Bury N R, Sturm A, Le Rouzic P, Lethimonier C, Ducouret B, Guiguen Y, Robinson-Rechavi M, Laudet V, Rafestin-Oblin M E, Prunet P
INRA SCRIBE, Campus de Beaulieu, 35042 Rennes-Cedex, France.
J Mol Endocrinol. 2003 Aug;31(1):141-56. doi: 10.1677/jme.0.0310141.
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 EC(50)=0.72+/-0.87 nM) compared with rtGR1 (cortisol EC(50)=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.
我们使用简并引物进行逆转录聚合酶链反应（RT-PCR），随后筛选虹鳟（Oncorhynchus mykiss）肠道cDNA文库，从虹鳟中分离出一种新的皮质类固醇受体，它与其他糖皮质激素受体（GRs）具有高度的序列同源性，但明显不同于先前的虹鳟GR（命名为rtGR1）。对这两个序列以及哺乳动物、两栖动物和鱼类中已知的其他GRs进行系统发育分析表明，GR复制在大多数硬骨鱼中可能很常见。这种新的虹鳟GR（命名为rtGR2）的开放阅读框编码一个669个氨基酸的蛋白质，体外翻译产生一个80 kDa的蛋白质，与rtGR1蛋白质（88 kDa）明显不同。使用rtGR2 cDNA作为探针，在各种组织中观察到一个7.3 kb的转录本，表明该基因会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。用重组rtGR1和rtGR2蛋白进行结合研究表明，这两种受体对地塞米松具有相似的亲和力（GR1 K(d)=5.05±0.45 nM；GR2 K(d)=3.04±0.79 nM）。将rtGR1或rtGR2表达载体与含有多个糖皮质激素反应元件共识序列的报告质粒共转染到CHO-K1或COS-7细胞中，结果表明，在皮质醇和地塞米松存在的情况下，两个克隆都能够诱导转录活性。此外，在10(-)(6 )M 11-脱氧皮质醇和皮质酮的作用下，rtGR2的反式激活活性部分被诱导，但对rtGR1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这些激素和皮质类固醇激素的分解产物，对这两种受体都没有主要影响。有趣的是，与rtGR1（皮质醇EC(50)=46±12 nM）相比，rtGR2在低得多的地塞米松或皮质醇浓度下（皮质醇EC(50)=0.72±0.87 nM）就能诱导反式激活活性。同样，尽管RU486抑制了rtGR1和rtGR2的反式激活活性，但rtGR1对这种GR拮抗剂更敏感。总之，这些结果表明，这两个GR序列编码两种功能不同的GRs，它们在虹鳟中作为配体诱导的转录因子发挥作用。
