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

肌肉烟碱型乙酰胆碱受体（nAChR）由5个亚基组成，它们在内质网中聚集。质量控制系统确保只有完全组装的复合物离开ER并被运输到质膜。未组装的亚基通过其第一跨膜结构域中的特异性ER保留/检索信号保留在ER中，但关于这种类型的ER保留/检索的分子细节和相互作用伙伴一无所知。我们的初步数据表明，Rer 1，一个假定的ER检索受体，在nAChR的组装中起着重要的作用。我们发现，siRNA介导的Rer 1在小鼠肌肉中的体内下调导致神经肌肉接头显著变小，这种作用在营养不良的肌肉中得到增强。此外，Rer 1与AChR α亚基共沉淀，表明这两种蛋白之间存在直接的相互作用。我们希望详细阐明nAChR组装和出口的ER的分子机制，并希望确定Rer 1在这些过程中的作用。为此，我们计划通过使用报告蛋白测定以及免疫共沉淀实验来生化分析Rer 1与AChR亚基的相互作用。组装和运输nAChR和Rer 1的作用，其中将在原位分析活的小鼠骨骼肌中使用双光子显微镜在营养不良的小鼠模型，以及在小鼠缺乏Rer 1。将在重症肌无力中分析Rer 1的潜在上调，其中nAChR的合成（因此组装）上调以补偿nAChR的损失。nAChR亚基中ER保留/恢复信号的突变可引起先天性肌无力综合征。将评估此类突变对nAChR组装和转运的影响。指导组装的神经元烟碱AChR，由亚基高度同源的肌肉nAChR亚基的机制，将进行研究。以前未表征的ER保留/检索信号在神经元AChR亚基将被确定和保留/检索这些受体的Rer 1的作用将使用相同的工具进行研究肌肉AChR亚基的分析。

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.