A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica):: identification, expression and binding activity
A novel G protein-coupled receptor for gonadotropin-inhibitory hormone in the Japanese quail (Coturnix japonica):: identification, expression and binding activity
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DOI：
10.1677/joe.1.05926
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发表时间：
2005-01-01
影响因子：
4
通讯作者：
Tsutsui, K
中科院分区：
文献类型：
--
作者：
Yin, H;Ukena, K;Tsutsui, K
我们最近发现了一种新的下丘脑十二肽抑制日本鹌鹑（Coturnix japonica）的促性腺激素释放。因此，这种新的肽被命名为促性腺激素抑制激素（GnIH）。GnIH前体编码一个GnIH和两个GnIH相关肽（GnIH-RP-1和GnIH-RP-2），它们具有相同的C-末端基序Leu-Pro-Xaa-Arg-Phe-NH 2（Xaa = Leu或Gln; LPXRF-酰胺肽）。GnIH受体的鉴定对于阐明GnIH的作用方式至关重要。因此，我们确定了鹌鹑间脑中GnIH的受体，并表征其表达和结合活性。我们首先通过3'和5'快速扩增cDNA末端（RACE）的组合，使用从大鼠RF-酰胺相关肽（RFRP）（GnIH的一种邻位肽）的受体的序列设计的PCR引物，克隆了编码推定的GnIH受体的cDNA。疏水性分析表明，推定的GnIH受体具有7个跨膜结构域，表明G蛋白偶联受体超家族的新成员。用推定的GnIH受体cDNA转染的COS-7细胞的粗膜部分以浓度依赖性方式特异性结合GnIH和GnIH-RPs。结合的Scatchard图分析表明，所鉴定的GnIH受体具有单一类别的高亲和力结合位点（Kd =0.752 nM，B-max = 24.8 fmol/mg蛋白）。逆转录酶介导的PCR产物的Southern印迹分析显示，在鹌鹑的垂体和几个脑区，包括间脑的GnIH受体mRNA的表达。这些结果表明，GnIH直接作用于垂体通过GnIH受体抑制促性腺激素释放。GnIH也可作用于下丘脑，抑制促性腺激素释放激素的释放。
We recently identified a novel hypothalamic dodecapeptide inhibiting gonadotropin release in the Japanese quail (Coturnix japonica). This novel peptide was therefore named gonadotropin-inhibitory hormone (GnIH). The GnIH precursor encoded one GnIH and two GnIH-related peptides (GnIH-RP-1 and GnIH-RP-2) that shared the same C-terminal motif, Leu-Pro-Xaa-Arg-Phe-NH2 (Xaa = Leu or Gln; LPXRF-amide peptides). Identification of the receptor for GnIH is crucial to elucidate the mode of action of GnIH. We therefore identified the receptor for GnIH in the quail diencephalon and characterized its expression and binding activity. We first cloned a cDNA encoding a putative GnIH receptor by a combination of 3' and 5' rapid amplification of cDNA ends (RACE) using PCR primers designed from the sequence for the receptor for rat RF-amide-related peptide (RFRP), an orthologous peptide of GnIH. Hydrophobic analysis revealed that the putative GnIH receptor possessed seven transmembrane domains, indicating a new member of the G protein-coupled receptor superfamily. The crude membrane fraction of COS-7 cells transfected with the putative GnIH receptor cDNA specifically bound to GnIH and GnIH-RPs in a concentration-dependent manner. Scatchard plot analysis of the binding showed that the identified GnIH receptor possessed a single class of high-affinity binding sites (K-d=0.752 nM, B-max = 24.8 fmol/mg protein). Southern blotting analysis of reverse transcriptase-mediated PCR products revealed the expression of GnIH receptor mRNA in the pituitary gland and several brain regions including diencephalon in the quail. These results suggest that GnIH acts directly on the pituitary via GnIH receptor to inhibit gonadotropin release. GnIH may also act on the hypothalamus to inhibit gonadotropin-releasing hormone release.