MUTATION ANALYSIS OF THYROID HORMONE FUNCTION

甲状腺激素功能突变分析

• 批准号: 6329404
• 负责人: JOHN D BAXTER
• 金额: $ 25.96万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-05-01 至 2003-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6329404
• 关键词: X ray crystallography; conformation; dimer; gene induction /repression; gene mutation; genetic regulatory element; hormone receptor; hormone regulation /control mechanism; nuclear receptors; protein protein interaction; protein structure function; receptor binding; retinoid binding proteins; site directed mutagenesis; thyroid hormones; tissue /cell culture; transcription factor; transfection

Thyroid hormones mediate responses through receptors (Trs) that are members of the nuclear receptor superfamily, including receptors that bind steroids, vitamins and fatty acids, each a major pharmaceutical target. We utilized X-ray crystallographic information about the TR ligand-binding domain (LBD) to guide placements of mutations on the TR LBD surface that block binding of proteins that interact with it. These are preferable to randomly placed mutations that often disrupt receptor folding and complicate interpretations. Using this scanning surface mutagenesis approach we defined the coactivator-binding surface, a distinct cleft which now provides a novel pharmaceutical target and provides information about ligand-induced conformational changes mediating receptor action. We defined dimer and heterodimer binding surfaces, which provide insights into formation and functions of TR homodimers vs. heterodimers with retinoid-X receptors (RXRs), and how receptors bind DNA. We defined a corepressor-binding surface. In the proposed studies we plan to use our large and growing bank of mutations to extend analyses to additional proteins and examine interactions of Trs with a selected group of proteins and their functional consequences in greater detail. Native and mutated Trs will be examined for protein binding in vitro and regulatory activities in cell culture. Proteins to be examined include: a novel coactivator, ligand-dissociated corepressors, a ligand-dependent inhibitor, and a receptor that blocks TR action. We will also use mutant receptors to: (i) define roles of the dimer surface and the hinge region connecting the LBD with the DNA-binding domain in formation of dimers and heterodimers with RXRs, mediation of allosteric influences, and influences on TR-DNA binding; and (ii) define TR surfaces involved in ligand-dependent repression and ligand-independent activation. Overall, these studies endeavor to provide a comprehensive understanding of the nuclear receptor LBD surface, with insights into receptor transmission of unliganded and hormone-mediated regulation of gene expression. The information gained will be relevant for understanding the nuclear receptor superfamily and gene regulation mechanisms in general.

甲状腺激素通过作为核受体超家族成员的受体（Trs）介导反应，所述核受体超家族包括结合类固醇、维生素和脂肪酸的受体，每种受体都是主要的药物靶标。 我们利用TR配体结合结构域（LBD）的X射线晶体学信息来指导TR LBD表面突变的位置，这些突变会阻止与之相互作用的蛋白质的结合，这比随机放置的突变更可取，这些突变通常会破坏受体折叠并使解释复杂化。 使用这种扫描表面诱变的方法，我们定义的辅激活剂结合表面，一个独特的裂缝，现在提供了一个新的药物靶点，并提供有关配体诱导的构象变化介导的受体作用的信息。 我们定义了二聚体和异二聚体结合表面，这提供了对TR同源二聚体与异源二聚体与维甲酸-X受体（RXR）的形成和功能以及受体如何结合DNA的见解。 我们定义了辅阻遏物结合表面。 在拟议的研究中，我们计划使用我们庞大且不断增长的突变库将分析扩展到其他蛋白质，并更详细地研究Trs与选定的一组蛋白质及其功能后果的相互作用。 将检查天然和突变Trs的体外蛋白结合和细胞培养物中的调节活性。 待检查的蛋白质包括：一种新的辅激活因子，配体解离的辅阻遏物，配体依赖性抑制剂，和受体，阻断TR行动。 我们还将使用突变体受体：（i）定义二聚体表面和连接LBD与DNA结合结构域的铰链区在与RXR形成二聚体和异二聚体、介导变构影响以及对TR-DNA结合的影响中的作用;（ii）定义参与配体依赖性抑制和配体非依赖性激活的TR表面。 总体而言，这些研究奋进提供一个全面的了解核受体LBD的表面，深入了解受体传输的unliganded和酶介导的基因表达调控。 所获得的信息将是相关的理解核受体超家族和基因调控机制一般。