975) have presented good evidence that such data accurately reflect the degradation rates of AChR-lZ51cy-BuTx complexes, and that AChR-a-BuTx complexes and AChR are degraded at equal or nearly equal rates. This conclusion is further supported by the results of Merlie, Changeux and Gros (1976). Thus we will consider that degradation of AChR‘*+LI-BuTx complex is operationally equivalent to degradation of AChR and use these terms interchangeably. We also note that a linear semi-logarithmic plot of such data is characteristic of an exponential process and is formally equivalent to a constant probability of degradation of any one AChR molecule with time. This may be expressed as an exponential first-order rate constant calculated from the expression k = In 2/t,,,.

Results Quantitative measurements of surface AChR in muscle cell cultures were performed by radioactive cu-BuTx binding to accessible AChR. To ensure that measurements of 1251-a-B~T~ binding represented specific and stoichiometric interaction with AChR, we have confirmed several parameters of the binding reaction (Vogel, Sytkowski and Nirenberg, 1972; Hartzell and Fambrough, 1973). The rate of binding of 1251-~-B~T~ to muscle cells in culture was saturable with increasing concentrations of radioactive toxin in the reaction mixture (see Experimental Procedures); the concentration of 1251-a-B~Tx which we used (2 x 1OmB M) was

Modulation of AChR Synthesis and Degradation in Normal Cultures: Effects of Culture Age, Embryo Extract and Fetal Bovine Serum Several reports have indicated age-dependent changes in the levels of surface AChR in muscle cell culture, reflecting systematic changes in the relative rates of AChR synthesis and degradation as a function of muscle cell maturation (Prives, Silman and Amsterdam, 1976; Spector and Prives, 1977). Under our routine conditions of culture (see Experimental Procedures), myogenic cells multiply rapidly and form confluent monolayers during the first 45 hr after plating. Cell fusion begins at approximately 45-50 hr and is completed by about 70

Acetylcholine 1269

Receptor:

Effect

of Oncogenic

Agents

hr after plating, and all cell division is terminated by the addition of cytosine arabinonucleoside (AraC) at 45-50 hr. Significant levels of surface AChR are measurable at 72 hr, generally increasing to a maximum around 5-7 days and declining slowly thereafter. In contrast to previous reports (Devreotes and Fambrough, 1975; Merlie, Changeux and Gros, 1978), we have found that there is a consistent increase in AChR half-life with increasing age of culture. While there was some variability among culture preparations in the absolute values at any given age, we observed that within any one cell preparation, AChR half-life is consistently longer in older cultures (see Figures 1 and 2). This is also evident upon examination of the data for normal control cultures presented in Tables 1 and 2. In 33 measurements of AChR half-life in normal cultures, unselected even for factors such as temperature (which are known to affect half-life), the correlation coefficient between age of culture in days and AChR half-life in hours was 0.72. Further indications of systematic changes in AChR metabolism with culture age are evident from the data in Table 1, in which rates of synthesis, half-life and surface levels of AChR were measured in companion cultures. Columns 7 and 8 in this table are the calculated degradation and net changes, in fmole/hr,

for each experiment. It is clear that both rates of synthesis and estimated net rate of increase of AChR were slower in older cultures, while the receptor half-life increased. These results are fully consistent with the measurements of surface AChR shown for normal cultures in Figure 3. These observations imply that while increases in surface AChR may be dominated by rate of synthesis of new receptors (Devreotes and Fambrough, 1976), there is also an age-dependent contribution from half-life affecting the net surface AChR levels in older cultures. While we are not able to explain in any detail the mechanism of the age-dependent changes in AChR metabolism, we have found that the metabolic parameters are affected by the composition of culture medium. For example, chick embryo extract and fetal bovine serum in culture medium affected AChR half-life (Tables 1 and 4). Both these supplements accelerated receptor metabolism: in comparison with cultures in which they were absent, embryo extract and fetal bovine serum increased both the rates of AChR synthesis and degradation, the higher levels of surface receptor achieved reflecting a greater incremental effect on synthesis relative to degradation. Effects of PMA and ts-66 RSV on AchR Metabolism in Muscle Cell Culture Effect of P MA on ACM Half-Life To study the effect of PMA on AChR turnover in differentiating muscle cultures, cells were pretreated with this drug (20 or 50 rig/ml, 32 or 81 nM

