Petersenn S, Rasch A C, Penshorn M, Beil F U, Schulte H M
IHF Institute for Hormone and Fertility Research, University of Hamburg, Germany.
Endocrinology. 2001 Jun;142(6):2649-59. doi: 10.1210/endo.142.6.8184.
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.
合成生长激素促分泌素通过与最近克隆的特定生长激素促分泌素受体(GHS-R)结合来刺激生长激素释放。该受体的内源性配体可能是控制生长激素分泌的新内分泌途径的一部分。已描述了两种不同的受体变体,即1a型和1b型,它们在3'末端氨基酸上有所不同。我们研究了人类GHS-R的基因组结构和转录调控。分离出一个18kb的基因组克隆,其中包含编码两种GHS-R变体的序列。测序显示这两种变体源自一个跨越约4.1kb的单基因的特定RNA加工。通过5'-反向PCR分析在-227位置确定了转录起始位点。RT-PCR分析表明存在差异转录起始和加工。1a型由两个外显子编码;相对于翻译起始位点,在796/797位置通过剪接去除了2152bp的内含子序列。1b型由单个外显子编码。在+4118位置鉴定出一个假定的聚腺苷酸化信号共有基序;对5'侧翼区域的2.7kb进行了测序,并鉴定出假定的转录因子结合位点。使用大小从168 - 1745bp不等的启动子片段进行瞬时转染来研究转录调控;GHS-R启动子的1745bp在GH(4)大鼠垂体细胞中指导了显著水平的荧光素酶表达,而在猴肾COS-7细胞、人子宫内膜Skut-1B细胞、小鼠下丘脑促性腺激素释放激素神经元GT1-7细胞或小鼠促肾上腺皮质激素垂体AtT20细胞中未检测到活性。一个最小的309bp启动子允许垂体特异性表达。在COS-7细胞中,通过共转染垂体特异性转录因子Pit-1可增强其活性。我们未发现福斯可林、生长抑素、胰岛素样生长因子I或12-O-十四酰佛波醇13-乙酸酯对GHS-R启动子有任何调控作用。甲状腺激素和雌激素可导致显著刺激;糖皮质激素可导致显著抑制。进一步的定位表明在- 309和翻译起始密码子之间存在一个甲状腺激素反应元件、一个雌激素反应元件和一个糖皮质激素反应元件。这些研究证明了人类GHS-R基因的性质并确定了其5'侧翼区域。此外,还证明了启动子的垂体特异性活性以及各种激素的调控作用。