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Volume 272, Number 16, Issue of April 18, 1997 pp. 10367-10371
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

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Identification of an Element Required for Acetylcholine Receptor-inducing Activity (ARIA)-induced Expression of the Acetylcholine Receptor  Subunit Gene

(Received for publication, January 21, 1997, and in revised form, February 25, 1997)

From the Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22908

cetylcholine eceptor (AChR)-nducing ctivity (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  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 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  subunit promoter. This element was named ARE for RIA-esponsive lement. 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.

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