Analysis of the first exon of the murine ACTH receptor gene

Our laboratory has reported the cloning of the promoter of the murine adrenocorticotropic hormone (ACTH) receptor gene. Although the sequences of the murine and human promoters have a high degree of homology, the transcriptional initiation site determined in the murine promoter lies sixty four nucleotides upstream of the corresponding site suggested for the human promoter, which consequently lies within the untranslated first exon of the murine gene. By performing a 3' deletion analysis on the ACTH-R promoter construct [-1805/+105] in the mouse adrenocortical cell line Y1, we have investigated the possibility of alternative transcriptional initiation downstream from the identified murine initiation site. Removing sequences from +105 to +87 causes a slight decrease in promoter activity, but further deletion to +49, removing the potential initiation site, causes no further decrease in activity, indicating that no transcription is initiating from this region. However, further deletions that impinge upon or remove a potential steroidogenic factor-1 (SF-1) binding site, lying between +31 and +39 in the first exon, cause a significant decrease in activity. Site-directed mutagenesis of this SF-1 binding site does not disable the promoter, suggesting that another factor overlapping this site may be responsible for maximal activity of the ACTH-R.