Carbohydrate requirement for expression and stability of - Europe PMC

Proc. Natl. Acad. Sci. USA Vol. 77, No. 9, pp. 5263-5267, September 1980

Cell Biology

Carbohydrate requirement for expression and stability of acetylcholine receptor on the surface of embryonic muscle cells in culture (glycosylation/protein degradation/tunicamycin/leupeptin)

JOAV M. PRIVES*t AND KENNETH OLDEN*§ *Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development; and tLaboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20205

Communicated by Eugene P. Kennedy, May 1, 1980

ABSTRACT We have investigated the significance of protein glycosylation for metabolism of acetylcholine receptors (AcChoR) in primary cultures of embryonic chicken muscle cells. Tunicamycin, a specific inhibitor of the glycosylation of asparagine residues on glycoproteins, decreased AcChoR accumulation and accelerated its degradation. In contrast, there was no evidence that tunicamycin treatment affected AcChoR biosynthesis, intracellular transport, or incorporation into surface membranes. Leupeptin, an inhibitor of intracellular proteases, markedly increased accumulation of AcChoR on the external surface of muscle cells treated with tunicamycin. Our findings indicate that impairment of protein glycosylation prevents accumulation of AcChoR by increasing its susceptibility to degradation by cellular proteases.

is strongly impaired by TM treatment. However, the proteolytic degradation of underglycosylated AcChoR is substantially increased under these conditions, resulting in marked diminution of AcChoR accumulation. The protease inhibitor leupeptin substantially reverses the decrease in AcChoR in TM-treated cultures, suggesting that glycosylation stabilizes AcChoR against proteolytic degradation. The possibility that the carbohydrate on AcChoR may function in regulating its stability is supported by our findings that: (i) impaired protein glycosylation results in accelerated degradation of AcChoR, and (ii) concanavalin A (Con A), a ligand of AcChoR (2,3), increases AcChoR stability in control cultures but not in TM-treated cells.

The acetylcholine receptor (AcChoR) of skeletal muscle cells is an integral membrane glycoprotein that is uniquely well characterized both pharmacologically and biochemically (for recent reviews see refs. 1-3). Thus AcChoR may serve as a model for detailed study of the function of the carbohydrate components of glycoproteins in the regulation of well-defined plasma membrane properties. AcChoR is of particular interest because its synthesis, distribution on the muscle cell surface, and degradation are tightly regulated during differentiation and upon innervation (3). The striking effects induced under neuronal influence include the disappearance of AcChoR from extrasynaptic regions of the muscle cell surface and a marked decrease in the degradation rate of synaptic AcChoR (3). The regulation of AcChoR metabolism has recently been intensively studied in cell cultures of differentiating skeletal muscle (3, 4) using 25I-labeled a-bungarotoxin ('25I-a-Bgt), a specific ligand that binds to AcChoR with high specificity and low reversibility (5). The function of carbohydrate components of glycoproteins can be conveniently studied in cultured cells by the use of tunicamycin (TM), an antibiotic that specifically inhibits protein glycosylation (6-17). Treatment of cells with TM results in the synthesis of glycoproteins deficient in asparagine-linked oligosaccharides (6, 18, 19). TM has been utilized to study the contribution of the oligosaccharide components to the posttranslational processing (16, 17), intracellular transport (9, 16, 17), secretion (7, 9, 13, 15-17, 20-23), and functional properties (9, 24) of specific glycoproteins. In the present study, we have used TM to determine the role of carbohydrate covalently attached to protein in the accumulation and degradation of AcChoR in cultured muscle cells. We have found that the synthesis and insertion of AcChoR into plasma membranes is not blocked when protein glycosylation

