Novel growth hormone releasing peptide, ghrelin: analyses of its structure and function

被称为生长激素分泌剂（GHSs）的小合成分子通过孤儿蛋白偶联受体GHS-R起作用，刺激垂体释放生长激素（GH）。为了寻找GHS-R的天然配体，我们构建了表达大鼠GHS-R的稳定CHO细胞系，用于监测大鼠组织提取物诱导的细胞内钙浓度（[Ca^<2+>]_i）的变化。利用这一细胞系，我们从胃中纯化了一种能有效激活GHS-R的肽。纯化的肽由28个氨基酸组成，其中Ser-3残基被正辛烷化。令人惊讶的是，Ser-3的正辛烷基修饰对活性至关重要，因为去酰基肽不能诱导（[Ca^<2+>]_i）变化。我们将这种释放gh的肽命名为ghrelin （ghre是“生长”一词的原始印欧语词根）。我们克隆了哺乳动物（人、大鼠、小鼠、犬、猪、羊和牛）的gbrelin cdna，并确定了它们的结构。动物胃饥饿素的前体结构具有高度的同源性。特别是，n端10个氨基酸是相同的，这支持辛烷基修饰对胃饥饿素活性的重要性。胃饥饿素在体外和体内均以剂量依赖性方式诱导生长激素释放。ICV注射胃促生长素也刺激生长激素的释放。胃内分泌细胞X/ a样细胞中可见胃生长素免疫反应细胞。Ghrelin存在于X/ a样细胞的秘书颗粒中，其激素产物和生理功能以前尚未阐明。在大鼠大脑中，胃饥饿素免疫反应神经元被发现位于下丘脑弓状核，这是一个调节食物摄入的区域。事实上，ICV注射胃饥饿素强烈刺激了大鼠的进食，增加了体重。另一方面，抗胃饥饿素IgG强烈抑制摄食。这些事实表明，胃饥饿素是一种新的营养稳态生理调节剂。最近，我们从鸡、蛙（牛蛙）和鱼（鳗鱼和鳟鱼）中纯化了胃饥饿素肽。在所有脊椎动物中，胃饥饿素肽的氨基酸序列都是保守的。此外，胃饥饿素肽均被辛酸修饰。这些结果表明胃饥饿素在脊椎动物中起着至关重要的作用。少

Small synthetic molecules termed growth hormone secretagogues (GHSs) act through an orphan 0protein coupled receptor, GHS-R, to stimulate growth hormone (GH) release from pituitary. To search for the natural ligand for GHS-R, we constructed a stable CHO cell line expressing rat GHS-R for monitoring intracellular calcium concentration ([Ca^<2+>]_i) changes induced by rat tissue extracts. Using this cell line, we purified a peptide from stomach that potently activated GHS-R. The purified peptide was consisted of 28 amino acids, in which the Ser-3 residue was n-octanoylated. Surprisingly, this n-octanoyl modification at Ser-3 was essential to the activity, since des-acyl-peptide could not induce ([Ca^<2+>]_i) changes. We named the GH-releasing peptide ghrelin (ghre is the Proto-Indo-European root of the word grow).We cloned mammalian gbrelin cDNAs (human, rat, mouse, canine, porcine, ovine and bovine) and determined their structures. Precursor structures of manimalian ghrelin are highly h … More omologous. In particular, N-terminal 10 amino-acids are identical, which supports the importance of octanoyl modification for ghrelin s activity.Ghrelin induced GH release in a dose-dependent manner both in vitro and in vivo. ICV injection of ghrelin also stimulated GH release. Ghrein immunoreactive cells in the stomach were found in X/A-like cells, an endocrine cell in stomach. Ghrelin is found in the secretary granules of X/A-like cells whose hormonal product and physiological functions have not been previously clarified. In rat brain, ghrelin immunoreactive neurons were found to be localized in the hypothalamic arcuate nucleus, a region where regulates food intake. In fact, ICV injection of ghrelin strongly stimulated feeding in rats and increased body weight gain. On the other hand, anti-ghrelin IgG robustly suppressed feeding. These facts suggest that ghrelin is a new physiological regulator of nutritional homeostasis.Recently we purified ghrein peptides from chicken, frog (bullfrog) and fish (eel and trout). Aminoacid sequences of ghrelin peptides in all vertebrates are well conserved. Moreover, the ghrelin peptides are all modified by octanoic acid. These results suggest that ghrelin plays a fundamentally important role in vertebrates. Less