Multiple functional Sp1 domains in the minimal promoter region of the neuronal nicotinic receptor α5 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 catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to similar to 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed 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, depending 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 activity by similar to 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC 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 nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27, Binding of Sp1 to the GC boxes 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 and beta 4 subunits, Since these three subunits have their genes tightly clustered 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.