Overexpression of rapsyn modifies the intracellular trafficking of acetylcholine receptors

Rapsyn is a protein that interacts with the cytoplasmic face of the nicotinic acetylcholine receptors (AChR) to cluster them within postsynaptic membrane of muscle. Here we show that intracellular AChRs are also affected by rapsyn. When rapsyn was co-transfected with AChR into QT-6 fibroblasts, (125)I-alpha-bungarotoxin binding indicated a reduction in the fraction of AChRs expressed on the cell surface, compared to cells expressing AChRs alone. Double fluorescent labeling showed that intracellular AChRs accumulated in patches at the cell periphery, beneath rapsyn-associated cell surface AChR clusters. These patches were observed even when cells were grown in medium containing excess unlabelled alpha-bungarotoxin to mask internalized AChRs, suggesting that they arose from hindered trafficking of newly formed AChRs to the cell surface. Similarly, in the muscle cell line, C2, overexpression of rapsyn resulted in the co-localization of aggregates of intracellular alpha-bungarotoxin binding sites with rapsyn beneath cell surface AChR microaggregates. The results indicate that rapsyn can modify the trafficking of AChRs within the cell and suggest a role in selectively targeting newly synthesized intracellular AChRs to the postsynaptic membrane.