Hormonal Regulation of Phosphodiesterases

描述(由申请人提供)：支持本建议的假设是磷酸二酯酶，即降解cAMP的酶，在cAMP信号转导中起关键作用。它们控制着cAMP瞬变的空间和时间维度，因此决定了激素刺激的特异性和结果。在过去的资助期间，在体外和体内研究了cAMP特异性磷酸二酯酶(PDE4)在激素诱导的环核苷酸信号转导中的作用。这些研究表明，PDE参与了内分泌细胞内cAMP水平的反馈调节。利用遗传学方法，进一步证明了体内PDE4的消融和反馈调节的失活扰乱了环核苷酸信号，并导致了激素依赖过程的广泛变化，包括基因表达的变化。消融PDE4影响生长、生育、免疫和中枢神经系统反应。最后，证实了PDE4与支架蛋白组装的PKA形成信号复合体，并且这些支架靶向于不同的亚细胞位置。这些研究将通过研究PDE4缺失小鼠表型变化的生化机制以及PDE4靶向在激素反应中的作用来扩展。这些实验是按照三个具体目标组织的。在第一个特定目标中，将通过颗粒细胞培养来研究导致PDE4D基因缺失小鼠排卵和卵泡功能受损的生化变化。此外，还将利用PDE4D缺陷小鼠在体内研究颗粒细胞基因的表达模式。第二个具体目标将致力于了解PDE4D和PDE4B基因在功能上的差异。这些实验将使用PDE4缺陷细胞系的重建实验。在最后一个具体目标中，将对PDE4靶向的机制和意义进行研究。PDE4与支架蛋白的相互作用将进一步研究，PDE/PKA信号复合体的功能将在无细胞系统和完整细胞中进行研究。最后，通过研究受体内化和通道功能，将在重建系统中评估靶向PDE4的影响。这些研究将使我们能够确定PDE在信号特异性和区隔中的作用。开发的概念将为研究PDE功能障碍在人类疾病中的作用提供基础，并为使用PDE4选择性抑制剂对内分泌疾病进行药物干预提供理论基础。

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.