Mechanistic Studies of Progesterone Receptor Function

这项研究的长期目标是阐明真核生物中的分子机制， 基因调控焦点集中在人类孕酮受体 (PR)协同结合到复杂的启动子，激素激动剂和拮抗剂的作用， 调节这些反应。另一个目标是确定相关的原则， 结构转变被传播到相邻域。公共关系作为两种功能共存 不同的同种型：83 kD A受体和99 kD B受体。两种亚型相同 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因子，而A受体通常作为弱转录激活因子。 活化剂。据推测，这种差异是通过B受体结合的能力而产生的。 在PR调节的启动子上协同作用。从机制上讲，B独特残基施加了一个 对受体其余部分的构象依赖性约束。此约束 导致公关结构和稳定性的变化-变化可能包括戏剧性的无序 转换-导致合作DNA结合。提出拮抗剂的作用是 通过稳定PR酶结合内的无效构象， 域这一假设和潜在的机制将通过执行 以下研究：目的1 -两种异构体在存在下的自组装的能量学， 将使用分析的方法确定是否存在促肾上腺皮质激素激动剂和拮抗剂 超离心目的2 -严格的热力学分析每个PR的相互作用 将使用以下方法确定具有多位点小鼠乳腺肿瘤病毒启动子的同种型 定量DNA酶足迹法。目的3 -异构体结构和相关稳定性的变化 与配体和DNA结合将映射使用羟基自由基蛋白水解足迹，CD 光谱学和微量热法。

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 goat 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.