Mutation Analysis of Thyroid Hormone Function

甲状腺激素(TH)信号由两个调节基因表达的相关甲状腺受体(TRs)传递，是核受体(NR)超家族的成员，该超家族还包括类固醇、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。目前约有20%的药物是与NRs结合的配体，这突显了NR家族的重要性。TH调节脂肪质量、胆固醇、心脏和其他功能；因此，开发有选择性地调节TRR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TRL配体结合(LBD)和DMA结合(DBD)结构域的X射线结晶学信息来指导突变在TRR上的定位，以分析TRR的作用机制。这种突变方法已经使我们能够定义LBD表面，用于结合介导基因表达变化的下游辅助调节因子，并与维甲酸X受体(RXR)形成TR-TR二聚体和异源二聚体。结果还确定了可能成为新型药物靶点的位置。在拟议的研究中，我们使用我们的大量突变和其他建议的突变来确定：受体如何区分不同的辅抑制子；TR与辅活化子(PGC-1)的一种新的结合模式的分子基础；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；盐桥在未连接模式与连接模式中影响几种不同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显示结合特定TR亚型的选择性。这些研究应该为深入了解转录激活剂的作用、其他核受体的作用机制、基因表达调控和药物设计提供实质性的见解。

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.