Multiple functional Sp1 domains in the minimal promoter region of the neuronal nicotinic receptor alpha 5 subunit gene

The alpha 5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catech olamine secretion process in bovine adrenomedullary chromaffin cells. The p romoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the s ite of transcription initiation (from -111 to -40). Deletion analysis and t ransient transfections showed that a 266-base pair region (-111 to +155) ga ve rise to similar to 77 and 100% of the maximal transcriptional activity o bserved in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-d irected mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha 5 gene, but in a different way, dep ending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha 5 promoter ac tivity by similar to 50%, whereas single mutations of the other GC boxes ha d no effect. In chromaffin cells, by contrast, modification of any of the G C boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nucl ear extracts from either chromaffin or SHSY-5Y cells showed the specific bi nding of Sp1 protein to fragment -111 to -27, Binding of Sp1 to the GC boxe s was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha 5 subunit expression, as has also been demonstrated previously for alpha 3 an d beta 4 subunits, Since these three subunits have their genes tightly clus tered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.