Evidence for two distinct functional glucocorticoid receptors in teleost fish
Evidence for two distinct functional glucocorticoid receptors in teleost fish
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DOI：
10.1677/jme.0.0310141
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发表时间：
2003-08-01
影响因子：
3.5
通讯作者：
Prunet, P
中科院分区：
文献类型：
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作者：
Bury, NR;Sturm, A;Prunet, P
利用简并引物RT-PCR技术，从虹鳟（Oncorhynchus mykiss）肠道cDNA文库中筛选出一个新的糖皮质激素受体（rtGR 1），该受体与其它糖皮质激素受体（GR）具有很高的同源性，但与先前的虹鳟GR（rtGR 1）明显不同。这两个序列和其他已知的哺乳动物，两栖动物和鱼类的GR的系统发育分析表明，GR重复可能是常见的大多数硬骨鱼。这种新的鳟鱼GR的开放阅读框架（命名为rtGR 2）编码669个氨基酸的蛋白质，体外翻译产生80 kDa的蛋白质，其明显不同于rtGR 1蛋白（88 kDa）。使用rtGR 2 cDNA作为探针，在各种组织中观察到7.3kb的转录本，表明该基因将导致类固醇受体的表达。体外研究用于进一步表征这种新的皮质类固醇受体。重组rtGR 1和rtGR 2蛋白的结合研究表明，这两种受体对地塞米松具有相似的亲和力（GR 1 K-d=5.05+/-0.45 nM; GR 2 K-d=3.04+/-0.79 nM）。将rtGR 1或rtGR 2表达载体与含有多个共有糖皮质激素反应元件的报告质粒一起共转染到CHO-K1或COS-7细胞中，沿着，表明两种克隆都能够在皮质醇和地塞米松存在下诱导转录活性。此外，在10（-6）M时，11-脱氧皮质醇和皮质酮部分诱导rtGR 2反式激活活性，但对rtGR 1没有影响。其他主要的硬骨鱼生殖激素，以及它们的一些前体或分解产物和皮质类固醇激素，对两种受体都没有重大影响。有趣的是，与rtGR 1（皮质醇EC 50 =46+/-12 nM）相比，在地塞米松或皮质醇（皮质醇EC 50 =0.72+/-0.87 nM）浓度低得多的情况下诱导rtGR 2反式激活活性。类似地，即使RU 486抑制rtGR 1和rtGR 2的反式激活活性，rtGR 1对这种GR拮抗剂更敏感。总之，这些结果表明，这两个GR序列编码两个功能不同的GR作为配体诱导型转录因子在虹鳟鱼。
Using RT-PCR with degenerated primers followed by screening of a rainbow trout (Oncorhynchus mykiss) intestinal cDNA library, we have isolated from the rainbow trout a new corticosteroid receptor which shows high sequence homology with other glucocorticoid receptors (GRs), but is clearly different from the previous trout GR (named rtGR1). Phylogenetic analysis of these two sequences and other GRs known in mammals, amphibians and fishes indicate that the GR duplication is probably common to most teleost fish. The open reading frame of this new trout GR (named rtGR2) encodes a protein of 669 amino acids and in vitro translation produces a protein of 80 kDa that appears clearly different from rtGR1 protein (88 kDa). Using rtGR2 cDNA as a probe, a 7.3 kb transcript was observed in various tissues suggesting that this gene would lead to expression of a steroid receptor. In vitro studies were used to further characterize this new corticosteroid receptor. Binding studies with recombinant rtGR1 and rtGR2 proteins show that the two receptors have a similar affinity for dexamethasone (GR1 K-d=5.05+/-0.45 nM; GR2 K-d=3.04+/-0.79 nM). Co-transfection of an rtGR1 or rtGR2 expression vector into CHO-K1 or COS-7 cells, along with a reporter plasmid containing multiple consensus glucocorticoid response elements, shows that both clones are able to induce transcriptional activity in the presence of cortisol and dexamethasone. Moreover, at 10(-6) M 11-deoxycortisol and corticosterone partially induced rtGR2 transactivation activity but were without effect on rtGR1. The other major teleost reproductive hormones, as well as a number of their precursors or breakdown products of these and corticosteroid hormones, were without major effects on either receptor, Interestingly, rtGR2 transactivational activity was induced at far lower concentrations of dexamethasone or cortisol (cortisol EC50=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC50=46+/-12 nM). Similarly, even though RU486 inhibited transactivation activity in both rtGR1 and rtGR2, rtGR1 was more sensitive to this GR antagonist. Altogether, these results indicate that these two GR sequences encode for two functionally distinct GRs acting as ligand-inducible transcription factors in rainbow trout.