We have used fibroblast clones expressing muscle nicotinic acetylcholine receptor α and γ, and γ and δ subunits to measure the kinetics of subunit assembly, and to study the properties of the partially assembled products that are formed. We demonstrate by coimmunoprecipitation that assembly intermediates in fibroblasts coexpressing α and δ subunits are formed in a time-dependent manner. The α and γ- and the α and δ-producing transfected cells form complexes that, when labeled with 125I-α-bungarotoxin, migrate in sucrose gradients at 6.3S, a value consistent with a hetero-dimer structure. An additional peak at 8.5S is formed from the α and γ subunits expressed in fibroblasts suggesting that γ may have more than one binding site for α subunit. The stability and specificity of formation of these partially assembled complexes suggests that they are normal intermediates in the assembly of acetylcholine receptor. Comparison of the binding of 125I-α-bungarotoxin to intact and detergent-extracted fibroblasts indicate that essentially all of the binding sites are retained in an intracellular pool. The fibroblast δ subunit has the electrophoretic mobility in SDS-PAGE of a precursor that does not contain complex carbohydrates. In addition, αγ and αδ complexes had lectin binding properties expected of subunits lacking complex oligosaccharides. Therefore, fibroblasts coexpressing α and γ or α and δ subunits produce discrete assembly intermediates that are retained in an intracellular compartment and are not processed by Golgi enzymes.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of Cell Biology|
|Issue number||6 PART 1|
|Publication status||Published - Dec 1990|
ASJC Scopus subject areas
- Cell Biology