Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein.
Constitutively active human estrogen receptors containing amino acid substitutions for tyrosine 537 in the receptor protein.
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DOI：
10.1210/mend.10.11.8923465
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发表时间：
1996-11
影响因子：
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通讯作者：
Karen E. Weis;K. Ekena;James A. Thomas;G. Lazennec;B. Katzenellenbogen
中科院分区：
文献类型：
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作者：
Karen E. Weis;K. Ekena;James A. Thomas;G. Lazennec;B. Katzenellenbogen
为了更好地了解人类雌激素受体（ER）的结构与活性关系，我们研究了酪氨酸537在受体转录反应中的作用，由于该残基靠近先前显示在激素中重要的酶结合结构域的区域，依赖性转录活性，并且因为该氨基酸已被提出是酪氨酸激酶磷酸化位点，其在以下活性中是重要的：急诊室我们取代5个氨基酸在这个位置（丙氨酸，苯丙氨酸，谷氨酸，赖氨酸，丝氨酸），并筛选这些突变体的生物活性的存在和不存在的雌二醇。两个ER突变体，Y 537 A和Y 537 S，显示雌激素非依赖性的组成型活性，分别约为20%或100%，野生型受体与雌二醇的活性，当在两个不同的细胞背景下使用三种不同的雌激素反应启动子进行评估。在某些情况下，Y 537 E和Y 537 K蛋白也表现出一定程度的低水平的组成型活性。突变体的组成活性，以及它们在E2存在下的活性，被抗雌激素完全抑制。组成型活性ER与类固醇受体共激活因子-1（SRC-1）的相互作用程度与受体转录活性的大小密切相关。而野生型ER仅在雌激素存在下显示与SRC-1的相互作用，Y 537 A和Y 537 S ER在不存在配体的情况下显示中度或完全相互作用，这种相互作用被抗雌激素阻断，并且在雌二醇处理后相互作用的程度增加到或保持在100%，这意味着ER与SRC-1结合的能力是受体转录活性构象状态的良好指示剂。我们的研究结果表明，酪氨酸537是在ER转录活性的配体调节中的重要区域，并且在该位置处的某些氨基酸的存在可以将ER转变为即使没有配体也是活性的构象。然而，酪氨酸是不需要在该网站的雌激素结合或转录反应雌激素的系统中调查。我们的研究结果，解释在最近发表的X-射线晶体结构的配体结合结构域的三个相关的受体的核受体超家族，表明，一些氨基酸取代引入位置537可能有利于移位的螺旋12的ER到一个活跃的构象和/或允许差异稳定的受体在其活性形式。
To better understand structure-activity relationships in the human estrogen receptor (ER), we examined the role of tyrosine 537 in the transcriptional response of the receptor, since this residue is close to a region of the hormone-binding domain shown previously to be important in hormone-dependent transcriptional activity and because this amino acid has been proposed to be a tyrosine kinase phosphorylation site important in the activity of the ER. We substituted five amino acids at this position (alanine, phenylalanine, glutamic acid, lysine, or serine) and screened these mutants for their biological activities in the presence and absence of estradiol. Two of the ER mutants, Y537A and Y537S, displayed estrogen-independent constitutive activity that was approximately 20% or 100%, respectively, of the activity of the wild type receptor with estradiol, when assessed in two different cell backgrounds using three different estrogen-responsive promoters. In some circumstances, the Y537E and Y537K proteins also exhibited some low level of constitutive activity. The constitutive activity of the mutants, as well as their activity in the presence of E2, was fully suppressed by antiestrogen. The extent of interaction of the constitutively active ERs with the steroid receptor coactivator-1 (SRC-1) closely parallel the magnitude of transcriptional activity of the receptor. Whereas wild type ER showed interaction with SRC-1 only in the presence of estrogen, Y537A and Y537S ER showed moderate or full interaction in the absence of ligand, an interaction that was blocked by antiestrogen, and the magnitude of interaction was increased to or remained at 100% upon estradiol treatment, implying that the ability of an ER to associate with SRC-1 is a good indicator of a transcriptionally active conformational state of the receptor. Our findings indicate that tyrosine 537 is in a region important in the ligand regulation of ER transcriptional activity and that the presence of certain amino acids at this position can shift ER into a conformation that is active even without ligand. However, tyrosine is not required at this site for estrogen binding or transcriptional response to estrogen in the systems investigated. Our findings, interpreted in light of the recently published x-ray crystal structure of the ligand-binding domains of three related receptors of the nuclear receptor superfamily, suggest that some of the amino acid substitutions introduced at position 537 may facilitate the shift of helix 12 of the ER into an active conformation and/or allow for differential stabilization of the receptor in its active form.