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. ? ? ?
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