IDENTIFICATION OF AN ELEMENT REQUIRED FOR ACETYLCHOLINE RECEPTOR-INDUCING ACTIVITY (ARIA)-INDUCED EXPRESSION OF THE ACETYLCHOLINE-RECEPTOR EPSILON-SUBUNIT GENE

Acetylcholine Receptor (AChR)-inducing activity (ARIA) is believed to be the trophic factor utilized by motoneurons to stimulate AChR synthe sis in the subsynaptic area. Among the four AChR subunit genes, the ep silon subunit gene is strictly expressed in nuclei localized to the sy naptic region of the muscle. To understand mechanisms of the regulatio n of synapse-specific transcription, we studied the promoter activity of the 5'-flanking region of the AChR epsilon subunit gene in response to ARIA. Transgenes containing the wild type or mutant 5'-flanking re gions upstream of a luciferase gene were transfected in C2C12 muscle c ells. The promoter activity of these transgenes was determined by assa ying activity of expressed luciferase. Analyzing a combination of 5' d eletion and site-directed mutants, we identified a 10-nucleotide eleme nt (position -55/-46), which was crucial for ARIA-induced expression f rom the epsilon subunit promoter. This element was named ARE for ARIA- responsive element, Mutation of ARE greatly diminished ARIA-induced tr ansgene expression and deletion of ARE abolished completely the ARIA r esponse. Electrophoretic mobility shift analyses revealed a DNA bindin g activity in muscle nuclear extract that interacted with ARE. Such in teraction was enhanced by ARIA stimulation of muscle cells and appeare d to be dependent on nuclear protein phosphorylation.