MATERIALS AND METHODS Cell Culture. Primary cultures of embryonic skeletal muscle were prepared from breasts of 12-day chicken embryos, plated on collagen-coated 6-cm culture dishes, and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) horse serum and 2% (vol/vol) chicken embryo extract as described (25, 26). Assay Procedures. To determine the effect of TM on the synthesis and glycosylation of protein, muscle cultures were preincubated for 6 hr in growth medium with or without TM at the specified concentration. After addition of radiolabeled precursor, incubation was continued for an additional 16 hr. Protein synthesis was measured by the incorporation of L[U-'4C]leucine (2 yCi/ml, specific activity 325 mCi/mmol; 1 Ci = 3.7 X 1010 becquerels) into the total 10% trichloroacetic acid-insoluble fraction. Protein glycosylation was monitored by D-[2-3H]mannose (0.5 ,uCi/ml, 2 Ci/mmol) incorporation into the acid-insoluble material. AcChoR on surface membranes of intact muscle cells was measured by the specific binding of 125I-a-Bgt as described (25, 26). Cultures were incubated with '25I-a-Bgt (10 nM) in DMEM containing bovine serum albumin (1 mg/ml) for 1 hr.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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Radioactivity that remained associated with the cells after five washes with 3-ml volumes of DMEM was measured by gamma spectroscopy. Specificity was established by the inhibition of 1251-a-Bgt labeling in the presence of the competitive inhibitor decamethonium (10 AM) as described (25). Total cellular AcChoR was also determined in detergent extracts of muscle cells, prepared by treatment with 1% Triton X-100 in phosphatebuffered saline of cells harvested from culture dishes by Abbreviations: AcChoR, acetylcholine receptor; Con A, concanavalin A; a-Bgt, a-bungarotoxin; TM, tunicamycin; DMEM, Dulbecco's modified Eagle's medium. t Present address: Dept. of Anatomical Sciences, Health Science Center, State Univ. of New York, Stony Brook, NY 11794. § Present address: Dept. of Oncology, Howard Univ. Cancer Center, Washington, DC 20060.

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Proc. Natl. Acad. Sci. USA 77 (1980)

Cell Biology: Prives and Olden

scraping. After a 4-hr incubation in detergent at 40C, the extracts were centrifuged at 100,000 X g for 1 hr and samples of the supernatant were labeled with 125I-a-Bgt (10 nM) for 1 hr. The '251-a-Bgt-AcChoR complexes were separated from the free toxin by precipitation with 40% saturated ammonium sulfate, and the precipitates were trapped on Whatman GLC filters and their radioactivities were measured by gamma spectroscopy. The degradation of 125I-a-Bgt-AcChoR complex was measured by the release of 125I into the medium by cultures previously incubated with '25I-a-Bgt (27, 28). After removal of unbound toxin by several washes with DMEM, 1 ml of fresh DMEM with or without Con A (0.05 mg/ml) was added to the cultures. After a 1-hr incubation, this medium was removed, pooled with a further 2-ml rinse for radioactivity measurement by gamma spectroscopy, and replaced with 2 ml of DMEM containing 10% horse serum. At specified intervals medium was removed for radioactivity measurement as above and replaced by new medium. Finally, the cells were removed from the culture dish by a 3-hr incubation with 1 M NaOH and the radioactivity that remained bound to the cells was determined. TM was a generous gift from Gakuza Tamura via the Drug Evaluation Branch of the National Cancer Institute. Radiochemicals- were purchased from New England Nuclear and leupeptin from Sigma. Protein determinations were performed according to the procedure of Lowry et al. (29) with bovine serum albumin as standard. RESULTS Effect of TM on Total Protein Synthesis and Glycosylation. TM was added to cultured muscle cells 1 day after plating, immediately after myoblast fusion, and shortly before the period of rapid accumulation of AcChoR (26). The effects of various concentrations of TM on the incorporation of leucine and mannose into the trichloroacetic acid-insoluble fraction are shown in Table 1. Glycosylation was inhibited by more than 90% at all three TM concentrations. The inhibition of protein synthesis was significantly less marked. At the lowest concentration of TM used, overall protein synthesis was inhibited by less than 20%. Consequently, the subsequent experiments reported here were performed at the lowest concentrations of TM (0.05 ,g/ml), unless stipulated otherwise. Table 1. Effects of TM on incorporation of leucine and mannose into total trichloroacetic acid-insoluble fractions of cultured embryonic muscle cells % Incorp. into inhibiprotein, TM, tion Precursor cpm/,g ,ug/ml

Table 2. Effect of TM on accumulation and degradation of AcChoR in embryonic muscle cells Degradation of 1251-a-Bgt-AcChoR 1251-a-Bgt bound % of fmol/ % of TM, culture control control ,ug/ml t1/2, hr 0 0.05 0.1 0.2

295 27 20 19

9.1 6.6 6.3

14 4.7 4.7 4.2

34 34 30

Cultured muscle cells were treated with TM for 1 day during the period of maximal AcChoR elaboration (see text), then labeled with 10 nM 1251-a-Bgt for 1 hr at 370C. The cells were washed five times in medium to remove unbound toxin. For the determinations of total 1251-a-Bgt bound, the cells were removed in 1 M NaOH immediately after these washes and their radioactivity was measured by gamma spectroscopy. For determination of AcChoR degradation rates, incubation was continued in fresh medium after the removal of unbound toxin. The amount of 1251 released into the medium was measured at various times. The results are expressed as half-time (t 1/2) of AcChoR degradation, the time at which the ratio 1251 released/1251 initially bound is 0.5. AcChoR half-times were estimated from plots such as those shown in Fig. 2. Each value is the mean of duplicate plates.

