Function of MAP2D in Granulosa Cells

描述（申请人提供）：卵巢卵泡成熟是一个动态的过程，需要精细的调节。卵泡在腹前（PA）阶段被抑制，直到它们被促卵泡激素（FSH）刺激。FSH信号通过cAMP及其靶蛋白激酶A （PKA）激活颗粒细胞表面的受体。在对FSH的反应中，颗粒细胞增殖并分化为成熟的表型，能够响应排卵前（PO）促黄体生成素（LH）的激增。然后，LH作用于这些PO细胞，激活PKA，促进颗粒最终分化为黄体细胞，停止细胞增殖和排卵。虽然FSH和LH都发出信号激活PKA，但PKA指导反应的途径在PA和PO滤泡颗粒细胞中既相似又不同。例如，FSH和LH通过被选择性PKA抑制剂多肽肉豆蔻酰基化（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依赖的方式促进ERK的激活（被Myr- PKI阻断），但FSH不促进PA细胞中上游ERK激酶MEK的激活，而LH促进PO细胞中MEK的激活。此外，在PO细胞中，FSH对PA和LH的调控是相反的，如抑制素- α、芳香化酶和LHR。PKA作用的特异性被认为是通过其与A激酶锚定蛋白（AKAPs）的结合将PKA及其底物靶向到特定的细胞位置而发生的。我们已经证明，PO卵泡的颗粒细胞与PA卵泡的颗粒细胞是通过80 kDa的AKAP的存在来区分的，我们最近将其鉴定为微管相关蛋白（MAP） 2D。我们假设MAP2D在PO颗粒细胞中作为信号支架，将LH信号从PKA引导到一组不同的底物。目的1将验证MAP2D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将验证MAP2D是LH向PO颗粒细胞信号选择途径所必需的假设；目的3将验证MAP2D与微管无关的假设，以及MAP2D在特定位点的磷酸化调节其与PKA的关联；和Aim 4将测试MAP2D是一个rl优先的AKAP的假设。了解卵泡成熟是如何被调节的，可以转化为更安全、更有效的生育治疗方法。

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.