METABOLIC EFFECTS OF THYROID HORMONE

甲状腺激素受体（c-erbAs）和视黄酸受体（RARs） 是密切相关的核受体蛋白亚组的成员。 c-erbA和RAR可以各自激活某些应答元件，并且两者都可以激活。 受体包含嵌入配体中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 七肽基序。 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，或 与其他因素的相互作用。 鸡c-erbA-α和人RAR-α 在E. coli中，并纯化至接近均一。 这些 受体以适当的亲和力结合配体，并形成同源和 允许二聚化的应答元件上的异源二聚体。 配体促进二聚体形成，表明配体介导 通过这种机制激活转录。 本申请是 关于界定所涉职能领域的综合建议 c-erbA、RAR及相关因子在转录激活中的作用。 为 这些研究我们构建了一个多功能的细菌/真核生物 表达载体（pEXPRESS），其允许定点诱变， 可用于真核细胞中受体的功能分析， 在E.杆菌 凝胶位移研究，使用 纯化的野生型和突变型受体蛋白以及多种天然的 并提出了综合反应元素来阐明“规则” 控制这些受体如何识别功能性反应元件。 这些研究还将确定对人类和哺乳动物的重要受体结构域。 异源二聚体形成，用于受体之间的协同相互作用 和其他因素，以及占主导地位的负面活动。 可能 c-erbA亚型（β 1和β 1）识别元件的差异 也将与研究一起进行沿着，以了解 以及c-erbA-alpha 2如何作为一种显性基因发挥作用 负调节器 这些研究将通过分析 甲状腺激素受体突变体 抵抗综合征 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 以及这些受体对转录增强的DNA需求。 最后，对mg量的纯化E.杆菌表达 野生型c-erbA及其DNA结合结构域将允许环状 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结合域）， 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特性。

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.