Familial glucocorticoid deficiency: one syndrome, but more than one gene.

Familial glucocorticoid deficiency is a rare autosomal recessive disease characterised by resistance to the action of ACTH. A number of mutations in the ACTH receptor have been demonstrated in patients with this disorder which are likely to lead to loss of receptor function and thus would account for the syndrome. Several patients, however, do not have mutations in the ACTH receptor gene coding region, and it can be demonstrated by segregation analysis that another distant gene must account for the disease in some of these cases. The nature of several candidate genes for this normal receptor form of the disease is discussed.

The mouse adrenocorticotropin receptor gene: cloning and characterization of its promoter and evidence for a role for the orphan nuclear receptor steroidogenic factor 1.

To elucidate the mechanism underlying the tissue-specific expression of the ACTH receptor/MC2 receptor (ACTH-R) in the adrenal cortex, we have cloned the mouse ACTH-R promoter. The analysis of the cDNA 5'-end showed an untranslated region of 321 bp, and the ACTH-R gene was demonstrated to be composed of two exons of 113 and 112 bp lying upstream of the single coding exon. S1 nuclease protection assay showed two major transcription start sites separated by 4 bp; 1.8 kb of the 5'-flanking region inserted in a luciferase reporter vector had cell-specific promoter activity because it was functional only in mouse adrenocortical Y1 cells but not in mouse Leydig TM3 cells or fibroblast L cells. There was no dramatic change in the promoter activity in Y1 cells for all the deletions tested up to -113 bp upstream of the transcription start site. In contrast, expression in TM3 cells was switched on from deletion -908 bp, while remaining undetectable in L cells. Site-directed mutagenesis of a steroidogenic factor 1 (SF1)-like site at position -25 bp resulted in a significant reduction in luciferase expression by the promoter in Y1 cells. Gel shift analysis of this site indicated specific binding of a protein in extracts of Y1 and TM3 cells. Moreover, expression of SF1 in L cells induced promoter activity of the construct p(908). In conclusion, cell-specific expression of the mouse ACTH-R appears to be controlled by at least two factors. One of them, most probably SF1, is responsible for steroidogenic cell-specific expression. The other as yet unknown factor binding between position -1236 bp and -908 bp acts as a strong inhibitory factor in nonadrenal steroidogenic cells, resulting in the adrenal-specific expression of ACTH-R.

Agonist and receptor binding properties of adrenocorticotropin peptides using the cloned mouse adrenocorticotropin receptor expressed in a stably transfected HeLa cell line.

The cloned mouse ACTH receptor was expressed in stably transfected human HeLa cells that lack an endogenous melanocortin receptor. ACTH[1-39] and several N- and C-terminally truncated analogues of ACTH were studied for their ability to stimulate cAMP generation and to displace bound 125I-ACTH. Only three of the peptides tested, ACTH[1-24], ACTH[1-39], and ACTH[1-17] were found to have agonist activity with EC50 values of 7.5, 57, and 49 x 10(-12) M respectively. Two peptides, ACTH[11-24] and ACTH[7-39], were devoid of agonist activity but had substantial competitive antagonist activity with IC50 values of approximately 10(-9) M. In binding studies, ACTH[1-39] and ACTH[1-24] were able to fully displace bound ligand, and Scatchard analysis indicated a dissociation constant (KD) of 0.84 and 0.94 x 10(-9) M for the two peptides, respectively. ACTH[1-17], ACTH[11-24], and ACTH[7-39] were only capable of displacing 60-70% of bound ligand. A three-site model for the interaction of ACTH and its receptor is proposed on the basis of these findings.

The ACTH receptor.

The ACTH-R is a receptor that has a prominent role in mammalian physiology, but which has been notoriously difficulty to study. The cloning of the gene encoding this receptor in 1992 should permit significant advances in the understanding of the physiology, pharmacology and pathophysiology of ACTH. Areas of particular interest that should be clarified in the next few years are the control of the tissue specific expression of the gene, and an understanding of the molecular determinants of the ligand-receptor interaction. This latter area raises the prospect of clinically useful, orally active ACTH agonists and antagonists.

Cloning, characterization and expression of a functional mouse ACTH receptor.

A polymerase chain reaction-generated mouse ACTH-R probe was used to screen a mouse genomic library, and a clone of 13kb containing the entire coding sequence was isolated. The coding sequence shows 84% homology with the human gene at the DNA level and encodes a peptide with 89% homology to the human ACTH-R. This gene is expressed as a major transcript of 1.8kb in the mouse adrenal gland. The gene was expressed in HeLa cells and cAMP production in response to either ACTH or alpha-MSH was measured. cAMP increased in an ACTH dose dependent manner suggesting an EC50 of 7 x 10(-10)M ACTH. alpha-MSH was without effect on this receptor. In conclusion we have cloned a mouse ACTH receptor gene and demonstrated for the first time its expression and functional effect in HeLa cells.