Effects of TM on the Accumulation and Degradation of AcChoR. We measured the effects of TM on the accumulation of AcChoR on the external surface of cultured muscle cells, as detected by the specific binding of 125I-a-Bgt (25, 26, 30, 31). Muscle cells were incubated with TM under the conditions described for Table 1. As shown in Table 2 and Fig. 1, treatment with TM at all concentrations strongly decreased the number of a-Bgt binding sites on the muscle cells. This effect was near maximal at a TM concentration of 0.05 ,gg/ml, under conditions in which protein glycosylation was almost abolished and protein synthesis was only slightly impaired (Table 1). A 5-fold increase in the 1251-a-Bgt concentration from 10 to 50 nM did not increase the specific binding to AcChoR in either TM-treated or control cultures. This observation suggests that the apparent decrease in '25I-a-Bgt binding in TM-treated cultures is not the result of reduced affinity of receptor for toxin but is due to reduced number of available receptor binding sites. The diminished population of AcChoR incorporated into surface membranes of TM-treated muscle cultures was distinguishable from AcChoR in untreated cells by an enhanced rate of degradation as shown in Table 2 and Fig. 2. Control

300 r

Eto0 200

-I

L-[U-14C]Leucine

D-[2-3H]Mannose

0 0.05 0.10 0.20 0 0.05 0.10 0.20

1683 + 176 1389+ 123 1125+ 69 785+ 57 298 ± 69 21± 7 15± 6 6± 4

0

17± 8 33 10 53± 4 0 93± 2 95± 2 98± 1

Cultured muscle cells were incubated with the radioactive precursor for 16 hr at 37°C after a 6-hr preincubation with or without TM. Incorporation was determined by precipitation of protein with 10% trichloroacetic acid, solubilization in 0.5 M NaOH, and dilution with Aquasol (New England Nuclear) of aliquots containing equal amounts of protein to a final volume of 20 ml. Radioactivity in these samples was measured by liquid scintillation spectrometry. The results represent the average of three experiments ± the range.

049 ._

'4-4

04 GQ

100 He

' +TM

72 24 48 Time in culture, hr FIG. 1. Effect of TM on accumulation of AcChoR in embryonic muscle cells. Cells were treated with TM and AcChoR was assayed as described in the legend to Table 2.



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mannosyl and glucosyl residues on glycoproteins. It binds to AcChoR in surface membranes of cultured muscle cells and decreases the lateral mobilit and degradation rate of AcChoR 40

1 60 -

-9 0.

0

5

10 Time, hr

15

2(D

FIG. 2. Effect of TM on the degradation rate of AcChoR in embryonic muscle cells. The release of 1251 into culture medi [um by TM-treated and untreated muscle cells labeled with 1251-a-E compared as described in the legend of Table 2 and Materic and Methods. The percent of total radioactivity initially bound ti hat was released with increasing intervals of incubation in fresh med.ium at 370C was plotted semilogarithmically versus incubation timte. The AcChoR half-times are shown in Tables 2 and 3, estimate(d from similar plots. Each point is the mean of duplicate plates. 0, C oesontrol cultures labeled with l25I-a-Bgt 2 days after plating; 0, cultu beled with 125I-a-Bgt 2 days after plating, after 24-hr exposure to X, cultures labeled with 1251-a-Bgt 3 days after plating, afterr 24-hr exposure to TM.

3gs

TMl

The kinetics of degradation are first-order (Fig. 2) Vwith a half-life of between 12 and 14 hr under control conditionLs (also see Table 4). AcChoR degradation was stimulated by inculbation of newly fused muscle cells with TM shortly before the rapid accumulation of AcChoR. Treatment of these cells with TM for 24 hr led to the incorporation into muscle cell membrai nes of AcChoR having a catabolic half-life of 4 hr, indicating

