McNaught R W, Raymoure W J, Smith R G
J Biol Chem. 1986 Dec 25;261(36):17011-7.
We have previously reported that the estrogen receptor exists in three distinct states in the oviduct of the estrogen-treated chick. Since two of these forms bind estrogen and possess a number of similar properties, the intrinsic relationship between these two receptor forms has been investigated. We now show that a quantitative conversion of the high affinity (Rx) to the lower affinity (Ry) state can be induced by mild heating (30 degrees C) in the presence of estradiol and ATP, or the synthetic analogue alpha,beta-methylene adenosine triphosphate and the divalent cation Mg2+. Other nucleotides, including ADP, GTP, CTP, cAMP, and cGMP, as well as the nonhydrolyzable analogues beta,gamma-methylene adenosine triphosphate and alpha,beta-methylene adenosine diphosphate are ineffective. The conversion occurs only partially in the absence of estradiol but completely in its presence. The process is not inhibited by the protease inhibitors phenylmethylsulfonyl fluoride and alpha 2-macroglobulin, and when conversion is induced by low concentrations of ATP (1 mM), a time dependent reequilibration back to Rx occurs. These observations and the fact that the pure hormone-binding peptides Rx and Ry have similar molecular weights on sodium dodecyl sulfate-polyacrylamide gels (66,000) confirm that proteolysis is not involved in the conversion. Moreover their physical properties suggest that Rx and Ry exist in alternate conformations, with Ry being favored as a result of an ATP-mediated event involving the gamma-phosphoryl moiety. The biological relevance of the receptor conversion is suggested by studies with the antiestrogen hydroxytamoxifen. This antiestrogen binds to Rx with higher affinity than either estradiol or diethylstilbestrol but with low affinity to Ry. Hydroxytamoxifen also inhibits the conversion of Rx to Ry. Since this antiestrogen is a complete antagonist in the chick oviduct and prevents estradiol-induced stimulation of ovalbumin gene transcription, it is speculated that Rx to Ry conversion is crucial for ovalbumin gene activation and that Rx may act as a transcriptional repressor. Furthermore, since Rx and Ry both bind to nuclei and DNA, it is proposed that the presence of Ry is a better predictor of ovalbumin gene activation than DNA binding alone.
我们之前报道过，雌激素受体在经雌激素处理的雏鸡输卵管中以三种不同状态存在。由于其中两种形式能结合雌激素并具有许多相似特性，因此对这两种受体形式之间的内在关系进行了研究。我们现在表明，在雌二醇和ATP存在的情况下，通过温和加热（30摄氏度），或者合成类似物α,β-亚甲基三磷酸腺苷和二价阳离子Mg2+，可以诱导高亲和力（Rx）状态向低亲和力（Ry）状态的定量转化。其他核苷酸，包括ADP、GTP、CTP、cAMP和cGMP，以及不可水解类似物β,γ-亚甲基三磷酸腺苷和α,β-亚甲基二磷酸腺苷均无效。在没有雌二醇的情况下，转化仅部分发生，但在其存在时则完全发生。该过程不受蛋白酶抑制剂苯甲基磺酰氟和α2-巨球蛋白的抑制，并且当由低浓度ATP（1 mM）诱导转化时，会发生时间依赖性的重新平衡回到Rx状态。这些观察结果以及纯激素结合肽Rx和Ry在十二烷基硫酸钠-聚丙烯酰胺凝胶上具有相似分子量（66,000）这一事实证实，蛋白水解不参与转化过程。此外，它们的物理性质表明Rx和Ry以交替构象存在，由于涉及γ-磷酸基团的ATP介导事件，Ry更受青睐。用抗雌激素他莫昔芬进行的研究表明了受体转化的生物学相关性。这种抗雌激素与Rx的结合亲和力高于雌二醇或己烯雌酚，但与Ry的亲和力较低。他莫昔芬还抑制Rx向Ry的转化。由于这种抗雌激素在雏鸡输卵管中是一种完全拮抗剂，并能阻止雌二醇诱导的卵清蛋白基因转录刺激，因此推测Rx向Ry的转化对于卵清蛋白基因激活至关重要，并且Rx可能作为转录抑制因子起作用。此外，由于Rx和Ry都能与细胞核和DNA结合，因此有人提出，Ry的存在比单独的DNA结合更能预测卵清蛋白基因的激活。
