CHARACTERIZATION OF THE HUMAN ACTH RECEPTOR GENE AND IN-VITRO EXPRESSION

The coding sequence of the human ACTH receptor, cloned in 1992, contai ns no intron, but the presence of one intron (of about 18 kb) separati ng the coding exon from an upstream exon has been demonstrated. One ma jor 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 polya denylation sites. We have isolated a 1 kb fragment of genomic DNA upst ream of exon 1 and studied its basal promoter activity. The sequence o f this region shows several putative CREs that could be responsible fo r the stimulation by ACTH of its own receptors as demonstrated on huma n adrenocortical cells. To functionally characterize the human ACTH re ceptor, we have prepared cells stably transfected with either the norm al receptor or a mutant receptor. This model allows the study of both binding to ACTH and coupling to adenylate cyclase. Two naturally mutat ed receptors, described in patients with Familial Glucocorticoid Defic iency, have been studied. Both mutations (C251F and D107N) strongly im paired the binding of ACTH to its receptors and are then responsible f or the absence of biological response to ACTH.