Although cyclic nucleotide phosphodiesterases (PDEs) were described soon after the discovery of cAMP, their complexity and functions in signaling is only recently beginning to become fully realized. We now know that at least 100 different PDE proteins degrade cAMP and cGMP in eukaryotes. A complex PDE gene organization and a large number of PDE splicing variants serve to fine-tune cyclic nucleotide signals and contribute to specificity in signaling. Here we review some of the major concepts related to our understanding of PDE function and regulation including: (a) the structure of catalytic and regulatory domains and arrangement in holoenzymes; (b) PDE integration into signaling complexes; (c) the nature and function of negative and positive feedback circuits that have been conserved in PDEs from prokaryotes to human; (d) the emerging association of mutant PDE alleles with inherited diseases; and (e) the role of PDEs in generating subcellular signaling compartments.

Before ovulation in mammals, a cascade of events resembling an inflammatory and/or tissue remodeling process is triggered by luteinizing hormone (LH) in the ovarian follicle. Many LH effects, however, are thought to be indirect because of the restricted expression of its receptor. Here, we demonstrate that LH stimulation induces the transient and sequential expression of the epidermal growth factor (EGF) family members amphiregulin, epiregulin, and beta-cellulin. Incubation of follicles with these growth factors recapitulates the morphological and biochemical events triggered by LH, including cumulus expansion and oocyte maturation. Thus, these EGF-related growth factors are paracrine mediators that propagate the LH signal throughout the follicle.

Phosphodiesterases (PDEs) regulate the local concentration of 3',5' cyclic adenosine monophosphate (cAMP) within cells. cAMP activates the cAMP-dependent protein kinase (PKA). In patients, PDE inhibitors have been linked to heart failure and cardiac arrhythmias, although the mechanisms are not understood. We show that PDE4D gene inactivation in mice results in a progressive cardiomyopathy, accelerated heart failure after myocardial infarction, and cardiac arrhythmias. The phosphodiesterase 4D3 (PDE4D3) was found in the cardiac ryanodine receptor (RyR2)/calcium-release-channel complex (required for excitation-contraction [EC] coupling in heart muscle). PDE4D3 levels in the RyR2 complex were reduced in failing human hearts, contributing to PKA-hyperphosphorylated, "leaky" RyR2 channels that promote cardiac dysfunction and arrhythmias. Cardiac arrhythmias and dysfunction associated with PDE4 inhibition or deficiency were suppressed in mice harboring RyR2 that cannot be PKA phosphorylated. These data suggest that reduced PDE4D activity causes defective RyR2-channel function associated with heart failure and arrhythmias.

Catecholamines signal through the beta2-adrenergic receptor by promoting production of the second messenger adenosine 3',5'-monophosphate (cAMP). The magnitude of this signal is restricted by desensitization of the receptors through their binding to beta-arrestins and by cAMP degradation by phosphodiesterase (PDE) enzymes. We show that beta-arrestins coordinate both processes by recruiting PDEs to activated beta2-adrenergic receptors in the plasma membrane of mammalian cells. In doing so, the beta-arrestins limit activation of membrane-associated cAMP-activated protein kinase by simultaneously slowing the rate of cAMP production through receptor desensitization and increasing the rate of its degradation at the membrane.

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A cAMP-specific phosphodiesterase (PDE4D3) is activated in rat thyroid cells by TSH through a cAMP-dependent phosphorylation (Sette, C., Iona, S., and Conti, M.(1994) J. Biol. Chem. 269, 9245-9252). This short term activation may be involved in the termination of the hormonal stimulation and/or in the induction of desensitization. Here, we have further characterized the protein kinase A (PKA)-dependent phosphorylation of this PDE4D3 variant and identified the phosphorylation site involved in the PDE activation. The PKA-dependent incorporation of phosphate in the partially purified, recombinant rat PDE4D3 followed a time course similar to that of activation. Half-maximal activation of the enzyme was obtained with 0.6 microM ATP and 30 nM of the catalytic subunit of PKA. Phosphorylation altered the Vmax of the PDE without affecting the Km for cAMP. Phosphorylation also modified the Mg2+ requirements and the pattern of inhibition by rolipram. Cyanogen bromide cleavage of the 32P-labeled rat PDE4D3 yielded two or three major phosphopeptide bands, providing a first indication that the enzyme may be phosphorylated at multiple sites in a cell-free system. Site-directed mutagenesis was performed on the serine residues present at the amino terminus of this PDE in the context of preferred motifs for PKA phosphorylation. The PKA-dependent incorporation of 32P was reduced to the largest extent in mutants with both Ser13 --> Ala and Ser54 --> Ala substitutions, confirming the presence of more than one phosphorylation site in rat PDE4D3. While substitution of serine 13 with alanine did not affect the activation by PKA, substitution of Ser54 completely suppressed the kinase activation. Similar conclusions were reached with wild type and mutated PDE4D3 proteins expressed in MA-10 cells, where the endogenous PKA was activated by dibutyryl cAMP. Again, the PDE with the Ser54 --> Ala substitution could not be activated by the endogenous PKA in the intact cell. These findings support the hypothesis that the PDE4D3 variant contains a regulatory domain target for phosphorylation at the amino terminus of the protein and that Ser54 in this domain plays a crucial role in activation.

Oocyte maturation, fertilization, and early embryonic development occur in the absence of gene transcription. Therefore, it is critical to understand at a global level the post-transcriptional events that are driving these transitions. Here we used a systems approach by combining polysome mRNA profiling and bioinformatics to identify RNA-binding motifs in mRNAs that either enter or exit the polysome pool during mouse oocyte maturation. Association of mRNA with the polysomes correlates with active translation. Using this strategy, we identified highly specific patterns of mRNA recruitment to the polysomes that are synchronized with the cell cycle. A large number of the mRNAs recovered with translating ribosomes contain motifs for the RNA-binding proteins DAZL (deleted in azoospermia-like) and CPEB (cytoplasmic polyadenylation element-binding protein). Although a Dazl role in early germ cell development is well established, no function has been described during oocyte-to-embryo transition. We demonstrate that CPEB1 regulates Dazl post-transcriptionally, and that DAZL is essential for meiotic maturation and embryonic cleavage. In the absence of DAZL synthesis, the meiotic spindle fails to form due to disorganization of meiotic microtubules. Therefore, Cpeb1 and Dazl function in a progressive, self-reinforcing pathway to promote oocyte maturation and early embryonic development.

To acquire the ability to fertilize, spermatozoa undergo complex, but at present poorly understood, activation processes. The intracellular rise of cAMP produced by the bicarbonate-dependent soluble adenylyl cyclase (sAC) has been suggested to play a central role in initiating the cascade of the events that culminates in spermatozoa maturation. Here, we show that targeted disruption of the sAC gene does not affect spermatogenesis but dramatically impairs sperm motility, leading to male sterility. sAC mutant spermatozoa are characterized by a total loss of forward motility and are unable to fertilize oocytes in vitro. Interestingly, motility in sAC mutant spermatozoa can be restored on cAMP loading, indicating that the motility defect observed is not caused by a structural defect. We, therefore, conclude that sAC plays an essential and nonredundant role in the activation of the signaling cascade controlling motility and, therefore, in fertility. The crucial role of sAC in fertility and the absence of any other obvious pathological abnormalities in sAC-deficient mice may provide a rationale for developing inhibitors that can be applied as a human male contraceptive