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.
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