Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation.
Estradiol-binding mechanism and binding capacity of the human estrogen receptor is regulated by tyrosine phosphorylation.
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DOI：
10.1210/mend.11.1.9876
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发表时间：
1997
影响因子：
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通讯作者：
S. F. Arnold;Michal Melamed;Daria P Vorojeikina;A. Notides;S. Sasson
中科院分区：
文献类型：
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作者：
S. F. Arnold;Michal Melamed;Daria P Vorojeikina;A. Notides;S. Sasson
我们研究了酪氨酸磷酸化对雌二醇结合机制和人雌激素受体（hER）结合能力的影响。在Sf 9昆虫细胞中表达野生型hER和点突变形式的hER，其中酪氨酸537突变为苯丙氨酸（Y 537 F hER）。野生型hER，而不是Y 537 F hER，与抗磷酸酪氨酸单克隆抗体反应，表明酪氨酸537是hER上唯一磷酸化的酪氨酸。Scatchard和Hill对[3 H]雌二醇与野生型hER结合相互作用的分析表明，加入毫摩尔磷酸酪氨酸，而不是酪氨酸、磷酸或磷酸丝氨酸，这些观察结果与磷酸酪氨酸阻断hER单体之间的二聚化和位点-位点相互作用的想法一致。野生型hER结合的[3 H]雌二醇比Y 537 F hER多10倍。用酪氨酸磷酸酶处理纯化的野生型hER使hER的结合能力降低约90%，而丝氨酸/苏氨酸磷酸酶则没有影响。酪氨酸去磷酸化hER的雌激素结合能力完全恢复酪氨酸537与p60 c-src，酪氨酸激酶的再磷酸化。这些结果表明，p60 c-src可以恢复雌激素结合酪氨酸去磷酸化的hER和二聚化和合作的位点-位点的相互作用的hER发生通过磷酸酪氨酸结合的相互作用。
We have investigated the effects of tyrosine phosphorylation on the estradiol-binding mechanism and binding capacity of the human estrogen receptor (hER). The wild type hER and a point mutant form of the hER, in which tyrosine 537 was mutated to phenylalanine (Y537F hER), were expressed in Sf9 insect cells. The wild type hER, but not the Y537F hER, reacted with a anti-phosphotyrosine monoclonal antibody, indicating that tyrosine 537 was the only tyrosine phosphorylated on the hER. Scatchard and Hill analyses of the the binding interaction of [3H]estradiol with the wild type hER indicated that the addition of millimolar phosphotyrosine, but not tyrosine, phosphate, or phosphoserine, abolished the cooperative binding mechanism of the hER. These observations are consistent with the idea that phosphotyrosine blocks dimerization and site-site interactions between the hER monomers. The wild type hER bound 10-fold more [3H]estradiol than the Y537F hER. Treatment of the purified wild type hER with a tyrosine phosphatase decreased the binding capacity of the hER by approximately 90%, whereas, a serine/threonine phosphatase had no effect. The estrogen-binding capacity of the tyrosine-dephosphorylated hER was completely restored by rephosphorylation of tyrosine 537 with p60c-src, a tyrosine kinase. These results indicate that p60c-src can restore estrogen binding to the tyrosine-dephosphorylated hER and that dimerization and cooperative site-site interaction of the hER occur via a phosphotyrosine-binding interaction.