Characterization of the human ACTH receptor gene and in vitro expression

The coding sequence of the human ACTH receptor, cloned in 1992, contains no intron, but the presence of one intron (of about 18 kb) separating the coding exon from an upstream exon has been demonstrated. One major transcription start site was located in this first exon. Northern blot analysis of cultured human adrenocortical cells revealed several transcripts that can be partly explained by the use of different polyadenylation sites. We have isolated a 1 kb fragment of genomic DNA upstream of exon 1 and studied its basal promoter activity. The sequence of this region shows several putative CREs that could be responsible for the stimulation by ACTH of its own receptors as demonstrated on human adrenocortical cells. To functionally characterize the human ACTH receptor, we have prepared cells stably transfected with either the normal receptor or a mutant receptor. This model allows the study of both binding to ACTH and coupling to adenylate cyclase. Two naturally mutated receptors, described in patients with Familial Glucocorticoid Deficiency, have been studied. Both mutations (C251F and D107N) strongly impaired the binding of ACTH to its receptors and are then responsible for the absence of biological response to ACTH.