a

i

rate

of degradation 3- to 4-fold higher than the degradation c)f AcChoR in control cultures. There was no significant incresase in the rate of degradation of total cellular protein, indicatingg that the more rapid turnover of AcChoR is not due to gener*al increase in cellular proteolytic activity. When TM was added for a 24-hr period 1 day later iin differentiation, when AcChoR in the membrane had rea ched near-maximal levels, a biphasic degradation curve waIS obtained, indicating the existence of two populations of rece ptors (Fig. 2). The rapidly degraded fraction (t1/2 4-5 hr) appar,ently corresponds to receptors made in the presence of TM, anid the slowly degraded fraction (t1/2 12-14 hr) represents Ac( ThoR made before the addition of TM. Effect of Con A on Degradation of AcChoR. To detenmine whether this change in AcChoR degradation is associated with altered glycosylation of cell surface AcChoR, we compare(d the effects of Con A on AcChoR degradation rates in controlI and TM-treated cultures. Con A is a lectin with high affinityy for

(28, 30, 32). As shown in Table 3, brief treatment of muscle cells with Con A reduced to l/2 the rate of AcChoR degradation in control cultures but had no effect on the accelerated AcChoR degradation induced by TM. These results support the possibility that TM treatment causes decreased glycosylation of AcChoR incorporated into the surface membranes of cultured muscle cells. Effect of Leupeptin on AcChoR Elaboration in Muscle Cells Treated with TM. A dramatic effect of TM in the present experiments is the strong inhibition of AcChoR accumulation on the external surface of cultured muscle cells. It was of interest to determine whether impairment of protein glycosylation by TM allows the rapid degradation of newly synthesized AcChoR by cellular protease activity, as had been suggested to occur with other glycoproteins in TM-treated cells (9, 16, 17, 24, 33, 34). Such an increase in enzymatic proteolysis of AcChoR could account for the major diminution in AcChoR detected on the surface of TM-treated muscle cells (Table 2). However, TM might also exert this effect by blocking the synthesis of AcChoR or by preventing the incorporation of newly synthesized AcChoR into plasma membranes of muscle cells. The possibility that AcChoR synthesis is blocked by TM seems unlikely in view of the disparity in magnitude of the effects on overall protein synthesis and AcChoR incorporation (Tables 1 and 2), unless it acts to inhibit the synthesis of specific cellular proteins. The detection of AcChoR with altered metabolic characteristics on plasma membranes of TM-treated muscle cells (Tables 2 and

3, Fig. 1) shows that AcChoR synthesis and incorporation into surface membrane were not abolished by TM. Significant increase in intracellular AcChoR might be anticipated in TM-treated cultures actively synthesizing AcChoR if TM

were

ceptors

into

to

selectively block the incorporation of the

plasma membranes. However,

we

found

no

re-

evi-

dence for accumulation of intracellular AcChoR in TM-treated muscle cells.. Treatment of cultured cells with TM for i day diminished the amount of AcChoR in Triton X-100 extracts of whole cells by approximately 85%-to the same extent as the depletion of membrane-bound AcChoR in intact muscle cells (data not shown). These findings rule out the possibility that TM exerts its inhibition on surface AcChoR levels by blocking AcChoR incorporation into cell membranes. We have observed that AcChoR synthesized and inserted into surface membranes of muscle cells in the presence of TM shows an enhanced rate of degradation (Table 2 and Fig. 2). This Table 3. Effect of Con A on AcChoR degradation in TM-treated and untreated embryonic muscle cells AcChoR degradation Conditions Additions Relative rate t 1/2, hr Control None 12 1 Con A 24 0.5 TM-treated None 5 2.4 Con A 5 2.4 TM-treated (0.05 Ag/ml) and control cultures were labeled with 10 nM I25-a-Bgt for 1 hr. After removal of unbound label, the cells were incubated for an additional hour with DMEM with or without Con A (50 ,g/ml). At the end of this period, this medium was replaced by DMEM containing 10%1 horse serum. All incubations were at 370C. The radioactivity released into the medium was measured at various times, and the half-time of AcChoR degradation was estimated as described for Table 2 and Fig. 2. Each value is the mean of duplicate plates.

