Implication of a multisubunit Ets-related transcription factor in synaptic expression of the nicotinic acetylcholine receptor

In adult muscle, transcription of the nicotinic acetylcholine receptor (AChR) is restricted to the nuclei located at the neuromuscular junction. The N-box, a new promoter element, was identified recently and shown to contribute to this compartmentalized synaptic expression of the AChR delta- and epsilon-subunits. We demonstrate that the N-box mediates transcriptional activation in cultured myotubes and identify the transcription factor that binds to the N-box as a heterooligomer in myotubes and adult muscle. The GAFF (GA-binding protein) alpha-subunit belongs to the Ets family of transcription factors, whereas the beta-subunit shares homology with I kappa B and Drosophila Notch protein. GABP binding specificity to mutated N-box in vitro strictly parallels the sequence requirement for beta-galactosidase targeting to the endplate in vivo, In situ hybridization studies reveal that the mRNAs of both GAFF subunits are abundant in mouse diaphragm, with preferential expression of the alpha-subunit at motor endplates, In addition, heregulin increases GABP alpha protein levels and regulates phosphorylation of both subunits in cultured chick myotubes. Finally, dominant-negative mutants of either GABP alpha or GABP beta block heregulin-elicited transcriptional activation of the AChR delta and epsilon genes. These findings establish the expected connection with a presynaptic trophic factor whose release contributes to the accumulation of AChR subunit mRNAs at the motor endplate.