Mechanistic Studies of Progesterone Receptor Function

黄体酮受体功能的机制研究

• 批准号: 7010717
• 负责人: DAVID L BAIN
• 金额: $ 22.56万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-03-15 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7010717
• 关键词: Mechanistic; Studies; Progesterone; Receptor; Function

DESCRIPTION (provided by applicant): The long-term goal of this research is to elucidate the molecular mechanisms underlying eukaryotic gene regulation. Focus is centered on the mechanisms by which human progesterone receptors (PR) cooperatively bind to complex promoters, and the role of hormone agonists and antagonists in regulating these reactions. A further goal is to determine the principles by which the associated structural transitions are propagated to neighboring domains. PR co-exist as two functionally distinct isoforms: an 83 kD A-receptor and a 99 kD B-receptor. The two isoforms are identical, except that the B-receptor has an additional 164 amino acids at its N-terminus. The B-receptor often functions as a strong transcriptional activator, while the A-receptor generally acts as a weak activator. It is hypothesized that this difference arises through the ability of the B-receptor to bind cooperatively at PR-regulated promoters. Mechanistically, the B-unique residues impose a hormone-dependent conformational constraint upon the remainder of the receptor. This constraint causes changes in PR structure and stability changes that can include dramatic disorder-order transitions, resulting in cooperative DNA binding. It is proposed that a role of antagonists is to decouple these linkages by stabilizing ineffective conformations within the PR hormone-binding domain. This hypothesis, and the underlying mechanism, will be examined by carrying out the following studies: Aim 1 - The energetics of self-assembly for both isoforms in the presence and absence of progestin agonists and antagonists will be determined using analytical ultracentrifugation. Aim 2 - A rigorous thermodynamic analysis of the interactions of each PR isoform with the multi-site mouse mammary tumor virus promoter will be determined using quantitative DNAse footprinting. Aim 3 - The changes in isoform structure and stability associated with ligand and DNA-binding will be mapped using hydroxyl radical proteolytic footprinting, CD spectroscopy and microcalorimetry.

描述（由申请人提供）：本研究的长期目标是阐明真核基因调控的分子机制。重点是集中在人类孕酮受体（PR）合作结合复杂的启动子的机制，以及激素激动剂和拮抗剂在调节这些反应的作用。另一个目标是确定相关结构转换传播到相邻域的原则。PR作为两种功能不同的同种型共存：83 kD A受体和99 kD B受体。这两种亚型是相同的，除了B-受体在其N-末端具有额外的164个氨基酸。B-受体通常作为强转录激活剂发挥作用，而A-受体通常作为弱激活剂发挥作用。据推测，这种差异是通过B受体在PR调节的启动子处协同结合的能力而产生的。从机制上讲，B-独特的残基对受体的其余部分施加了依赖于构象的约束。这种约束导致PR结构的变化和稳定性的变化，其中可能包括戏剧性的无序-有序转变，导致合作DNA结合。有人提出，拮抗剂的作用是通过稳定PR酶结合结构域内的无效构象来解耦这些连接。将通过进行以下研究来检验这一假设和潜在的机制：目的1 -使用分析性超离心来确定在存在和不存在胱抑素激动剂和拮抗剂的情况下两种亚型的自组装的能量学。目的2 -使用定量DNA酶足迹法对每个PR亚型与多位点小鼠乳腺肿瘤病毒启动子的相互作用进行严格的热力学分析。目的3 -异构体结构和稳定性的变化与配体和DNA结合将映射使用羟基自由基蛋白水解足迹，CD光谱和微量热法。