Function of MAP2D in Granulosa Cells

描述（由申请人提供）：卵巢卵泡成熟是一个动态过程，需要精细的调节。卵泡在窦前（PA）阶段被抑制，直到它们被卵泡刺激素（FSH）刺激。FSH通过cAMP及其靶蛋白激酶A（PKA）激活颗粒细胞表面的FSH受体来发出信号。为了响应FSH，颗粒细胞增殖并分化为成熟的表型，能够对促黄体生成素（LH）的排卵前（PO）激增做出反应。然后LH作用于这些PO细胞，激活PKA，促进颗粒终末分化为黄体细胞，停止细胞增殖和排卵。虽然FSH和LH信号激活PKA，PKA指导反应的途径在PA与PO卵泡的颗粒细胞中既相似又不同。例如，FSH和LH均通过被选择性PKA抑制剂肽豆蔻酰化（Myr-）PKI阻断的途径促进组蛋白H3磷酸化。然而，虽然FSH和LH都以PKA依赖性方式促进ERK的活化（被Myr-PKI阻断），但FSH不促进PA细胞中上游ERK激酶MEK的活化，而LH促进PO细胞中MEK的活化。此外，有许多基因在PA中受FSH的调节与在PO细胞中受LH的调节是相反的，如α-胡萝卜素、芳香酶和LHR。PKA作用的特异性被认为是通过PKA及其底物与A激酶锚定蛋白（AKAP）结合而靶向特定细胞区域而发生的。我们已经表明，颗粒细胞的PO卵泡是区别于PA卵泡的存在下，我们最近确定为微管相关蛋白（MAP）2D的80 kDa AKAP。我们假设MAP 2D在PO颗粒细胞中作为一个信号传导支架，将LH信号从PKA引导到一组不同的底物。目的1验证MAP 2D在PO颗粒细胞中作为支架协调信号转导通路的假设，目的2验证MAP 2D是LH在PO颗粒细胞中选择信号转导通路的必需条件的假设，目的3验证MAP 2D与微管无关，MAP 2D在特定位点的磷酸化调节其与PKA的结合的假设，目的4验证MAP 2D与微管蛋白的结合。并且目标4将检验MAP 2D是RI-优先AKAP的假设。了解卵泡成熟是如何调节的可以转化为更安全，更有效的生育治疗。

DESCRIPTION (provided by applicant): Ovarian follicular maturation is a dynamic process that requires exquisite regulation. Follicles are restrained at the preantral (PA) stage until they are stimulated by follicle stimulating hormone (FSH). FSH signals by activating its receptor on the surface of granulosa cells via cAMP and its target protein kinase A (PKA). In response to FSH, granulosa cells proliferate and differentiate to a mature phenotype competent to respond to the preovulatory (PO) surge of luteinizing hormone (LH). LH then acts on these PO cells to activate PKA to promote terminal differentiation of granulosa into luteal cells, cessation of cell proliferation, and ovulation. While both FSH and LH signal to activate PKA, the pathways by which PKA directs responses are both similar and different in granulosa cells of PA versus PO follicles. For example, both FSH and LH promote histone H3 phosphorylation by a pathway that is blocked by the selective PKA inhibitor peptide myristoylated (Myr-) PKI. However, while both FSH and LH promote activation of ERK in a PKA-dependent manner (blocked by Myr- PKI), FSH does not promote activation of the upstream ERK kinase MEK in PA cells while LH promotes activation of MEK in PO cells. Additionally, there are numerous genes whose regulation by FSH in PA versus LH in PO cells is opposite, such as inhibin-alpha, aromatase, and LHR. The specificity of PKA action is thought to occur by the targeting of PKA and its substrates to specific cellular locales by virtue of its binding to A kinase anchoring proteins (AKAPs). We have shown that granulosa cells of PO follicles are distinguished from those of PA follicles by the presence of an 80 kDa AKAP that we recently identified as microtubule associated protein (MAP) 2D. We hypothesize that MAP2D functions as a signaling scaffold in PO granulosa cells to direct LH signals from PKA to a distinct set of substrates. Aim 1 will test the hypothesis that MAP2D functions as a scaffold to coordinate signal transduction pathways in PO granulosa cells; Aim 2 will test the hypothesis that MAP2D is obligatory for LH to signal to select pathways hi PO granulosa cells; Aim 3 will test the hypothesis that MAP2D is not associated microtubules and that the phosphorylation of MAP2D at specific sites regulates its association with PKA; and Aim 4 will test, the hypothesis that MAP2D is an Rl-preferential AKAP. Understanding how follicular maturation is regulated can translate into safer and more effective treatments for fertility.