Genomic structure and transcriptional regulation of the human growth hormone secretagogue receptor.
Synthetic GH secretagogues stimulate GH release through binding to a recently cloned specific GH secretagogue receptor (GHS-R). The endogenous ligand of this receptor may be part of a new endocrine pathway controlling GH secretion. 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 genomic clone including sequences encoding for the two GHS-R variants was isolated. Sequencing revealed that the two variants originate from specific RNA processing of a single gene that spans approximately 4.1 kb. The transcription start site was defined by 5'-inverse PCR analysis at position -227. RT-PCR analysis points to differential transcriptional initiation and processing. Type 1a is encoded by two exons; 2152 bp of intronic sequence are removed by splicing at position 796/797 relative to the translation start site. Type 1b is encoded by a single exon. A putative polyadenylation signal consensus motif was identified at position +4118; 2.7 kb of the 5'-flanking region were sequenced, and putative transcription factor binding sites were identified. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 168-1745 bp; 1745 bp of the GHS-R promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, whereas no activity was detected in monkey kidney COS-7 cells, human endometrium Skut-1B cells, mouse hypothalamic LHRH neuronal GT1-7 cells, or mouse corticotroph pituitary AtT20 cells. A minimal 309-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells was enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHS-R promoter by forskolin, somatostatin, insulin-like growth factor I, or 12-O-tetraphorbol 12-myristate 13-acetate. Thyroid hormone and estrogen lead to a significant stimulation; glucocorticoids lead to a significant inhibition. Further mapping suggests a thyroid hormone-responsive element, an estrogen-responsive element, 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-specific activity of the promoter and regulation by various hormones are demonstrated.
Petersenn
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IHF Institute for Hormone and Fertility Research, University of Hamburg, Germany. petersenn@uni-essen.de
Rasch
A C
AC
Penshorn
M
M
Beil
F U
FU
Schulte
H M
HM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003062:Codon; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004958:Estradiol / Q000494:pharmacology; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects; D005938:Glucocorticoids / Q000494:pharmacology; D000882:Haplorhini; D006801:Humans; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics* / Q000502:physiology; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D050817:Transcription Factor Pit-1; D014157:Transcription Factors / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014284:Triiodothyronine / Q000494:pharmacology
D003062:Codon; D004268:DNA-Binding Proteins; D004958:Estradiol; D005938:Glucocorticoids; D008156:Luciferases; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D054440:Receptors, Ghrelin; D050817:Transcription Factor Pit-1; D014157:Transcription Factors; D014284:Triiodothyronine; C494187:POU1F1 protein, human; C494185:Pou1f1 protein, mouse; C494186:Pou1f1 protein, rat