Multiple Functional Sp1 Domains in the Minimal Promoter Region of the Neuronal Nicotinic Receptor α5 Subunit Gene

Antonio Campos-Caro,Carmen Carrasco-Serrano,Luis M Valor,Salvador Viniegra,Juan J Ballesta,Manuel Criado

doi:10.1074/jbc.274.8.4693

Antonio Campos-Caro, Carmen Carrasco-Serrano + Show 4 more

Open Access

https://doi.org/10.1074/jbc.274.8.4693

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Abstract

The alpha5 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 approximately 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 alpha5 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 alpha5 promoter activity by approximately 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 alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 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.

Highlights

We have shown that Nicotinic acetylcholine receptors (nAChRs) formed by ␣7 subunits are differentially expressed in adrenergic cells [10], probably as the result of transcriptional regulation, whereas ␣3, ␣5, and ␤4 subunits have a less restricted distribution in adrenergic and noradrenergic cells [7]
Since the ␣3 promoter is the target of Sp1 [22], we suggest the possible involvement of this transcription factor in a regulatory mechanism common to the ␣3, ␤4, and ␣5 subunits
Clone ␭␣5–21 contained ϳ16 kilobases of bovine genomic sequence including exons 1 and 2 and ϳ1.6 kilobases of 5Ј-flanking region. This region was further subcloned and sequenced (Fig. 1A). Comparison of this sequence to a data base of binding sequences of known transcription factors revealed the main features of the promoter/regulatory region of the ␣5 gene: the lack of a TATA box and the presence of several GC boxes, all of them concentrated into ϳ110 bases located 5Ј to the transcription initiation site

Summary

Introduction

Deletion analysis of the most promoter-proximal region was performed by generating polymerase chain reaction fragments with suitable sense oligonucleotides and an antisense primer (5Ј-CTTTATGTTTTTGGCGTCTTCC-3Ј) that anneals to the pGL2-Basic vector downstream of the site of transcription initiation. In SHSY-5Y cells, the construct containing 111 bp of ␣5 promoter sequence plus 155 bp of 5Ј-noncoding region (p111␣5LUC) showed the maximal activity.

Results

Conclusion