Altered glycosylation sites of the delta subunit of the acetylcholine receptor (AChR) reduce alpha delta association and receptor assembly

We have used mutagenesis to investigate the potential N-glycosylation sites in the delta subunit of the mouse muscle acetylcholine receptor (AChR). Of the three sites, Asn(76), Asn(143), and Asn(169), only the first two were glycosylated when the delta subunit was expressed in COS cells. Because the heterologously expressed delta subunit was similar in its properties to th at expressed in C2 muscle cells, the sites of glycosylation are likely to b e the same in both cases. In COS cells, mutations of the delta subunit that prevented glycosylation at either of the sites did not change its metaboli c stability nor its steady-state level. These results are in contrast to th ose found previously for the alpha subunit, in which glycosylation at a sin gle site metabolically stabilized the polypeptide (Blount, P., and Merlie, J, P. (1990) J. Cell Biol. 111, 2613-2622). Mutations of the delta subunit that prevented glycosylation, however, decreased its ability to form an alp ha delta heterodimer when the alpha and delta subunit were expressed togeth er. When all four subunits of the AChR (alpha, beta, delta, and epsilon) we re coexpressed, mutation of the delta subunit to prevent glycosylation resu lted in a reduced amount of fully assembled AChR and reduced surface AChR l evels, consistent with the role of the heterodimer in the assembly reaction . These results suggest that glycosylation of the delta subunit at both Asn (76) and Asn(143) is needed for its efficient folding and/or its subsequent interaction with the alpha subunit.