Abstract
We have studied the effect of tunicamycin (TM), an antibiotic which inhibits the glycosylation of nascent proteins, on the properties of the acetylcholine receptor (AChR) at the surface of embryonic chick skeletal muscle cells. The use of two separate assays, specific binding of 125I-alpha-bungarotoxin and carbamylcholine-activated 22Na+ uptake, has allowed us to monitor the effects of impaired glycosylation on the metabolic and functional properties of AChR. A significant decrease in the amounts of surface AChR elaborated in the presence of TM is detected by both measurements. This decrease has been found to reflect an enhanced proteolytic degradation of the underglycosylated AChR. The underglycosylated AChR, expressed on the cell surface in the presence of TM, retains the capability of mediating agonist-activated ionic permeability changes, but displays quantitatively altered interactions with receptor ligands. We conclude that the carbohydrate moiety on AChR may play a role in determining the folding of newly synthesized polypeptides to form a conformation compatible with the metabolic properties and ligand interactions characteristic of glycosylated AChR.
Highlights
From the Cellular and Deuelopmental Biology Program andDepartment ofAnatomical Sciences, Health Sciences Center, State University of New York at Stony Brook, Stony Brook,Long Island, New York 11 794
Fig. 1shows the time course of expression of AChR duringmuscle differentiation as monitored by these twoassays.Highlysimilarkinetics of developmental appearance are observed. These results confirm that thelinear uptake of 'lNaf induced by carbamylcholine reflects the amount of cell surface AChR
AChR was monitored by the specific binding of '"I-a-Bgt and by the linear uptakeof T'Na+ activated by carbamylcholine as described in Fig. 1 andunder"Experimental Procedures." Where specified, cultures were pretreated with T M (0.05 pg/ml) andtheprotease inhibitorsleupeptin (100 p ~ a)nd chloroquine (10 PM) for a 24-h period
Summary
Effect of Tunicamycin, an Inhibitorof Protein Glycosylation, on the Biological Propertiesof Acetylcholine Receptor in Cultured Muscle. Antibiotic which inhibits the glycosylation of nascent Studies utilizing TM have indicated a role for carbohydrate proteins, on the properties of the acetylcholine recempotioerties in regulating the processing and turnoverof specific (AChR) at the surface of embryonic chsickkeletal mus- glycoproteins [10,11,12,13,14,15,16,17,18,19,20,21]. We provide evidence that theinhibition of protein glycosylation by T M treatment results in the expression of in the presence oTfM, retains the capability of mediat- functionally altered AChR on thesurface of cultured muscle ing agonist-activated ionic permeability changes, but cells. Displays quantitatively altered interactionws ith receptor ligands.We conclude that the carbohydrate moiety
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