l 0 +EE . 0 -EE

0.25

I 10

I 20

I 30

I 40

I 50

1 60

I

70

Hours Figure 1. Effect of Culture Degradation of AChR

Age

and

Medium

Composition

on

Cultures were incubated at 41°C. On day 3. medium was changed and half the cultures were fed medium without embryo extract. All cultures were then shifted to 37%. At days 4 and 7, cultures in medium with embryo extract and companion cultures maintained without embryo extract were tested for AChR degradation as indicated in Experimental Procedures. Results are presented as log fraction of initial radioactivity which was cell-associated at each sampling time. Half-lives in hours are shown adjacent to corresponding curves. (O--O) 4 day culture, embryo extract in medium; (O-O) 7 day culture, embryo extract in medium; (Lm) 4 day culture, embryo extract omitted; (0-O) 7 day culture, embryo extract omitted. The initial binding of ‘*sl-a-B~Tx for these cultures was 710, 637, 746 and 479 fmole per culture, respectively.

Figure

2. Degradation

of AChR

in Control

and PMA-Treated

Cul-

tu res Cultures were incubated at 41°C and embryo extract was omitted on day 3. Some cultures were shifted to 37% and/or treated with F’MA (20 rig/ml) for 16 hr (3d) or 12 hr (7d) before labeling of AChR with radioactive a-BuTx. Degradation of AChR was determined as described in Experimental Procedures. (Left panels) Culture age 3 days at the time of AChR labeling; upper at 41”C, lower at 3PC. (Right panels) Culture age 7 days at time of labeling: upper at 41”C, lower at 37°C. (O---O) Control: (A-A) WA-treated. Initial binding of radioactive a-BuTx was, respectively, 63, 76, 230 and 114 fmole per culture at 3 days, and 172. 157, 179 and 166 fmole per culture at 7 days.

Cell 1290

Table 1. Effect Muscle Cultures

of Culture

Experiment Number

Culture Pays)

1

Age, Embryo

and Fetal Bovine

Serum

on Synthesis,

State Level

of AChR

666

26

16

+2

22

520

34

IO

+12

+EE

32

666

26

18

+14

-EE

19

603

33

13

+6

+EE

14

502

39

9

+5

Steady (fmole

4

-EE

20

2

4

+EE

3

4

-EE

5

and Steady

VW

Synthesis (fmole hrr’)

4

Half-Life

Calculated Degradation (fmole hrr’)

Medium Supplement

7

Age

Extract

State Culture-‘)

Half-Life

Net (fmole

3

184

59

2

+1

+FBS

24

595

36

11

+13

3

-EE

42

718

26

19

+23

6

-EE

8

430

56

5

+3

3

-EE

12

184

21

6

+6

7

-EE

3

185

56

2

+1

in Normal

hr-‘)

Cultures were prepared and incubated at 41°C as described in Experimental Procedures. On day 3, fresh culture medium contained either 1.5% embryo extract (+EE) or no embryo extract (-EE), as indicated. In experiment 3. 5% FBS replaced embryo extract at the medium change on day 3. Cultures were shifted to 37°C between 5 and 16 hr before the initiation of experiments. All determinations were performed at 37°C. For all experiments presented in this report, culture age denoted the day after seeding on which cultures were exposed to 1*51-~ BuTx; cultures were initiated on day 0.

