Mutation Analysis of Thyroid Hormone Function

甲状腺激素（TH）信号由两个相关的调节基因表达的甲状腺受体（TRs）转导，是核受体（NR）超家族的成员，该家族还包括类固醇、维生素和脂肪酸以及各种胆固醇和脂肪酸及其代谢物的受体。目前约20%的药物是与NR结合的配体，强调了NR家族的重要性。TH调节脂肪量、胆固醇、心脏等功能；因此，开发选择性调节TR作用的方法可能导致具有选择性作用的有用药物。我们利用关于TR配体结合（LBD）和dma结合（DBD）结构域的x射线晶体学信息来指导TR上突变的位置，以分析TR的作用机制。这种诱变方法已经允许我们定义LBD表面，以结合介导基因表达变化的下游共调节因子，并与类视黄醇x受体（RXR）形成TR-TR二聚体和异源二聚体。研究结果还确定了可能成为新型药物靶点的位点。在拟议的研究中，我们使用我们的大突变库和额外的拟议突变来确定：受体如何区分各种辅抑制因子；TR与辅激活剂（PGC-1）结合的新模式的分子基础；TR和RXR如何适应识别其DMA结合位点的不同方向；TH调节受体与DMA结合的机制；盐桥在非配体与配体模式下影响几种不同的TR功能的作用；以及新型配体如何通过在配体结合腔外的结构之间插入侧基来表现出结合特定TR异构体的选择性。这些研究将为理解TR的作用、其他nr的作用机制、基因表达调控和药物设计提供重要的信息。

Thyroid hormone (TH) signals are transduced by two related thyroid receptors (TRs) that regulate gene expression and are members of the nuclear receptor (NR) superfamily, which also includes receptors for steroids, vitamins and fatty acids and a variety of cholesterol and fatty acids and their metabolites. About 20% of current Pharmaceuticals are ligands that bind to NRs, underlining the importance of the NR family. TH regulates fat mass, cholesterol, heart and other functions; thus developing means to selectively regulate TR actions could lead to useful Pharmaceuticals with selective actions. We have utilized X-ray crystallographic information about the TR ligand-binding (LBD) and DMA-binding (DBD) domains to guide placements of mutations on the TR to analyze the mechanism of TR action. This mutagenesis approach has already allowed us to define LBD surfaces for binding downstream coregulators that mediate changes in gene expression, arid for forming TR-TR dimers and heterodimers with the retinoid X-receptor (RXR). The results also identified sites that might be targets for novel Pharmaceuticals. In the proposed studies we use our large bank of mutations and additional proposed mutations to determine: how receptors discriminate between various corepressors; the molecular basis of a novel mode of binding of TR to a coactivator (PGC-1); how the TR and RXR adapt to recognize diverse orientations of their DMA binding sites; mechanisms whereby TH regulates receptor binding to DMA; the role of salt bridges that influence several different TR functions in the unliganded vs. the liganded modes; and how a novel ligand exhibits selectivity for binding a particular TR isoform by inserting a side group between structures outside the ligand-binding cavity. These studies should provide substantial insight into TR function that is relevant for understanding TR action, mechanism of action of other NRs, regulation of gene expression and pharmaceutical design.