Novel conformation-based nuclear receptor activity assay

基于新型构象的核受体活性测定

• 批准号: 6691855
• 负责人: JOHN ARCHDEACON
• 金额: $ 9.95万
• 依托单位: ACTIVE MOTIF, INC.
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-08-01 至 2004-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6691855
• 关键词: binding proteins; conformation; estrogen receptors; hormone receptor; ligands; method development; nuclear receptors; receptor binding; receptor expression; retinoid binding proteins; technology /technique development; transcription factor; vitamin D receptors

DESCRIPTION (provided by applicant): The objective of this proposal is the development of novel methods for the study of the Class II Nuclear Receptors Thyroid hormone receptor (TR), Retinoic Acid Receptor (RAR), and Vitamin D3 Receptor (VDR). Like other members of the nuclear receptor superfamily, these transcription factors directly activate or repress target genes by binding to DNA response elements in the regulatory regions of target genes. A ligand-mediated conformational change in the nuclear receptor serves as the basic mechanism for switching gene repression to activation. Liganded receptors have a high affinity for proteins that activate transcription (co-activators), while un-liganded receptors have a high affinity for transcriptional repressors. Unlike many of the members of the nuclear receptor superfamily, un-liganded TR, RAR, and VDR are not sequestered in the cytoplasm via an interaction with HSP90. In the presence or absence of ligand, these proteins are bound to their response elements on nuclear DNA as heterodimers with Retinoid X Receptor (RXR). In addition, there are no reliable post-translation modifications (such as acetylation or phosphorylation) associated with activation of these receptors. Thus, there are no currently available methods to rapidly quantify ligand-mediated conversion of TR, RAR, and VDR from transcription repressors to transcriptional activators. Recent studies have identified the peptide sequence (LXXLL) of the transcriptional activator SCR-1, which is responsible for this protein's ligand-dependent interaction with nuclear receptors. In this study, we will use this information to develop a peptide-based assay for the quantification of active-conformation receptors contained in nuclear extracts. Further, the peptide sequence (LXX(I/H)IXXX(I/L)) of the transcriptional repressor SMRT (silencing mediator for retinoid and thyroid receptors) responsible for its interaction with non-liganded nuclear receptor has been identified. We will use this peptide to develop an assay to quantify receptors that is in the non-activating conformation.

描述（由申请人提供）：本提案的目的是开发研究 II 类核受体甲状腺激素受体 (TR)、视黄酸受体 (RAR) 和维生素 D3 受体 (VDR) 的新方法。与核受体超家族的其他成员一样，这些转录因子通过与靶基因调控区域中的 DNA 反应元件结合，直接激活或抑制靶基因。核受体中配体介导的构象变化是将基因抑制转变为激活的基本机制。配体受体对激活转录的蛋白质（共激活因子）具有高亲和力，而未配体受体对转录阻遏蛋白具有高亲和力。与核受体超家族的许多成员不同，未配体的 TR、RAR 和 VDR 不会通过与 HSP90 相互作用而隔离在细胞质中。在存在或不存在配体的情况下，这些蛋白质以类视黄醇 X 受体 (RXR) 的异二聚体形式与核 DNA 上的响应元件结合。此外，不存在与这些受体的激活相关的可靠的翻译后修饰（例如乙酰化或磷酸化）。因此，目前没有可用的方法来快速定量配体介导的 TR、RAR 和 VDR 从转录抑制因子到转录激活因子的转化。最近的研究已经确定了转录激活剂 SCR-1 的肽序列 (LXXLL)，该序列负责该蛋白与核受体的配体依赖性相互作用。在本研究中，我们将利用这些信息开发一种基于肽的测定方法，用于定量核提取物中所含的活性构象受体。此外，还鉴定了负责与非配体核受体相互作用的转录阻遏物SMRT（类维生素A和甲状腺受体的沉默介体）的肽序列（LXX(I/H)IXXX(I/L)）。我们将使用这种肽来开发一种测定方法来量化处于非激活构象的受体。