Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells.

Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina. IMAGES:

发育过程中，乙酰胆碱受体（acetylcholine receptor, AChR）ε亚基基因的表达，受到位于神经肌肉接头（neuromuscular junction）处的肌细胞核的精确调控。该调控过程的关键作用由突触基膜（synaptic basal lamina）发挥；该结构已知含有集聚蛋白（agrin）——一种负责介导突触后特化结构形成的组分。本研究探究了集聚蛋白是否在突触AChR基因表达中发挥功能。从天然成年肌肉中分离的突触基膜，以及结合于多种底物的重组集聚蛋白，均可在培养的大鼠肌管中诱导出与ε亚基mRNA共定位的AChR簇集结构。通过对总RNA进行Northern印迹杂交（Northern hybridization）分析以评估转录本水平，结果显示：当肌管培养于浸有集聚蛋白的底物时，转录本水平显著升高；而当集聚蛋白添加至培养基中时，转录本水平无明显变化。该效应与所使用的集聚蛋白亚型的受体聚集活性无关，且集聚蛋白至少部分通过调控ε亚基基因的转录水平发挥作用。上述研究结果支持集聚蛋白在神经肌肉接头处调控AChR亚基基因表达的功能，而这一功能依赖于其与突触基膜的结合。图片：