for 5-16 hr) after completion of fusion to form myotubes. Following this pretreatment, 1251-~BuTx was bound to surface AChR, and the time course of AChR degradation and receptor half-life were determined in the continued presence of PMA. Typical results are presented in Figures 2 and 4. In the experiments shown in Figure 2, receptor half-life was estimated for PMA-treated and for companion control cultures at two temperatures (37 and 41°C) and for two culture ages [3 days (left panels) and 7 days (right panels)]. Relative to the appropriate controls, PMA treatment accelerated AChR degradation, thereby shortening receptor half-life at both incubation temperatures and for both ages of cultures. As already noted above for control cultures and consistent with a previous report (Devreotes and Fambrough, 1975) the numerical value for AChR half-life was dependent upon both culture age and temperature of incubation. In the experiment shown in Figure 4, the effect of PMA treatment was examined at day 7 in companion cultures maintained from day 3 to day 7 in medium either with or without embryo extract. Again, for identical culture media, PMA treatment increased AChR degradation relative to control rates. As already shown for untreated control cultures (Figure l), however, the absolute rates of AChR degradation in both control and PMA-treated cultures were slower in cultures deprived of embryo extract. Results of other experiments demonstrating the effects of PMA on AChR half-life are presented in Table 2; the values in column 6 are

the ratios of the half-lives for PMA and control cultures, reflecting the increase in AChR degradation resulting from PMA treatment. These results indicate that PMA treatment consistently increased the rate of AChR degradation over that found in companion control cultures under all conditions tested. We also note that at least two variables, culture age (Figure 2) and embryo extract supplement (Figure 4), affected AChR half-life in both control and PMA-treated cultures; furthermore, a consideration of absolute rates of receptor degradation in PMA-treated cultures suggests that PMA acts by modulating the “normal” rate of degradation characteristic of control cultures at a given age or nutritional history rather than itself determining a unique “PMA-induced” rate of AChR degradation. The preceding experiments were performed at PMA concentrations of 20 or 50 rig/ml (32 or 81 nM), corresponding to the levels that were effective in increasing secretion of plasminogen activator (PA) by chicken embryo fibroblasts (Wilson and Reich, 1979). Recent experiments measuring PA synthesis in PMA-treated muscle cultures indicate some variability in the sensitivity of individul culture preparations to PMA at concentrations of 20 rig/ml or lower, but not at 50 rig/ml or higher (our unpublished observations). This result may be relevant to the minimal effect of PMA on AChR degradation which we observed in 4 of 23 separate experiments. For some experiments presented in Table 2, the half-life of AChR in control cultures at 41°C was

Acetylcholine 1291

Table

2. Effect

Receptor:

Effect

of PMA Treatment

on Half-Life

Agents

of AChR

Experiment Number

Culture (Days)

Incubation Temperature

1

3

2

3

4

4

6

3

7

Age

of Dncogenic

VW

Control

(W

Ratio of Half-Life Control/PMA

37

26

17

1 .7

41

24

16

1 .5

(“C)

Half-Life

PMA Half-Life

37

26

18

1.4

41

34

32

1 .l

37

56

24

2.3

41

53

29

1.0

37

21

16

1.3

41

24

15

1.6

37

56

39

1.4

41

45

35

1.3 1.5 Average 1.6 Average

All cultures were initially incubated at 41°C. Cultures were shifted to 37°C and/or treated with PMA (32 or 61 nM) 5-16 to ‘251-~-B~T~. Determination of AChR half-life was performed as described in Experimental Procedures.

longer than for companion control cultures at 3PC (see Devreotes and Fambrough, 1975). In those apparently anomalous cultures, this seems to be related to lonoer times of PMA pretreatment and temperature shift preceding exposure to 1251-~BuTx. We have not, however, specifically and systematically investigated any relation between the duration of pretreatment at different temperatures and subsequent AChR half-lifes at 37 and 4l”C, either for control or PMA-treated cultures. In several experiments, the radioactivity released into culture medium from PMA and from companion control cultures, representing degradation of AChR-a-BuTx complexes, was analyzed by gel filtration on Bio-Gel P-2 columns as described by Devreotes and Fambrough (1975). The recovery of radioactivity from columns was lOO%, and 80-90% of the radioactivity was found at elution volumes greater than the elution volume for cy-BuTx. The profiles of eluted radioactivity for PMA-treated cultures were indistinguishable from those for companion control cultures, indicating that AChR degradation rates observed in PMA-treated cultures were not attributable to altered AChR binding properties for a-BuTx and subsequent release of intact c~-BuTx into culture supernatants, and were also unlikely to be due to extracellular proteolysis and release of radioactive CX-BuTx fragments. As a further indication that extracellular proteolysis in PMA-treated cultures did not account for observed degradation of cr-BuTx, PMA-treated cultures and companion control cultures were incubated for 16 hr in excess (3 x 10m9 M) 1251-a-B~T~. Under these conditions, most of the radioactivity (>97%) recovered from culture supernatants and analyzed by gel filtration co-eluted with authentic

