Hormonal Regulation of Phosphodiesterases

描述（由申请人提供）：本提议的假设是磷酸二酯酶（降解cAMP的酶）在cAMP信号传导中具有关键作用。它们控制cAMP瞬变的空间和时间维度，因此定义了激素刺激的特异性和结果。在过去的资助期间，cAMP特异性磷酸二酯酶（PDE4）在激素环核苷酸信号转导中的作用进行了体外和体内研究。这些研究表明PDE参与内分泌细胞cAMP水平的反馈调节。使用遗传学方法，进一步证明了体内PDE4的消融和反馈调节的失活破坏了环核苷酸信号传导，并导致了包括基因表达变化在内的依赖性过程的广泛改变。PDE4的消融影响生长、生育力以及免疫和CNS反应。最后，已经确定PDE4与由支架蛋白组装的PKA形成信号复合物，并且这些支架将PDE4靶向不同的亚细胞位点。这些研究将通过研究PDE4缺失小鼠表型变化的生化机制和PDE4靶向在激素反应中的作用来扩展。这些实验是按照沿着三个具体目标组织的。在第一个特定目的中，将使用颗粒细胞培养物研究PDE 4D敲除小鼠中诱导排卵和卵泡功能受损的生化变化。此外，将使用PDE4D缺陷小鼠在体内研究颗粒细胞基因表达模式。第二个具体目标将致力于了解PDE4D和PDE4B基因之间的功能差异。使用PDE 4缺陷细胞系的复溶实验将用于这些实验。在最后的具体目标中，将研究PDE4靶向的机制和意义。将进一步表征PDE 4与支架蛋白的相互作用，并在无细胞系统和完整细胞中研究PDE/PKA信号复合物的功能。最后，通过研究受体内化和通道功能，在重建系统中评估靶向PDE 4的影响。这些研究将使我们能够确定PDE在信号特异性和区室化中的作用。开发的概念将提供PDE功能障碍在人类疾病中的作用的研究的基础和使用PDE 4-选择性抑制剂的内分泌疾病的药理学干预的理由。

DESCRIPTION (provided by applicant): The hypothesis underlying the present proposal is that phosphodiesterases, the enzymes that degrade cAMP, have a critical role in cAMP signaling. They control the spatial and temporal dimensions of the cAMP transients, therefore defining the specificity and outcome of the hormone stimulation. During the past funded period, the role of the cAMP-specific phosphodiesterases (PDE4s) in cyclic nucleotide signaling by hormones was investigated in vitro and in vivo. These studies show that PDEs are involved in the feedback regulations of cAMP levels in endocrine cells. Using a genetic approach, it was further demonstrated that ablation of PDE4 and inactivation of the feedback regulation in vivo disrupts cyclic nucleotide signaling and results in broad alterations of hormone-dependent processes including changes in gene expression. Ablation of PDE4 affects growth, fertility, and immune and CNS responses. Finally, it was established that PDE4s form signaling complexes with PKAs assembled by scaffold proteins, and that these scaffolds target PDE4 to different subcellular sites. These studies will be extended by investigating the biochemical mechanisms underlying the phenotypic changes of the PDE4 null mice and the role of PDE4 targeting in hormone responses. The experiments are organized along three Specific Aims. In the first Specific Aim, the biochemical changes that induce impaired ovulation and follicle function in the PDE4D null mice will be studied using granulosa cell cultures. In addition, the pattern of granulosa cell gene expression will be investigated in vivo using the PDE4D-deficient mice. The second Specific Aim will be devoted to understanding the differences in function between the PDE4D and PDE4B genes. Reconstitution experiments using PDE4-deficient cell lines will be used for these experiments. In the last Specific Aim, the mechanism and significance of PDE4 targeting will be investigated. The interaction of PDE4 with scaffold proteins will be further characterized and the function of PDE/PKA signaling complexes will be studied in cell-free systems and in intact cells. Finally, the impact of targeting PDE4 will be evaluated in a reconstitution system by investigating receptor internalization and channel functions. These studies will allow us to determine the role of PDEs in signal specificity and compartmentalization. The concepts developed will provide the basis for studies on the role of PDE dysfunction in human diseases and a rationale for pharmacological intervention in endocrine disorders using PDE4-selective inhibitors.