Skafar Debra F, Koide Shohei
Department of Physiology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201, USA.
Mol Cell Endocrinol. 2006 Feb 26;246(1-2):83-90. doi: 10.1016/j.mce.2005.12.015. Epub 2006 Jan 18.
The estrogen receptor-alpha is a wonderfully complex protein important in normal biology, breast cancer, and as a target for anti-cancer agents. We are using the available structures of the hERalpha as well as secondary structure predictions to guide site-directed mutagenesis in order to test the importance of specific interactions and regions in the ligand-regulated activity of the protein. In one area of interest, we are investigating the role of the F domain in the ligand-stimulated activity of the hERalpha. Results from our laboratory and others suggest that the F domain modulates the activity of the hERalpha. In order to better understand the role of the F domain in the hERalpha, we have constructed mutants within this region. Mutations within a predicted alpha-helical region alter the response of the ER to estradiol (E2), eliminate or impair the agonist activity of 4-hydroxytamoxifen (4-OHT), and alter the ability of E2 to overcome 4-OHT's antagonist activity. Deleting the F domain increases the affinity of the receptor for E2; by contrast, mutating a residue in the middle of the predicted helix to a proline does not alter the affinity for E2, but does change the binding mechanism from a positive cooperative to a noncooperative interaction. These and other results show the F domain exhibits substantial functional complexity, and support the idea that this domain modulates the activity of the hERalpha. In a second area of interest, we are investigating the role of hydrophobic and hydrogen-bonding interactions at the start of helix 12 in the activity of the hERalpha. Leucine-536 (L536) has been proposed to participate in hydrophobic interactions that form part of a capping motif stabilizing the start of helix 12. When mutated, the resulting receptors exhibit a reduced response, or even an inverted response, to E2 and 4-OHT on both ERE-driven and AP-1-driven promoters. Interestingly, these mutated receptors also exhibit altered interactions with probes that recognize the agonist-bound and 4-OHT-bound conformations of the ERalpha. Thus, L536 couples the binding of ligand with the conformation of the receptor. Overall, these results show that combining structure-based hypotheses with functional tests of the ER's activity can identify regions and interactions that are important in the ligand-stimulated activity of the protein.
雌激素受体α是一种极其复杂的蛋白质，在正常生物学、乳腺癌以及作为抗癌药物的靶点方面都具有重要意义。我们正在利用人雌激素受体α（hERα）的现有结构以及二级结构预测来指导定点诱变，以测试特定相互作用和区域在该蛋白质配体调节活性中的重要性。在一个感兴趣的领域，我们正在研究F结构域在hERα配体刺激活性中的作用。我们实验室及其他研究结果表明，F结构域可调节hERα的活性。为了更好地理解F结构域在hERα中的作用，我们在该区域构建了突变体。预测的α螺旋区域内的突变会改变雌激素受体（ER）对雌二醇（E2）的反应，消除或损害4-羟基他莫昔芬（4-OHT）的激动剂活性，并改变E2克服4-OHT拮抗剂活性的能力。删除F结构域会增加受体对E2的亲和力；相比之下，将预测螺旋中间的一个残基突变为脯氨酸不会改变对E2的亲和力，但会将结合机制从正协同相互作用变为非协同相互作用。这些及其他结果表明F结构域表现出显著的功能复杂性，并支持该结构域调节hERα活性的观点。在另一个感兴趣的领域，我们正在研究螺旋12起始处的疏水和氢键相互作用在hERα活性中的作用。亮氨酸-536（L536）被认为参与了疏水相互作用，这些相互作用构成了稳定螺旋12起始处的封端基序的一部分。突变后，所得受体在雌激素反应元件（ERE）驱动和激活蛋白-1（AP-1）驱动的启动子上对E2和4-OHT的反应降低，甚至出现反向反应。有趣的是，这些突变受体与识别ERα激动剂结合和4-OHT结合构象的探针的相互作用也发生了改变。因此，L536将配体的结合与受体的构象联系起来。总体而言，这些结果表明，将基于结构的假设与ER活性的功能测试相结合，可以确定在蛋白质配体刺激活性中重要的区域和相互作用。
