Identification of an element required for acetylcholine receptor- inducing activity (ARIA)-induced expression of the acetylcholine receptor ε subunit gene

Acetylcholine Receptor (AChR)-inducing activity (ARIA) is believed to be the trophic factor utilized by motoneurons to stimulate AChR synthesis in the subsynaptic area. Among the four AChR subunit genes, the ε subunit gene is strictly expressed in nuclei localized to the synaptic region of the muscle. To understand mechanisms of the regulation of synapse-specific transcription, we studied the promoter activity of the 5'-flanking region of the AChR ε subunit gene in response to ARIA. Transgenes containing the wild type or mutant 5'-flanking regions upstream of a luciferase gene were transfected in C2C12 muscle cells. The promoter activity of these transgenes was determined by assaying activity of expressed luciferase. Analyzing a combination of 5' deletion and site-directed mutants, we identified a 10-nucleotide element (position -55/-46), which was crucial for ARIA-induced expression from the ε subunit promoter. This element was named ARE for ARIA-responsive element. Mutation of ARE greatly diminished ARIA-induced transgene expression and deletion of ARE abolished completely the ARIA response. Electrophoretic mobility shift analyses revealed a DNA binding activity in muscle nuclear extract that interacted with ARE. Such interaction was enhanced by ARIA stimulation of muscle cells and appeared to be dependent on nuclear protein phosphorylation.