Characteristics of a putative steroid membrane receptor

假定的类固醇膜受体的特征

• 批准号: 7142649
• 负责人: PETER THOMAS
• 金额: $ 33.49万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7142649
• 关键词: G protein; chemical binding; genetic transcription; hormone regulation /control mechanism; myometrium; nuclear receptors; progesterone receptors; protein localization; protein structure function; receptor coupling; receptor expression; recombinant proteins; tissue /cell culture

DESCRIPTION (provided by applicant): Long term goals are to determine the mechanisms of nonclassical steroid actions mediated by a novel family of putative membrane progesterone (P4) receptors (mPRs) we recently discovered in human cells and their functional significance in health and disease. Nonclassical P4 signaling pathways mediated via mPRa and mPRb in human myocytes will be investigated. Preliminary studies suggest these mPRs are upregulated in human myocytes during labor and activate multiple inhibitory G-proteins and second messenger pathways, resulting in down-regulation of nuclear progesterone receptor (nPR) transcriptional activity as well other conditions favoring myometrial contraction at term. Therefore, the hypothesis that P4 acts via the novel putative mPRa and mPR(3 receptors in human myocytes to activate multiple inhibitory G-protein/second messenger pathways and to modulate nPR transcriptional activity will be tested. Aims are to: (1) Determine steroid binding characteristics and localization of human wild type and recombinant mPRa and mPRb. Binding of progestins to cell membranes from human myocytes in the presence or absence of siRNA for the mPRs, and to mammalian cells transfected with human mPRs will be examined; (2) Investigate coupling of the mPRs to G-proteins and their associated second messenger pathways. Coupling of G-proteins, their identities and identities of second messengers activated will be investigated in these mPR cell expression models and in myometrial biopsy tissues (3) Investigate functional domains of hmPRa for steroid binding and G-protein coupling. Receptor domains of mPRa required for ligand binding and G-protein coupling will be investigated by developing pharmacophore and receptor models, followed by site-directed mutagenesis and functional analyses. (4) Explore modulation of nPR transcriptional activity and co-activator functions via mPR-dependent pathways. Cross-talk will be investigated using several reporter assays and nPR phosphorylation and co-activator function by immunological methods. Preterm birth is a major medical problem, occurring with 12% of births, but the mechanism of a functional progestin withdrawal in humans resulting in the onset of labor is unclear, The present study will characterize previously unrecognized multiple signaling cascades initiated by progesterone activation of mPRs that are likely involved in functional progesterone withdrawal at term, shifting the balance from a quiescent state to a contractile one. Although many of the specific experiments were difficult to follow, it appears that the overall organization attacks the issue on a broad front, such that a lot of information about the receptors will be forthcoming. It could be argued that this is just the type of characterization that has been done with many receptors. However, it is important to establish these points for the mPR. In addition, the studies should provide indications of important regulatory events that might occur in human cells.

描述（由申请人提供）：长期目标是确定我们最近在人体细胞中发现的一种新型推定膜孕酮（P4）受体（mPR）家族介导的非经典类固醇作用机制及其在健康和疾病中的功能意义。将研究人心肌细胞中通过mPRa和mPRb介导的非经典P4信号通路。初步研究表明，这些mPR在分娩过程中在人肌细胞中上调，并激活多种抑制性G蛋白和第二信使途径，导致核孕酮受体（nPR）转录活性下调以及有利于足月子宫肌层收缩的其他条件。因此，将检验P4通过人肌细胞中新的推定mPR α和mPR β受体激活多种抑制性G蛋白/第二信使途径并调节nPR转录活性的假设。目的：（1）确定人野生型和重组mPRa和mPRb的类固醇结合特性和定位。将检查在存在或不存在针对mPR的siRNA的情况下孕激素与来自人肌细胞的细胞膜的结合，以及与用人mPR转染的哺乳动物细胞的结合;（2）研究mPR与G蛋白的偶联及其相关的第二信使途径。将在这些mPR细胞表达模型和子宫肌层活检组织中研究G蛋白的偶联、它们的身份和激活的第二信使的身份。（3）研究hmPR α的类固醇结合和G蛋白偶联的功能域。受体结构域的mPRa所需的配体结合和G-蛋白偶联将通过开发药效团和受体模型，然后通过定点诱变和功能分析进行研究。(4)探索通过mPR依赖性途径调节nPR转录活性和辅激活因子功能。将使用几种报告基因测定和免疫学方法研究nPR磷酸化和共激活因子功能。早产是一个主要的医学问题，发生在12%的出生，但在人类中的功能性孕酮撤退导致分娩的发生机制尚不清楚，本研究将表征以前未被识别的多个信号级联启动的孕酮激活的mPR可能参与功能性孕酮撤退在足月，从静止状态的平衡转移到收缩的。虽然许多具体的实验很难遵循，但似乎整个组织都在广泛的战线上攻击这个问题，因此关于受体的许多信息即将到来。可以说，这只是对许多受体进行的表征类型。然而，重要的是要为mPR建立这些点。此外，这些研究应该提供可能在人类细胞中发生的重要调节事件的指示。