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Table 4. Effects of TM and leupeptin on accumulation and degradation of AcChoR in embryonic muscle cells AcChoR degradation AcChoR accumulation % of % of fmol/mg % of fmol/ Additions t1/2, hr control control protein control culture to control 100 13.5 100 1300 100 240 None 111 15 92 1200 104 250 Leupeptin 30 4 29 382 27 65 TM 67 9 59 764 59 141 TM + leupeptin 1 day. The for medium growth Cultured muscle cells were treated with TM (0.05 ,ug/ml), leupeptin (0.5 mM), or both, in cells were labeled with 1251-a-Bgt and accumulation and degradation of AcChoR were measured. Each value is the mean of two plates.

suggested that an increase in degradation of newly synthesized AcChoR may underlie the diminished amount of this component in surface membranes of TM-treated muscle cells. To determine whether increased proteolytic degradation is responsible for the diminution of AcChoR caused by TM, we compared AcChoR accumulation in control and TM-treated cultures with companion cultures incubated with TM plus leupeptin, an inhibitor of several proteolytic enzymes (35). Under conditions similar to those used in the present study, leupeptin has been reported to reduce overall intracellular protease activity to approximately half (36). Table 4 shows that the inhibition of AcChoR accumulation that results from a 1-day exposure of the cultured muscle cells to TM is significantly reversed by the simultaneous presence of leupeptin. These results strongly suggest that the impairment of protein glycosylation by TM increases the susceptibility of newly synthesized AcChoR to degradation by cellular proteases in embryonic muscle cell cultures. The altered AcChoR can be partially rescued from intracellular proteolysis by treatment with the protease inhibitor leupeptin. Therefore, we conclude that the major mechanism responsible for the decrease in surface receptors is increased degradation. However, our results do not completely eliminate the possibility of a small impairment in receptor synthesis, processing, or both as contributing factors. DISCUSSION AcChoR on skeletal muscle cell surfaces is an integral membrane glycoprotein that mediates transmembrane signaling at the neuromuscular synapse. Although AcChoR has been purified to homogeneity and extensively characterized, no specific function has as yet been identified for the carbohydrate components of these receptors. The present study suggests that the carbohydrate moieties profoundly affect the metabolic properties of AcChoR. We have used TM to examine the role of asparagine-linked oligosaccharides in the elaboration and degradation of AcChoR in cultures of embryonic muscle. We have found that treatment of muscle cells with TM resulted in three marked effects: First, the glycosylation of protein was almost abolished. Second, a comparably large inhibition was observed in accumulation of AcChoR, as detected by the specific binding of 125I-a-Bgt to utact muscle cells, as well as to detergent extracts of the muscle cells. Third, AcChoR incorporated into surface membranes of TM-treated muscle cells displayed a markedly enhanced rate of degradation. In contrast to these changes in its properties, part or all of the altered AcChoR incorporated into surface membranes of TM-treated muscle cells retained the capacity for the specific binding of a-Bgt. The antibiotic TM inhibits the glycosylation of proteins by blocking the formation of N-acetylglucosaminyldiphosphorylpolyisoprenol, essential for the glycosylation of asparagine residues of proteins (6, 18, 19, 37-39). Previous studies have

shown that these carbohydrate moieties are not required for the synthesis, intracellular processing, secretion, or biological activity of a variety of glycoproteins (7-9, 11, 14-17, 20, 21, 24). We find that protein glycosylation protects newly synthesized AcChoR from cellular degradation, thus allowing the accumulation of these receptors on the surface membranes of differentiating muscle cells. Our clearest evidence that the inhibition of AcChoR elaboration observed in TM-treated muscle cells (40) results from enhanced enzymatic proteolysis, rather than a block in AcChoR biosynthesis or translocation to the cell surface, was the observation that this inhibition can be significantly reversed by the protease inhibitor leupeptin. Several proteolytic enzymes, including cathepsin B, a lysozomal protease found in muscle (36), are inhibited by leupeptin. This inhibitor acts at low concentrations on muscle cells in vitro (36, 41) to selectively decrease protein degradation, without affecting protein synthesis (36). The extent of reversal by leupeptin of AcChoR depletion in TM-treated cultured muscle observed in the present study is quantitatively similar to the inhibition by leupeptin of overall protein degradation in muscle (36). It is noteworthy that leupeptin increased the steady-state level of AcChoR on the surface of TM-treated cells but not control cultures (Table 4). This suggests a difference in the degradation process itself for fully glycosylated and underglycosylated receptors. A possible explanation for this difference is that proteolytic degradation was rate-limiting in TM-treated cultures, whereas internalization of AcChoR was the ratelimiting event in untreated cells. If this is true, then the internalization, as well as proteolytic degradation of AcChoR, may be accelerated by the impairment of protein glycosylation. An additional possibility is that distinct enzymatic processes accomplish the proteolytic degradation of AcChoR in control and TM-treated cultures, with the "normal" degradation process being markedly less sensitive to inhibition by leupeptin. A more general interpretation of these results, that glycoproteins synthesized under conditions of impaired protein glycosylation show enhanced susceptibility to proteolytic degradation, is consistent with several recent studies that utilized TM-treated cells. For example, it has been reported that the nonglycosylated forms of invertase (24), fibronectin (14), and the corticotropin-endorphin precursor (16, 17) are more highly sensitive to protease-catalyzed hydrolysis in vitro. Our findings indicate that the protection from accelerated degradation conferred to AcChoR by glycosylation is not restricted to the intracellular precursor pool but applies as well to cell surface AcChoR. However, these results suggest that glycosylation is not essential for translocation of newly synthesized AcChoR to the cell periphery, its insertion into the surface membrane, or its subsequent internalization. The lectin Con A, a known ligand of the AcChoR glycoprotein (2, 3), is shown here to slow the degradation of AcChoR in control cultures of muscle cells, but not in cultures grown in TM.

