Nakao Nobuhiro, Higashimoto Yoshifumi, Ohkubo Takeshi, Yoshizato Hideo, Nakai Naoya, Nakashima Kunio, Tanaka Minoru
Department of Biochemistry, Faculty of Medicine, Mie University, Tsu, Mie 514-8507, Japan.
J Endocrinol. 2004 Jul;182(1):157-64. doi: 10.1677/joe.0.1820157.
Growth hormone receptor (GHR) cDNA and gene of the Japanese flounder (Paralicthys olivaceus) were cloned and their molecular structures were characterized. The 641 amino acid sequence predicted from the cDNA sequence showed more than 75% overall sequence similarity with GHRs of other teleosts such as turbot and goldfish, and contained common structural features of vertebrate GHRs. The extracellular domain of flounder GHR had three pairs of cysteines and an FGEFS motif with a replacement E to D. The cytoplasmic domain contained two conserved motifs referred to as box 1 and box 2. The flounder GHR gene was cloned by PCR using primers designed from the sequence of the GHR cDNA. The GHR gene was composed of 10 exons. The sequence of exon 1 corresponded to the 5'-untranslated region of the cDNA, and exons 2-6 encoded most parts of the extracellular domain. The transmembrane domain was found in exon 7, and the intracellular domain was encoded in exons 8-10. Exon 10 also encoded the 3'-untranslated region. Comparison of the flounder GHR gene with the human GHR gene shows that the flounder gene contains no exons corresponding to exon 3 of the human GHR gene, and that the region corresponding to exon 10 in the human GHR gene is encoded by exons 9 and 10 in the flounder GHR gene. These findings indicate that the flounder GHR gene diverged from those of mammalian and avian GHR genes, especially in the organization of the exons encoding the cytoplasmic domain. In addition to the regular form of GHR mRNA, a 3'-truncated form lacking the region derived from exons 9 and 10 was detected as a minor species in the liver by RT-PCR and by RNase protection assay. RT-PCR analysis showed that both the regular and the 3'-truncated GHR mRNAs are expressed in a wide range of flounder tissues with the highest levels being found in the liver. The 5'-flanking region of the flounder GHR gene was cloned by inverse PCR, and three transcription start points were identified with similar frequency by RNase protection assay.
克隆了牙鲆（Paralicthys olivaceus）的生长激素受体（GHR）cDNA和基因，并对其分子结构进行了表征。从cDNA序列预测的641个氨基酸序列与其他硬骨鱼类如大菱鲆和金鱼的GHR具有超过75%的总体序列相似性，并包含脊椎动物GHR的共同结构特征。牙鲆GHR的细胞外结构域有三对半胱氨酸和一个FGEFS基序，其中E被D取代。细胞质结构域包含两个保守基序，称为框1和框2。利用根据GHR cDNA序列设计的引物通过PCR克隆了牙鲆GHR基因。GHR基因由10个外显子组成。外显子1的序列对应于cDNA的5'-非翻译区，外显子2-6编码细胞外结构域的大部分。跨膜结构域在外显子7中发现，细胞内结构域在外显子8-10中编码。外显子10还编码3'-非翻译区。牙鲆GHR基因与人GHR基因的比较表明，牙鲆基因不包含与人GHR基因外显子3相对应的外显子，并且人GHR基因中与外显子10相对应的区域由牙鲆GHR基因的外显子9和10编码。这些发现表明，牙鲆GHR基因与哺乳动物和鸟类GHR基因不同，特别是在编码细胞质结构域的外显子组织方面。除了常规形式的GHR mRNA外，通过RT-PCR和核糖核酸酶保护试验在肝脏中检测到一种缺少外显子9和10区域的3'-截短形式，其为次要类型。RT-PCR分析表明，常规和3'-截短的GHR mRNA在牙鲆的多种组织中均有表达，在肝脏中表达水平最高。通过反向PCR克隆了牙鲆GHR基因的5'-侧翼区，并通过核糖核酸酶保护试验以相似的频率鉴定了三个转录起始点。
