Characteristics of a putative steroid membrane receptor

描述(申请人提供)：长期目标是确定由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(MPR)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。我们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在分娩期间在人的心肌细胞中上调，并激活多个抑制性G蛋白和第二信使途径，导致核孕酮受体(NPR)转录活性下调，以及其他有利于足月子宫肌层收缩的条件。因此，P4通过人类心肌细胞中新的mPRa和MPR(3个受体)激活多个抑制性G蛋白/第二信使通路并调节NPR转录活性的假说将得到验证。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。(2)研究MPR与G蛋白及其相关的第二信使通路的偶联。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hmPRa与类固醇结合和G蛋白偶联的功能域。配体结合和G蛋白偶联所需的mPRa受体结构域将通过开发药效团和受体模型来研究，随后将进行定点突变和功能分析。(4)探索MPR依赖的途径对NPR转录活性和共激活蛋白功能的调控。串扰将使用几种报告分析方法和NPR磷酸化和共激活功能的免疫学方法进行研究。早产是一个主要的医学问题，有12%的新生儿发生早产，但人类功能性孕激素退出导致分娩开始的机制尚不清楚，本研究将表征先前未知的由孕酮激活的MPR启动的多个信号级联，这些信号级联可能与足月功能性孕酮退出有关，将平衡从静止状态转变为收缩状态。尽管许多具体的实验很难跟踪，但整个组织似乎从广泛的角度来攻击这个问题，因此关于受体的许多信息将会出现。可以争辩说，这正是许多受体所做的表征类型。然而，为MPR确立这些要点是很重要的。此外，这些研究应该提供可能发生在人类细胞中的重要调控事件的迹象。

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.