Differentiation of granulosa cells from an immature to a mature phenotype is regulated by FSH-stimulated activation of cAMP-dependent protein kinase (PKA). While some early and many late response genes have been identified downstream of PKA in immature granulosa cells, the signaling pathways beyond PKA which lead to activation of these genes are poorly understood. We have determined in immature cells that FSH-activated PKA directly phosphorylates the nuclear protein histone H3 and that PKA promotes the unique activation of both the p38 and p42/44 mitogen activated protein kinase (MAPK) pathways; these pathways are inhibited by cAMP in most other cells. Activation of PKA in mature granulosa cells (by LH) similarly promotes activation of the p42/44 MAPK pathway and phosphorylation of histone 3, but as shown by others, leads to down-regulation of some of the late response genes activated in immature granulosa cells and up- regulation of other proteins. Recent studies suggest that the cellular location of PKA (a) is a critical determinant for phosphorylation of appropriate target proteins and (b) is regulated by association with A- kinase anchoring proteins (AKAPs). We hypothesize that the unique responses of granulosa cells to PKA, like MAPK activation, and distinct responses between immature versus mature cells are mediated in part by specific expression of one or more AKAPs. In support of this hypothesis, we have demonstrated that FSH promotes the induction of an 80 kDA AKAP which preferentially binds RIIalpha, and that expression of AKAP 80 promotes the redistribution of PKAIIalpha in mature granulosa cells.
Aims of this proposal will test the hypotheses that actions of FSH in immature granulosa cells require anchoring of PKA to AKAPs; that the FSH-inducible 80 kDA AKAP is a unique protein which co-localizes with PKAIIalpha in preovulatory granulosa cells; and that AKAP 80 is required for PKA to activate responses characteristic of mature granulosa cells; This basic knowledge of how cAMP/PKA signals in granulosa cells is expected to provide insights towards understanding pathways which regulate ovulation and luteinization and which can translate into safer and more effective treatments of infertility and early pregnancy loss.
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