The role of Rer1 in assembly of the nicotinic acetylcholine receptor and its transport to the plasma membrane

• Applicant: Dr. Christoph Kaether
• Subject Area: Cell Biology
• Term: from 2012 to 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 58099525

Muscle nicotinic acetylcholine receptor (nAChR) is composed of five subunits that assemble in the ER. A quality control system ensures that only fully assembled complexes leave the ER and are transported to the plasma membrane. Unassembled subunits are retained in the ER by specific ER-retention/retrieval signals in their first transmembrane domain, but nothing is known about the molecular details and interaction partners of this type of ER retention/retrieval. Our preliminary data suggest that Rer1, a putative ER retrieval receptor, plays an important role in the assembly of nAChR. We found that siRNAmediated down-regulation of Rer1 in vivo in mouse muscle leads to significantly smaller neuromuscular junctions, an effect that is enhanced in dystrophic muscle. Furthermore, Rer1 co-precipitated with AChRα subunit, suggesting a direct interaction between these two proteins. We want to elucidate in detail the molecular mechanisms that govern nAChR assembly and export from the ER and want to determine the role of Rer1 in these processes. To this end we plan to biochemically analyze the interaction of Rer1 with AChR subunits by using reporter protein assays as well as co-immunoprecipitation experiments. Assembly and transport of nAChR and the role of Rer1 therein will be analyzed in situ in live mouse skeletal muscle using two-photon microscopy in dystrophic mouse models as well as in mice lacking Rer1. Potential upregulation of Rer1 will be analyzed in myasthenia gravis, where synthesis (and therefore assembly) of nAChR are upregulated to compensate the loss of nAChR. Mutations in ER-retention/retrieval signals in subunits of nAChR can cause congenital myasthenic syndromes. The effects of such mutations on assembly and transport of nAChR will be assessed. The mechanisms steering the assembly of the neuronal nicotinic AChR, consisting of subunits highly homologous to muscle nAChR subunits, will be studied. Previously uncharacterized ER-retention/retrieval signals in neuronal AChR subunits will be determined and the role of Rer1 in retention/retrieval of these receptors will be studied using the same tools as for the analysis of muscular AChR subunits.

• DFG Programme: Research Grants