Regulation of gene expression of a novel G-protein coupled receptor involved in growth hormone secretion

1996年，默克研究实验室报道了一种新的g蛋白偶联受体的分子克隆，称为生长激素（GH）分泌激素受体（GHS-R），从猪和人的垂体，结合合成GH释放肽和非肽基GH分泌剂（GHSs）。尽管其内源性配体尚不清楚，但该受体在刺激和放大搏动GH分泌中起作用。GHS- r的发现表明，生长激素的分泌受三个因素的调节，一个假定的天然GHS和两种下丘脑肽，GH释放激素（GRH）和GH释放抑制激素（生长抑素）。利用RT-PCR和RACE技术，于1997年克隆了一个大鼠垂体GHS-R同源基因，该基因属于7个跨膜g蛋白偶联受体家族，在氨基酸水平上与猪和人的GHS-R同源性为94- 95%。地高辛标记的cRNA探针原位杂交显示，下丘脑弓形核和腹内侧核均大量表达GHS-R mRNA。GHRP已被证明能刺激动物体内的GRH释放。这一发现为GHS和GHRP作用于下丘脑弓状核刺激GRH释放从而增加GH脉动分泌的观点提供了形态学支持。1998年，我们尝试建立一种能够定量GHS-R mRNA水平的分析系统。可能由于GHS-R mRNA是一种极为罕见的转录物，因此Northern blotting无法检测到GHS-R mRNA，这促使我们开发了一种更敏感和特异性的检测方法，即RNase保护实验。通过该实验，我们成功地检测和定量了大鼠下丘脑和垂体中的GHS-R mRNA，发现下丘脑内侧基底区每单位总RNA的GHS-R mRNA水平大约是垂体前叶的2.5倍。人重组生长激素给药5天后，垂体GHS-R mRNA水平显著降低，下丘脑GHS-R mRNA水平无显著降低。这一发现提示GHS-R在垂体中的表达受GH的负调控，其调控可能与下丘脑中的不同。少

In 1996, Merck research laboratory reported molecular cloning of a novel G-protein coupled receptor, termed growth hormone (GH) secretagogue receptor (GHS-R), from porcine and human pituitaries, that binds synthetic GH-releasing peptides and nonpeptidyl GH secretagogues (GHSs). This receptor functions in stimulating and amplifying pulsatile GH secretion although its endogenous ligand remains unknown. The discovery of GHS-R indicates that secretion of GH is regulated by three factors, a putative natural GHS and the two hypothalamic peptides, GH-releasing hormone (GRH) and GH release-inhibiting hormone (somatostatin). Using RT-PCR and a RACE protocol, we cloned a rat homolog of pituitary GHS-R in 1997, which belonged to a family of 7-transmembrane G-protein coupled receptors and showed 94-95 % homology to porcine and human GHS-Rs at amino acid level. In situ hybridization with digoxigenin-labeled cRNA probe revealed that both arcuate and ventromedial nuclei in the hypothalamus expressed … More GHS-R mRNA abundantly. GHRP has been shown to stimulate GRH release in animals. This finding provides a morphological support to the views that GHS and GHRP act on the hypothalamic arcuate nucleus to stimulate GRH release thereby amplifying GH pulsatile secretion. We then tried to set up an assay system capable of quantifying GHS-R mRNA levels in1998. Probably because of being a extremely rare transcript, GHS-R mRNA was not detected with Northern blotting, which lead us to develop a more sensitive and specific assay, i.e. RNase protection assay. With this assay we succeeded in detecting and quantitating rat GHS-R mRNA in both the hypothalamus and the pituitary and found that GHS-R mRNA levels per unit total RNA was approximately 2.5 times greater in the medial basal hypothalamus than in the anterior pituitary. Administration of human recombinant GH for 5 days caused a significant decrease on GHS-R mRNA levels in the pituitary but not in the hypothalamus. This finding suggests that GHS-R expression in the pituitaryis negatively regulated by GH and its regulation might be different from that in the hypothalamus. Less