MUTATION ANALYSIS OF THYROID HORMONE FUNCTION

甲状腺激素通过核受体超家族成员受体(Trs)来调节反应，包括与类固醇、维生素和脂肪酸结合的受体，每一个受体都是主要的药物靶点。我们利用关于TR配体结合域(LBD)的X射线结晶学信息来指导突变在TRLBD表面的放置，这些突变阻止了与其相互作用的蛋白质的结合。这些突变比随机放置的突变更可取，后者往往会扰乱受体折叠，使解释复杂化。使用这种扫描表面突变方法，我们定义了共激活子结合表面，这是一个独特的裂隙，现在提供了一个新的药物靶点，并提供了有关配体诱导的构象变化介导受体作用的信息。我们定义了二聚体和异源二聚体结合面，它们提供了对同源二聚体和异源二聚体与视黄醇-X受体(RXRs)的形成和功能的见解，以及受体如何与DNA结合。我们定义了一个辅阻遏子结合表面。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来扩展对其他蛋白质的分析，并更详细地检查Trs与选定的一组蛋白质的相互作用及其功能后果。天然和突变的Trs将在体外检测蛋白质结合和细胞培养中的调节活性。待检测的蛋白质包括：一种新的辅活化子、配体解离的辅抑制子、一种配体依赖的抑制物和一种阻断tr作用的受体。我们还将使用突变受体来：(I)确定二聚体表面和连接LBD与DNA结合域的铰链区域在与RXRs形成二聚体和异源二聚体中的作用，变构影响的中介，以及对tr-DNA结合的影响；以及(Ii)确定参与配体依赖的抑制和非配体依赖的激活的tr表面。总体而言，这些研究致力于提供对核受体LBD表面的全面了解，并深入了解未连接的受体传递和激素介导的基因表达调节。所获得的信息将与了解核受体超家族和基因调控机制有关。

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.