Identification of an upstream pituitary-active promoter of human somatostatin receptor subtype 5.

Somatostatin receptor subtype 5 (sst5) has been linked to inhibition of PRL and insulin secretion. We characterized the genomic structure of the human sst5. The transcription start site was located 94 nucleotides upstream of the initiator ATG codon. Sequence analysis of 5'-inverse PCR products revealed the presence of a 6.1-kb intron in the 5'-untranslated region. RT-PCR analysis indicated tissue-specific activation of the newly identified upstream promoter in pituitary, but not in small intestine, lung, or placenta. A -1741 promoter directed significant levels of luciferase expression in GH(4) rat pituitary cells, Skut-1B endometrium cells, and JEG3 chorion carcinoma cells, which was absent in COS-7 monkey kidney cells. A minimal -101 promoter was sufficient to allow tissue-specific expression. Its activity in COS-7 cells was not enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. Analysis of deletion constructs revealed a GC-rich region immediately upstream of the transcription start site, which is necessary for promoter activity. Somatostatin led to a significant inhibition, and forskolin and thyroid hormone to a significant stimulation of pituitary-specific promoter activity. Further mapping suggested a cAMP-responsive element located between -101 and the transcription start site, and thyroid hormone-responsive elements between -1741 and -1269 and between -317 and -101. These studies identified an upstream promoter of the sst5 gene with tissue-specific activity.

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.

Genomic structure and transcriptional regulation of the human somatostatin receptor type 2.

Somatostatin exerts inhibitory effects on virtually all endocrine and exocrine secretions. The somatostatin receptor subtype 2 (sst2) acts as a critical molecule for growth hormone regulation and cell proliferation. We investigated the structure and regulation of the human sst2 gene. A genomic clone including the sst2 gene was isolated, 1.5 kb of the promoter was sequenced and putative transcription factor binding sites were identified. The transcription start site was located 93 nucleotides upstream of the translation start site. The nucleotide sequences of the complete gene and 0.5 kb of 3' region were determined. A possible polyadenylation signal was identified. Transcriptional regulation was investigated by transient transfections using various promoter fragments. A -1100 sst2 promoter directed significant levels of luciferase expression in GH4 rat pituitary cells and Skut1-B endometrium cells whereas only low activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal -252 promoter allowed cell specific expression. We did not find any regulation of the sst2 promoter by somatostatin, forskolin, TRH, TPA, T3, and 17beta-estradiol. Glucocorticoids lead to a significant inhibition of sst2 promoter activity. Further mapping suggest a glucocorticoid-responsive element between -905 and -707 and between -252 and -163. These studies demonstrate the nature of the human sst2 gene and identify its 5' and 3' flanking regions. Furthermore, specific activity of the promoter and regulation by various hormones is demonstrated.

Structure and regulation of the human growth hormone-releasing hormone receptor gene.

The GHRH receptor (GHRH-R) acts as a critical molecule for proliferation and differentiation of somatotrophic pituitary cells. A role in the pathogenesis of GH hypersecretion and GH deficiency has been implicated. We investigated structure and regulation of the human GHRH-R gene. A genomic clone including approximately 12 kb of 5'-flanking region was isolated. The gene is of complex structure consisting of more than 10 exons. Two kilobase pairs of the promoter were sequenced, and putative transcription factor binding sites were identified. The transcription start site was defined by ribonuclease protection assay. Transcriptional regulation was investigated by transient transfections using promoter fragments ranging in size from 108-1456 bp. GHRH-R promoter (1456 bp) directed high levels of luciferase expression in GH4 rat pituitary cells whereas no activity was detected in JEG3 chorion carcinoma cells or COS-7 monkey kidney cells. A minimal 202-bp promoter allowed pituitary-specific expression. Its activity in COS-7 cells is enhanced by cotransfection of the pituitary-specific transcription factor Pit-1. We did not find any regulation of the GHRH-R promoter by forskolin, phorbol-myristate-acetate, or T3. Glucocorticoids lead to a significant stimulation, and estrogen leads to a significant inhibition. Further mapping suggests a glucocorticoid-responsive element between -1456 and -1181 and an estrogen-responsive element between -202 and -108. These studies demonstrate the complex nature of the human GHRH-R gene and identify its 5'-flanking region. Furthermore, specific activity of the promoter and regulation by various hormones are demonstrated.