THE PRIMARY STRUCTURE OF THE ALPHA SUBUNIT OF A STARFISH GUANOSINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN INVOLVED IN 1-METHYLADENINE-INDUCED OOCYTE MATURATION
THE PRIMARY STRUCTURE OF THE ALPHA SUBUNIT OF A STARFISH GUANOSINE-NUCLEOTIDE-BINDING REGULATORY PROTEIN INVOLVED IN 1-METHYLADENINE-INDUCED OOCYTE MATURATION
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DOI：
10.1111/j.1432-1033.1992.tb17114.x
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发表时间：
1992-08-01
期刊：
影响因子：
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通讯作者：
HOSHI, M
中科院分区：
文献类型：
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作者：
CHIBA, K;TADENUMA, H;HOSHI, M
微量注射百日咳毒素（PTX）可抑制1-甲基腺嘌呤（米德）诱导的海星卵母细胞成熟。这种抑制似乎是由于卵母细胞中39 kDa鸟苷酸结合调节蛋白（G蛋白）的PTX催化ADP核糖基化所致。这些结果有力地支持了MeAde诱导的信号通过膜受体起作用并由PTX敏感的G蛋白携带的假设。当PTX注射的卵母细胞与二硫苏糖醇处理，85%的人重新启动减数分裂，这表明二硫苏糖醇不作用于米德受体。我们构建了海星未成熟卵巢的cDNA文库，并用抑制性大鼠G蛋白α亚基（Gi-2）的cDNA进行筛选。阳性克隆含有1062个碱基的开放阅读框，与大鼠G（i-2）cDNA的同源性为74%。推导的氨基酸序列与大鼠G（i-1）和大鼠G（i-1）的同源性分别为85%和89%。从海星卵母细胞皮质中纯化的G蛋白α亚基经胰蛋白酶消化，并对所得的四个肽段进行微测序。将这些氨基酸序列与预测的氨基酸序列进行比较表明，分离的cDNA克隆编码卵母细胞中PTX敏感性G蛋白的α亚基。C端序列KNNLKDCGLF与G（i）相同，表明半胱氨酸残基是ADP核糖基化位点。
Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha-subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat G(i-2) cDNA. The deduced amino acid sequence was 85% and 89% identical to rat G(i-1), and rat G(i-1), respectively. The alpha-subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha-subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of G(i), suggesting that the cysteine residue is the site of ADP-ribosylation.