Agrin Can Mediate Acetylcholine Receptor Gene Expression in Muscle by Aggregation of Muscle-Derived Neuregulins

The neural isoforms of agrin can stimulate transcription of the acetylcholine receptor (AChR) ε subunit gene in electrically active muscle fibers, as does the motor neuron upon the formation of a neuromuscular junction. It is not clear, however, whether this induction involves neuregulins (NRGs), which stimulate AChR subunit gene transcription in vitro by activating ErbB receptors. In this study, we show that agrin-induced induction of AChR ε subunit gene transcription is inhibited in cultured myotubes overexpressing an inactive mutant of the ErbB2 receptor, demonstrating involvement of the NRG/ErbB pathway in agrin-induced AChR expression. Furthermore, salt extracts from the surface of cultured myotubes induce tyrosine phosphorylation of ErbB2 receptors, indicating that muscle cells express biological NRG-like activity on their surface. We further demonstrate by RT-PCR analysis that muscle NRGs have Ig-like domains required for their immobilization at heparan sulfate proteoglycans (HSPGs) of the extracellular matrix. In extrasynaptic regions of innervated muscle fibers in vivo, ectopically expressed neural agrin induces the colocalized accumulation of AChRs, muscle-derived NRGs, and HSPGs. By using overlay and radioligand-binding assays we show that the Ig domain of NRGs bind to the HSPGs agrin and perlecan. These findings show that neural agrin can induce AChR subunit gene transcription by aggregating muscle HSPGs on the muscle fiber surface that then serve as a local sink for focal binding of muscle-derived NRGs to regulate AChR gene expression at the neuromuscular junction.