Conformational Analysis of the Progesterone Receptor

描述(申请人提供)：黄体酮是一种关键的生殖激素，它与核孕酮受体(PR)结合以调节它们的作用。PR有两种亚型，即85 kDa A受体(PR-A)和103 kDa B受体(PR-B)。它们除了在PR-B的N端有164个氨基酸延伸外，其余都是相同的。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性基因的基因芯片分析表明，这两种异构体的生物学活性不同。特别值得注意的是，人类乳腺癌表达的PR-A和PR-B的比例非常不同，而不同于正常乳腺的两个受体是等摩尔的。这项工作的目的是确定导致PR-A和PR-B功能差异的N-末端的结构元件。为此，将使用圆二色谱和荧光光谱等技术分析PR-A、PR-B和PR的功能改变突变体之间的构象差异。此外，免疫共沉淀试验将用于鉴定与PR-B的N-末端有差异结合的蛋白质，并评估辅助调节因子结合如何影响N-末端结构。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，是如何解释它们之间的功能差异的。

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.