Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor

Synthetic GH secretagogues stimulate GH release through binding to a recent ly cloned specific GH secretagogue receptor (GHS-R). The endogenous ligand of this receptor may be part of a new endocrine pathway controlling GH secr etion. Two different receptor variants, type 1a and 1b, have been described that differ in their 3'-terminal amino acids. We investigated the genomic structure and transcriptional regulation of the human GHS-R. An 18-kb genom ic clone including sequences encoding for the two GHS-R variants was isolat ed. Sequencing revealed that the two variants originate from specific RNA p rocessing of a single gene that spans approximately 4.1 kb. The transcripti on start site was defined by 5'-inverse PCR analysis at position -227. RT-P CR analysis points to differential transcriptional initiation and processin g. Type la is encoded by two exons; 2152 bp of intronic sequence are remove d by splicing at position 796/797 relative to the translation start site. T ype Ib is encoded by a single exon. A putative polyadenylation signal conse nsus motif was identified at position +4118; 2.7 kb of the 5'-flanking regi on were sequenced, and putative transcription factor binding sites were ide ntified. Transcriptional regulation was investigated by transient transfect ions using promoter fragments ranging in size from 168-1745 bp; 1745 bp of the GHS-R promotor directed significant levels of luciferase expression in GH, rat pituitary cells, whereas no activity was detected in monkey kidney COS-7 cells, human endometrium Skut-1B cells, mouse hypothalamic LHRH neuro nal GT1-7 cells, or mouse corticotroph pituitary AtT20 cells. A minimal 309 -bp promoter allowed pituitary-specific expression. Its activity in COS-7 c ells was enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHS-R promoter by fors kolin, somatostatin, insulin-like growth factor I, or 12-O-tetraphorbol 12- myristate 13-acetate. Thyroid hormone and estrogen lead to a significant st imulation; glucocorticoids lead to a significant inhibition. Further mappin g suggests a thyroid hormone-responsive element, an estrogen-responsive ele ment, and a glucocorticoid-responsive element located between -309 and the translation start codon. These studies demonstrate the nature of the human GHS-R gene and identify its 5'-flanking region. Furthermore, pituitary-spec ific activity of the promoter and regulation by various hormones are demons trated.