of 23 trials hr before

exposure

cu-BuTx. We have observed that in cultures in which cell fusion is not completed, PMA treatment prevents further myotube formation, as previously reported by Cohen et al. (1977). In view of this complication, we have not examined the effects of PMA on AChR metabolism in cultures less than 3 days old-that is, before myotube formation was essentially complete. We have also tested three PMA analogs-4-aphorbol-12,13-didodecanoate, 1,2-dihydro-12-0tetradecanoyl-phorbol-13-acetate-p-oxide and phorbol-for effects on AChR half-life in muscle cells. In contrast to PMA, these analogues, which lack tumor promoter activity in vivo (Suss, Kreibich and Kinzel, 1972; Schmidt and Hecker, 1975), did not affect either half-life or cell surface levels of AChR when tested at 100 rig/ml. We surmise from these results that the effect of PMA on AChR turnover is probably related to the tumor-promoting activity of this agent. Effect of PMA on Surface Levels of ACM To study the consequences of PMA exposure for levels of surface AChR, a series of replicate uninfected cultures was prepared and maintained at 41°C for 3 days to allow completion of fusion and myotube formation. The cultures were first divided into two groups, one of which received medium supplemented with embryo extract (1.5%) with the other receiving no embryo extract; each group was then subdivided so that half of the cultures received PMA, whose concentration was maintained by daily renewal, and the remainder served as controls. The level of surface AChR in individual cultures from each group was measured daily by binding of ‘251-(u-B~T~. The results are summa-

Cell 1292

I

1

3

I 4

I 5

I 6

1 7

1 8

Days Figure 3. Changes in Surface Levels of AChR with Age of Culture in Control, WA-Treated and ts6Elnfected Muscle Cells (Upper panel) Cultures were incubated at 41°C and maintained as described in Experimental Procedures. On day 3, cultures were refed medium with either 1.5% or no embryo extract and reincubated at 3PC. Some cultures also received PMA (50 rig/ml); for the duration of the experiment, these cultures received fresh PMA daily, either during medium changes (50 rig/ml) on days 5 and 7 or by additions of PMA (25 rig/ml) to culture medium on days 4 and 6. a-BuTx binding to AChR was determined first on day 3 just before the temperature shift and at 24 hr intervals thereafter. The binding reaction was performed as described in Experimental Procedures, with the following changes: after unbound toxin was removed, cells were gently scraped with a teflon policeman and suspended in 4 ml of cold PBS, followed by centrifugation at 2000 x g for 5 min. The radioactivity of the pellet was counted and the pellet was resuspended in 0.2 ml of 0.5% Triton X-100. The amount of pellet protein was determined fluorimetrically (Bohlen et al., 1973) using bovine serum albumin as a standard. Results are expressed as fmole of ‘V-CC-BuTx bound per mg protein. (60) Control cultures with embryo extract; (00) control cultures without embryo extract; (A-A) PMA-treated cultures with embryo extract; (A-A) MA-treated cultures without embryo extract. (Lower panel) In a separate experiment, cultures were prepared from uninfected or tsb&infected muscle cells and incubated at 41°C. On day 3, all cultures were refed medium with 1.5% embryo extract and part of the cultures were shifted to 3PC. WBUTX binding was performed as described above. (GO) Control cultures at 41°C; (G 0) control cultures at 3PC; (A-A) ts6Sinfected cultures at 41°C; (A-A) tsB&infectad cultures at 3PC. Inset: (A-A) and (A-A) WBUTX binding to ts-66 infected cultures incubated at 41 or 3PC, respectively, expressed as the percentage of binding to companion uninfected cultures also at 41 or 3PC.

