Characteristics of a putative steroid membrane receptor

描述（由申请人提供）：长期目标是确定我们最近在人类细胞中发现的一种新的假定的膜孕酮（P4）受体（mPRs）家族介导的非经典类固醇作用的机制及其在健康和疾病中的功能意义。通过mPRa和mPRb介导的非经典P4信号通路将在人类肌细胞中进行研究。初步研究表明，这些mPRs在分娩过程中在人肌细胞中上调，激活多种抑制性g蛋白和第二信使通路，导致核孕酮受体（nPR）转录活性下调，以及其他有利于子宫内膜收缩的条件。因此，P4通过新的假定的mPRa和mPR(3)受体在人肌细胞中激活多种抑制性g蛋白/第二信使通路并调节nPR转录活性的假设将被验证。目的：(1)确定人野生型和重组型mPRa和mPRb的类固醇结合特性和定位。在存在或不存在用于mPRs的siRNA的情况下，孕激素与人肌细胞细胞膜的结合，以及与转染了人mPRs的哺乳动物细胞的结合将被检查；(2)研究mPRs与g蛋白的偶联及其相关的第二信使通路。g蛋白的偶联、它们的身份和第二信使的身份将在这些mPR细胞表达模型和子宫肌活检组织中被研究。(3)研究hmPRa的功能域对类固醇结合和g蛋白偶联的影响。通过建立药效团和受体模型，以及位点定向突变和功能分析，研究mPRa配体结合和g蛋白偶联所需的受体结构域。(4)探索通过mpr依赖通路调控nPR转录活性和共激活子功能。我们将使用几种报告基因分析和免疫方法研究nPR磷酸化和共激活因子的功能。早产是一个主要的医学问题，有12%的新生儿发生早产，但人类功能性黄体酮停药导致分娩的机制尚不清楚。本研究将描述以前未被认识到的多种信号级联反应，这些信号级联反应由黄体酮激活的mPRs启动，可能涉及足月功能性黄体酮停药，将平衡从静止状态转移到收缩状态。虽然许多具体的实验很难遵循，但似乎整个组织在广泛的战线上攻击这个问题，这样就会有很多关于受体的信息。可以说，这只是对许多受体所做的表征。然而，确定mPR的这些要点是很重要的。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。

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.