Substrate-bound agrin induces expression of acetylcholine receptor e-subunit gene in cultured mammalian muscle cells ( synapse development )

Content Provider	Semantic Scholar
Author	Jones, Graham Lichtsteiner, Marianne Brenner, Hans Rudolf
Abstract	Expression of the e-subunit gene of the ace-tylcholine receptor (AChR) by myonuclei located at the neu-romuscular 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 e-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 E-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. The acetylcholine receptor (AChR) channel in mammalian skeletal muscle fibers is composed of five different subunits termed a, /3, y, 6, and E that may combine in two different stoichiometries (a2P3yS8 or a23E8) to form two different sub-types of functional AChR channels in the muscle fiber membrane (1). Transcription of the AChR E-subunit gene in the myonuclei at the neuromuscular junction is regulated almost exclusively by factors of neural origin and is associated with the synaptic portion of the basal lamina (BL) (2-4). Two factors thought to be involved in synapse formation have been identified in synaptic BL: neuregulin-like immunoreactivity (5, 6) and agrin (7). ARIA, recently identified as a member of the neuregulin family (8) of growth factors that are generated by alternative mRNA splicing (9, 10), increases the level of AChR e-subunit mRNA (11) in cultured mouse myotubes and is expressed in embryonic spinal cord motor neurones. ARIA and one of its human homologs, heregulin (31 (hrg31), like the physiological factor, stimulates transcription of the e-subunit gene (6) and has been proposed to mediate neural regulation of AChR expression at the neuromuscular synapse (11). Another BL-associated protein, agrin, directs the clustering of AChRs to synaptic sites (7). Agrin is expressed in different isoforms arising from alternative mRNA splicing (12, …
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