Prostaglandin E2 (PGE2) is a major cyclooxygenase metabolite of arachidonic acid and a potent modulator of a wide variety of cellular responses including vascular tone, febrile response as well as water and ion transport in the kidney. There is now firm evidence that most of PGE2's effects are mediated via specific guanine nucleotide regulatory protein coupled receptors designated EP1, EP2, EP3, and EP4. The EP3 receptor is the unique among the EP receptor family in that it is represented by multiple alternatively spliced variants generated by alternative splicing from a single gene of a common precursor mRNA. The principal hypothesis of this proposal is that differential expression of EP3 receptor splice variants on individual cell types results in differential activation of signaling pathways that determine the physiologic response of a target cell to PGE2. Implicit in this hypothesis is the notion that the EP3 receptor variants have functionally distinct tissue distribution, cellular expression, and intracellular compartmentalization as well as distinct signal transduction properties. To test this hypothesis, in Specific Aim 1 we will determine the physiologic role of the EP3 receptor splice variants using an genetically modified mice strategy expressing a restricted repertoire of EP3 receptor splice variants. We will characterize phenotypic changes focusing on blood pressure, and vascular reactivity. Although the EP3 receptor has classically been characterized as a Gi coupled receptor that signals by lowering intracellular cAMP levels, recent studies suggest that EP3 alternative splice variants couple through other non-Gi signal transduction pathways.
In Specific Aim 2, studies will focus on the characterization of the EP3 receptor-mediated mechanism of cAMP independent signal transduction and its effect on gene transcription. We will examine the potential for this pathway to modulate gene expression using specific inhibitors and phosphorylation-specific antibodies in Western blot analysis, and promoter analysis.
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