METABOLIC EFFECTS OF THYROID HORMONE

甲状腺激素受体(c-Erbas)和维甲酸受体(RARs) 是亲缘关系密切的核受体蛋白亚群的成员。 C-Erbas和RAR各自可以激活某些反应元件，并且两者 受体含有一个嵌入在配体内的高度保守的结构域 结合区含有一系列“亮氨酸拉链样”疏水物质 七元图案。功能研究表明，七肽重复结构域 介导c-erba和RAR的同源和异源二聚体相互作用 与其他因素的相互作用。鸡c-erba-α和人RAR-α 已经在大肠杆菌中表达，并纯化到接近均一的水平。这些 受体以适当的亲和力结合配体，形成同源异构体 允许二聚化的响应元件上的杂二聚体。 配体促进了二聚体的形成，提示配体介导 通过这种机制进行转录激活。此应用程序是一个 全面的建议书，以定义 转录激活中的c-erba、RAR及相关因子。为 这些研究我们构建了多功能细菌/真核生物 表达载体(PEXPRESS)，允许定点突变和 可用于对真核细胞中的受体进行功能分析和 在大肠杆菌中高效表达受体。凝胶漂移研究使用 纯化的野生型和突变型受体蛋白以及多种天然的 并提出了合成反应要素来解释规则。 它控制着这些受体如何识别功能反应元件。 这些研究还将定义对Homo-And至关重要的受体结构域 异源二聚体的形成，用于受体之间的协同作用 和其他因素，以及占主导地位的消极活动。可能的 C-erba亚型(字母和贝塔尔)对元素识别的差异 也将与了解差异的研究一起进行检查 V-erba和c-erba之间的关系以及c-erba-alpha2如何发挥支配作用 负调节器。这些研究将通过分析 来自甲状腺激素患者的受体突变 耐药综合征。哺乳动物细胞的功能研究将是 扩展到体外转录，目的是定义蛋白质 以及这些受体转录增强所需的DNA。 最后，对纯化后的大肠杆菌中镁的利用率进行了研究 野生型c-erba及其DNA结合域将允许循环 二向色性、荧光和紫外可见光谱(两者 蛋白质)和核磁共振研究(DNA结合结构域)以 提供结构信息以阐明功能和物理 这些蛋白质在分子水平上的性质。

Thyroid hormone receptors (c-erbAs) and retinoic acid receptors (RARs) are members of a subgroup of closely related nuclear receptor proteins. c-erbAs and RAR can each activate certain response elements and both receptors contain a highly conserved domain embedded within the ligand binding region containing a series of "leucine-zipper-like" hydrophobic heptad motifs. Functional studies suggest that the heptad repeat domain mediates homo- and heterodimeric interactions of c-erbA and RAR or interactions with other factors. Chick c-erbA-alpha and human RAR-alpha have been expressed in E. coli and purified to near homogeneity. These receptors bind ligand with appropriate affinity and form homo- and hetero-dimers on response elements which are permissive for dimerization. Dimer formation is enhanced by ligand suggesting that ligand mediates transcriptional activation by this mechanism. This application is a comprehensive proposal to define the functional domains involved in c-erbA, RAR, and related factors in transcriptional activation. For these studies we constructed a multifunctional bacterial/eucaryotic expression vector (pEXPRESS) which permits site directed mutagenesis and can be used to functionally analyze receptor in eucaryotic cells and to express receptor at high levels in E. coli. Gel shift studies using purified wild-type and mutant receptor proteins and a variety of native and synthetic response elements are proposed to elucidate the "rules" which govern how these receptors recognize functional response elements. These studies will also define receptor domains critical for homo- and hetero-dimer formation, for cooperative interactions between receptors and other factors, and for dominant negative activities. Possible differences in element recognition by c-erbA subtypes (alphal and betal) will also be examined along with studies to understand differences between v-erbA and c-erbA and how c-erbA-alpha2 functions as a dominant negative regulator. These studies will be complemented by analyzing receptor mutants derived from patients with the thyroid hormone resistance syndrome. Functional studies in mammalian cells will be extended to in vitro transcription with the goal of defining the protein and DNA requirements for transcriptional enhancement by these receptors. Finally, the availability of mg amounts of purified E. coli expressed wild-type c-erbA and its DNA binding domain will allow circular dichroism, fluorescence, and ultraviolet-visible spectroscopy (both proteins), and nuclear magnetic resonance studies (DNA binding domain) to provide structural information to elucidate the functional and physical properties of these proteins at the molecular level.