Conformational Analysis of the Progesterone Receptor

描述（由申请人提供）：黄体酮是一种关键的生殖激素，7与核黄体酮受体（PR）结合，介导其作用。PR有两种亚型，85 kDa a受体（PR- a）和103 kDa b受体（PR- b）。除了PR-B的n端有164个氨基酸延伸外，它们是相同的。虽然PR-A和PR-B与激素和DNA的结合相似，但体外转录研究和内源基因的cDNA阵列分析表明，这两种亚型的生物活性存在差异。特别值得注意的是，人类乳腺癌表达PR-A和PR-B的比例差异很大，这与正常乳腺中这两种受体是等摩尔的情况不同。这项工作的目的是确定PR-A和PR-B的n端结构元件，这些结构元件导致了它们的功能差异。为此，将使用圆二色性和荧光光谱等技术对PR- a、PR- b和PR的功能改变突变体之间的构象差异进行分析。此外，共免疫沉淀法将用于鉴定与PR-B的n端不同结合的蛋白质，并评估共调节因子结合如何影响n端结构。这些研究的成功完成将解释两种PR之间的结构差异以及与受体相互作用的蛋白质之间的差异如何解释它们之间的功能差异。

DESCRIPTION (provided by applicant): Progesterone is a key reproductive hormone that7 binds to nuclear progesterone receptors (PR) to mediate their effects. There are two isoforms of PR, 85 kDa A-receptors (PR-A) and 103 kDa B-receptors (PR-B). They are identical except for a 164 amino acid extension at the N-terminus of PR-B. Although the binding of PR-A and PR-B to hormone and DNA are similar, in vitro transcriptional studies and cDNA array analyses of endogenous genes have demonstrated differences in the biological activities of these two isoforms. Of particular note is the fact that human breast cancers express widely differing ratios of PR-A vs. PR-B, unlike the normal breast in which the two receptors are equimolar. The goal of this work is to define the structural elements in the N-termini of PR-A and PR-B that that give rise to their functional differences. To do so, an analysis of the conformational differences between PR-A, PR-B, and functionally-altered mutants of PR will be performed, using techniques such as circular dichroism and fluorescence spectroscopy. Additionally, co-immunoprecipitation assays will be used to identify proteins that bind differentially to the N-terminus of PR-B and assess how coregulator binding affects the N-terminal structure. Successful completion of these studies will explain how structural differences between the two PR, and differences among the proteins that interact with the receptors, account for the function differences between them.