Cell Biology: Prives and Olden This suggests that Con A-specific carbohydrate residues either are not present or are not reactive because of alteration in AcChoR molecular structure or membrane surface exposure. Moreover, these findings indicate that the stability of AcChoR on the surface of muscle cells can be increased as well as diminished by modification of the associated carbohydrate. Recent work has shown that the degradation rate of AcChoR in muscle cells is an important parameter in that it can be modulated by a variety of signals, including anti-AcChoR antibodies and other external ligands (28, 42-44), differentiation and transformation of cultured muscle (45), and interaction with neurons (46, 47). The present study suggests that several of these effects might be mediated by the oligosaccharide portions of AcChoR. It is possible that, as with the TM-treated cells, altered glycosylation and a consequent increase in proteolytic degradation underlie the decreased accumulation and accelerated degradation of AcChoR observed with muscle cell transformation (45, 48, 49). The stabilization of synaptic AcChoR by innervation might be analogous to the inhibition of AcChoR degradation elicited in the absence of neuronal influence by treatment of cultured muscle cells with Con A. The localized increase in AcChoR stability could involve the interaction of externally exposed sugar residues on AcChoR with a specific ligand located in the synaptic cleft. The use of TM to inhibit glycosylation of proteins provides a means to define the role of the carbohydrate components of AcChoR and other membrane glycoproteins in the formation and maintenance of synapses between neurons and muscle cells. We thank Drs. Phillip Nelson and Kenneth M. Yamada for valuable discussions, and Valerie A. Hunter and Marie Neal for technical assistance. J.P. thanks the Muscular Dystrophy Association for support. 1. Karlin, A. (1977) in Pathogenesis of Human Muscular Dystrophies, ed. Rowland, L. P. (Excerpta Medica, AmsterdamOxford), pp. 73-84. 2. Heidmann, T. & Changeux, J.-P. (1978) Annu. Rev. Biochem. 47,317-357. 3. Fambrough, D. M. (1979) Physiol. Rev. 59, 165-227. 4. Patrick, J., Heinemann, S. & Schubert, D. (1978) Annu. Rev. Neurosci. 1, 417-443. 5. Lee, C. Y. (1972) Annu. Rev. Pharmacol. 12,265-286. 6. Struck, D. K. & Lennarz, W. J. (1977) J. Biol. Chem. 252, 1007-1013. 7. Leavitt, R., Schlesinger, S. & Kornfeld, S. (1977) J. Virol. 21, 375-385. 8. Hickman, S., Kulczycki, A., Lynch, R. G. & Kornfeld, S. (1977) J. Biol. Chem. 252,4402-4408. 9. Olden, K., Pratt, R. M. & Yamada, K. M. (1978) Cell 13,461473. 10. Pratt, R. M., Yamada, K. M., Olden, K., Ohanian, S. H. & Hascall, V. C. (1979) Exp. Cell Res. 118, 245-252. 11. Olden, K., Pratt, R. M., Jaworski, C. J. & Yamada, K. M. (1979) Proc. Natl. Acad. Sci. USA 76,791-795. 12. Olden, K., Pratt, R. M. & Yamada, K. M. (1979) Int. J. Cancer 24,60-66. 13. Olden, K. & Olden, A. T. (1979) in Recent Advances in Cancer and Molecular Biology, ed. Guillory, W. A. (Utah Univ. Press, Salt Lake City, UT), pp. 19-41. 14. Olden, K., Pratt, R. M. & Yamada, K. M. (1979) Proc. Natl. Acad. Sci. USA 76,3343-3347.


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