rized in Figure 3 (upper panel) and show the following. First, PMA treatment decreased the level of surface AChR to less than one eighth of that in the control cultures after 4 days of exposure. Second, neither this effect of PMA nor the level of surface AChR in control cultures was substantially influenced by the presence or absence of embryo extract. Third, the magnitude of the PMA-stimulated increase in AChR degradation rate could not by itself have accounted for the profound loss in surface receptor; hence PMA must have concurrently depressed the rate of receptor synthesis, a conclusion that is documented below. A point of some interest for the actions of embryo extract on AChR metabolism emerges from consideration of the data in Figure 3 in relation to those in Figure 4 and Table 4. The presence of embryo extract increased the rate of receptor degradation but did not significantly alter the quantity of surface AChR; hence the rate of receptor synthesis must have increased correspondingly. By coupling the rates of AChR synthesis and degradation, the cultures regulated the level of their surface receptors, and they did so independently of the absolute rates of receptor metabolism. It follows that the dynamic regulation of surface AChR levels is probably achieved under normal conditions by adjustment of the rates of both synthesis and degradation, rather than of either parameter alone. This is in contrast to the uncoupling of receptor synthesis and degradation caused by PMA, the depression of the former being matched with an increase in the latter, thereby leading to a sharp drop in surface receptor levels. Effect of h-68 RSV on ACM Half-Life To study the effect of Rous sarcoma virus (RSV) transformation on AChR metabolism, we have used a mutant of the Schmidt-Ruppin A RSV that is temperature-sensitive for expression of transforming function. This mutant, designated ts-68, replicates well at both 37 and 41”C, but transforms infected chick embryo fibroblasts only at 37% (Kawai and Hanafusa, 1971). We have found that primary chick embryo muscle cells infected in suspension by ts-68 (5-10 ffu per cell) before plating and then maintained at 41°C yield cultures in which the time course of myotube formation closely parallels uninfected control cultures. In contrast, infection with ts-68 after cell attachment to culture dishes is less conducive to subsequent culture development. The effect on the rate of degradation of AChR in muscle cells infected and transformed by the ts-68 mutant of Rous sarcoma virus is shown in Figure 5. The upper panel shows results for RSV-infected and control uninfected muscle cells at 41X, a nonpermissive temperature for expression of virus transforming function. In this experiment, the halffife for AChR in infected cells was slightly longer

Acetylcholine 1293

Receptor:

Effect

of Oncogenic

Agents

-EE

t e tL

l

0

+EE

Control

A A PMA I 10

I 20

I 3c

I 40

$ A

I 50

29\* 0.50-

\*.

\

5

Hours Figure 4. Degradation of AChR in Control and PMA-Treated ture in Medium with or without Embryo Extract

7 c

Cul-

Cultures were incubated at 41°C in medium with 3% embryo extract. Cultures were refed on day 3 and half of them received medium without embryo extract. At day 6, half the cultures were treated with PMA for 14 hr before labeling of AChR. Degradation of AChFl was measured at 3PC as described in Experimental Procedures: (C--0) control, without embryo extract; (A-A) PMA-treated, without embryo extract; (M) control, with embryo extract; (A-A) PMA-treated, with embryo extract. Surface AChR (fmole per culture) was 176. 148, 460 and 384, respectively.

than in control cells; the difference (33 hr versus 28 hr), although small, is significant (p

Metabolism of acetylcholine receptor in chick embryo muscle cells: effects of RSV and PMA.

Cell. Vol. 15, 1287-1300, December 1978, Copyright 0 1978 by MIT Metabolism of Acetylcholine Receptor in Chick Embryo Muscle Cells: Effects of RS...
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