Function of MAP2D in Granulosa Cells

描述(申请人提供)：卵泡成熟是一个动态的过程，需要精细的调控。卵泡在腔前(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受体来传递信号。颗粒细胞对卵泡刺激素(FSH)的反应是增殖和分化为成熟的表型，能够对黄体生成素(LH)的排卵前(PO)激增作出反应。然后，黄体生成素作用于这些PO细胞，激活PKA，促进颗粒细胞向黄体细胞的终末分化，停止细胞增殖和排卵。虽然FSH和LH都是激活PKA的信号，但在PA和PO卵泡的颗粒细胞中，PKA引导反应的途径既相似又不同。例如，卵泡刺激素和黄体生成素都通过选择性PKA抑制素肉豆蔻酰化(Myr-)PKI阻断的途径促进组蛋白H3的磷酸化。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并不促进PA细胞中ERK上游激酶MEK的激活，而LH促进PO细胞中MEK的激活。此外，在PO细胞中，还有许多受FSH调节的基因，如抑制素α、芳香化酶和LHR等，它们在PA中的调节与在PO细胞中的LH相反。PKA的特异性作用被认为是通过与A激酶锚定蛋白(AKAP)结合，将PKA及其底物靶向于特定的细胞位置。我们已经证明，PO卵泡的颗粒细胞与PA卵泡的颗粒细胞是不同的，因为存在一个80 kDa的AKAP，我们最近发现它是微管相关蛋白(MAP)2D。我们假设MAP2D在PO颗粒细胞中起信号支架的作用，将来自PKA的黄体生成素信号引导到一组不同的底物上。Aim 1将测试MAP2D作为支架的功能以协调PO颗粒细胞中的信号转导途径的假设；Aim 2将测试MAP2D对于黄体生成素在PO颗粒细胞中的信号选择是必需的假设；Aim 3将测试MAP2D不是相关微管以及MAP2D在特定位置的磷酸化调节其与PKA关联的假设；以及Aim 4将测试MAP2D是RL偏好的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.