The ACTH receptor gene has one untranslated exon (exon one), an 18kb intron, and the coding exon (exon two). The existence of different ACTH-R transcripts in human adrenal cortical cells suggests the presence of multiple transcription initiation sites. Recent evidence indicated that an alternate exon 1 (exon1f) is transcribed in adipose tissue but not in the adrenals. This exon appears to be transcribed by a different promoter region from that reported in the adrenal, thus conferring tissue specificity (24). Studies on ACTH-R promoter polymorphisms revealed a single nucleotide polymorphism close to the transcriptional initiation site (-2C/T) resulted in inhibition of ACTH-R transcription and accounted for reduced ACTH-R levels even in the heterozygous state. This allele is present in 10% of the population. The relevance of this and other polymorphisms in disease remains to be evaluated (25).

The ACTH receptor promoter contains binding sites for several transcription factors. Transcription factors are nuclear proteins modifying the expression of genes by binding to specific DNA sequences usually located upstream of gene promoters. Phosphorylation of a transcription factor results in its activation and modulation of the transcriptional activity of a promoter containing response elements for the specific factor. The most important transcription factors affecting the expression of ACTH receptor gene are the following:

SF-1 (Steroidogenic Factor-1 binding sites): SF-1 is an orphan nuclear receptor regulating the transcription of genes involved in steroidogenesis. It also plays a role in adrenal organogenesis. Indeed, SF-1 knockout mice lack adrenal glands and gonads. SF-1 is also essential for the compensatory adrenal growth following unilateral adrenalectomy (26). SF-1 plays a key role in steroidogenesis in both adrenal cortical and gonadal cells. Thus, the transcription of CYP11A1, a gene that encodes the P450scc cholesterol side-chain cleavage enzyme, the first step in steroidogenesis, has several SF-1-binding sites modulating its transcription rate (1). As for the ACTH receptor gene, it has been shown that it contains three SF-1 binding sites in the proximity of the transcription initiation site. It should be noted that the nuclear receptor Dax-1 (dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene-1) inhibits SF-1-mediated steroidogenesis. Absence of Dax-1 results in an increased adrenal responsiveness to ACTH most probably mediated by upregulation of the ACTH receptor via SF-1 (27). Furthermore, cAMP-dependent PKA augments the SF-1-mediated stimulation of steroidogenic enzymes (28). It should be noted however, that it has been recently reported that during ovine late-gestation the ACTH ligand does not appear to affect the expression of either SF-1 or the ACTH receptor genes in fetal adrenals (29).

DAX-1 (Dosage-sensitive sex reversal, Adrenal hypoplasia congenital critical region on the X chromosome, gene 1): DAX-1 is a transcription factor expressed in the adrenal gland and in the gonads. In fact, DAX-1 encodes an orphan member of the nuclear hormone receptor super family. DAX-1 is a suppressor of the transcription of several genes involved in the steroidogenic pathway. However, the physiological roles of DAX-1 are far from being elucidated. Thus, while DAX-1 inhibits SF-1-mediated induction of the steroidogenic genes including MIS, inactivating mutations of DAX-1 results in the X-linked form of adrenal hypoplasia congenital (AHC) with associated hypogonadotropic hypogonadism. AHC usually reveals itself as adrenal failure in early infancy, although a wide range of phenotypic expression has been reported. Interestingly, the ACTH-R promoter contains DAX-1 sites (27). DAX-1 represses basal ACTH-R levels when transfected in adrenocortical Y-1 cells. In adrenocortical tumors there is a definite negative correlation between DAX-1 and ACTH-R (27).

StAR (steroidogenic acute regulatory protein): StAR, which promotes intramitochondrial cholesterol transfer in the adrenal cortical cells, is the only major adrenal transcription factor, which has not been associated to the expression of the ACTH-R gene. StAR is induced by ACTH via the ACTH-R to regulate steroidogenesis but no direct effect or binding element on the ACTH-R promoter has been described (30).

AP-1 (Activator Protein-1 regulatory element): Activation of several signaling pathways, including that of PKA and PKC, cause hetero-dimerization of the proto-oncogenes Fos and Jun to form the AP-1 transcription factor. Two AP-1 binding sites have been identified upstream of the ACTH-R gene modulating its response to cAMP. Thus, deletion of the AP-1 binding sites on the ACTH-R gene abolishes the effect of cAMP. The effect of glucocorticoids and Angiotensin II on the expression of ACTH receptor may be accomplished via glucocorticoid-mediated inhibition of AP-1binding sites on the ACTH receptor promoter (31).

ACTH: Several studies have shown that the ACTH-receptor gene is up regulated by its own ligand, ACTH (32). Indeed, ligand-induced up-regulation of ACTH-receptor expression may be an important adaptive process directed towards optimizing adrenal responsiveness to ACTH. The effect of ACTH on ACTH-receptor expression is dependent on cAMP, probably mediated through AP-1. SF-1 plays an important role in ACTH-induced upregulation of ACTH-R expression. Indeed, the ACTH receptor promoter has three SF-1-binding sites. Suppression of ACTH secretion by endogenous or exogenous glucocorticoids results in reduction of the ACTH-R gene expression (33). On the other hand, DAX-1 suppresses the expression of the ACTH receptor gene. The mRNA for MC2R and SF-1 do not appear to be regulated by ACTH in the late-gestation ovine fetus, although a pituitary-dependent factor may be involved in the regulation of SF-1 mRNA abundance (29).

Glucocorticoids: Six glucocorticoid regulatory elements (GRE) in the ACTH receptor gene indicate that glucocorticoids are major regulators of its expression. Glucocorticoids exert an enhancing effect on basal, ACTH- and cAMP-induced ACTH-R expression (34).

Angiotensin II: Angiotensin II stimulates the expression of ACTH receptor gene in the adrenal cortex (35, 36). Promoter deletion studies revealed that the two AP1 binding elements on the ACTH-R (MC2-R) promoter mediate the Angiotensin II stimulatory signals. Indeed, Angiotensin II rapidly activates Fos and Jun to promote ACTH-R transcription (31).