MINIREVIEW
Cyclic AMP-specific PDE4 Phosphodiesterases as Critical Components of Cyclic AMP Signaling*

Marco ContiDagger, Wito Richter, Celine Mehats, Gabriel Livera, Jy-Young Park, and Catherine Jin

From the Division of Reproductive Biology, Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford, California 94305

    INTRODUCTION
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Presence of a PDE4D-PKA...
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REFERENCES

In the five decades that have elapsed since the identification of the second messenger cAMP, most of the components involved in this signaling pathway have been identified, and many of their functions are understood at the molecular and atomic levels. Yet an unexpected and often disconcerting outcome of this progress is the realization that apparently identical cAMP signals induce divergent physiological responses. Countless reports indicate that G protein-coupled receptors (GPCRs)1 that activate the cAMP pathway have distinct and often opposing effects on cell replication/differentiation. Signal compartmentalization, coupling to and activation of additional signaling pathways, and the cellular context in which the cAMP signal develops may account for these divergent biological effects. In view of their role in cAMP signal inactivation and compartmentalization, it is likely that the different phosphodiesterases (PDEs) expressed in any given cell contribute to the properties of cAMP signaling. Here we will review the PDE4 family of enzymes, as well as their role in desensitization, feedback regulation, and signal compartmentalization.

    PDE4 Genes and Transcripts
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PDE4 Genes and Transcripts
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Of the 11 families of PDEs thus far identified, PDE4, PDE7, and PDE8 are specific for cAMP (1, 2). However, isoforms of the PDE4 family often account for most of the cAMP-hydrolyzing activity of a cell. These isoenzymes were initially identified as a chromatography peak of PDE activity and in early reports were referred to as PDEIV, PDEIII, cAMP-specific PDE, or rolipram-sensitive PDE. The Drosophila dunce gene was the first PDE gene to be characterized (3). Soon thereafter, the rodent orthologous genes were identified (4-6), demonstrating that four PDE4 paralogs are present on different chromosomes in the mammalian genomes. Whereas only one PDE4 gene is found in Caenorhabditis elegans, Drosophila, and the sponge Ephytadia fluviatidis, the presence of multiple genes can be traced back to the zebrafish (Danio rerio). The Drosophila and mammalian PDE4 genes are composed of multiple transcriptional units and multiple promoters (2, 7). For instance, the human PDE4D locus that maps to 5q12 encompasses 150 kb of genomic sequence with at least four transcriptional units. Introns functioning as promoters are often more than 20 kb long. Similar complexity has been reported for other PDE4 genes (7), with some minor differences. As a result of this elaborate architecture, there are 4 or 5 transcripts for PDE4A, 4 for PDE4B, possibly 3 for PDE4C, and 5 or more for PDE4D, for a total of at least 16 open reading frames in humans. Undoubtedly, the presence of multiple promoters is necessary for appropriate tissue- and development-specific expression, as well as for regulation by different extracellular stimuli (see below).

    Structural/Functional Properties of PDE4s
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From the characterization of PDE4 mRNAs and their corresponding protein products, it is established that 16-18 different PDE4 isoenzymes or variants are expressed in mammalian cells (2, 7). These variants have closely related kinetic properties and ion requirements and are all inhibited by rolipram. Structurally, they are composed of a highly conserved catalytic domain flanked by domains with regulatory functions. These domains at the carboxyl- and amino-terminal end of the PDE4 are often variant-specific (Fig. 1). The structure of the PDE4B catalytic domain has been solved at the atomic level (8), paving the way for the modeling of the catalytic domain of other PDE4s or even members of other PDE families. This domain is composed of 17 alpha  helices connected by loops, with helices 6-13 containing residues critical for substrate binding and coordination of two metal ions involved in catalysis. Helices 1-7, 8-11, and 12-16 are clustered in subdomains allowing different conformational states of the catalytic center (8). Although the binding to metal ion binding site 1 (Me1) probably occupied by Zn2+ appears to be stable, the Mg2+ or Mn2+ binding to Me2 may be subject to rapid exchange, a finding consistent with multiple conformation of the catalytic domain (9). Structural analysis and mutagenesis (10, 11) have identified several residues involved in substrate or inhibitor binding and for cAMP hydrolysis as well as ion coordination.


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Fig. 1.   Domain organization of the PDE4 short and long forms. Domains are depicted as barrels connected by wires (putative linker regions). Phosphorylation sites are in blue circles. The domain arrangement reported is shared by all PDE4 splicing variants derived from the four genes.

Unfortunately, the spatial relationship between the catalytic domain and the surrounding domains is still unknown because the physicochemical properties of full-length proteins have prevented the generation of useful crystals. Nevertheless, it is widely accepted that domains at the amino and carboxyl termini of the PDE4 protein exert important constraints on the conformation and therefore on the function of the catalytic core. On the amino-terminal side of the catalytic domain, two highly conserved regions, termed upstream conserved regions 1 and 2 (UCR1 and -2), have been identified in PDE4 (12). Depending on the presence of UCR1 and UCR2, PDE4 variants can be distinguished into two major subgroups, the long and short forms (Fig. 1). The long forms include UCR1 and UCR2, whereas the short forms contain only UCR2 or a portion of this domain. The UCR1/UCR2 cassette functions as a regulatory domain that controls the conformation of the catalytic domain. Long PDE4s are phosphorylated at a site present in the amino terminus of UCR1, and this post-translational modification increases the Vmax of the enzyme up to 4-fold (see below). In addition, nested deletions or controlled proteolysis of PDE4D, which cleaves UCR1 and UCR2 away from the catalytic domain, cause an increase in catalysis (10, 13). Together with the finding that an antibody that binds UCR2 induces an increase in Vmax (13), these properties of the long forms have led to a model whereby UCR1 phosphorylation modulates the interactions of UCR2 with the catalytic domain, ultimately altering its conformation and activity. Using yeast two-hybrid assays, it has been determined that UCR1 and UCR2 interact with each other (13, 14). This interaction, thought to be intramolecular, is most likely relevant to the mechanism of enzyme activation by phosphorylation (13, 14). However, we have recently provided evidence for a major role for the UCR1-UCR2 domain in the quaternary structure of PDE4. Splicing variants containing both modules behave as dimers, whereas variants with one of the two UCRs missing behave as monomers (15). This dimerization is most likely critical for transmitting the conformational changes at the amino terminus to changes in conformation of the catalytic domain.

It is well established that a subgroup of PDE4 inhibitors, the prototype being rolipram, bind to the enzyme with kinetics that indicates the presence of multiple conformational states (16). Although it was initially thought that this anomalous behavior is due to the presence of an allosteric site, it is now accepted that rolipram binds to two or more conformers of the catalytic domain. Because detection of the high affinity conformation state (termed high affinity rolipram binding state (16)) requires the presence of Mg2+, and in view of fluorescence resonance energy transfer data, it has been proposed that the high affinity state is due to the occupancy of the Me2 binding site by Mg2+, whereas the low affinity binding site reflects inhibitor binding to an apoenzyme (17). Although the hypothesis is quite appealing, additional data suggest that either more than two conformers are present, or more likely, the UCR1/UCR2 domain is the primary determinant of multiple conformations of the catalytic domain. This latter hypothesis is consistent with the differences in rolipram affinity observed in PDE4 upon phosphorylation (18), in PDE4s in complex with other proteins (19), and in PDE4s exposed to thiol reagents (20).

The presence of multiple conformations of PDE4 is relevant for drug design because it has been proposed that the low affinity conformation is associated with the therapeutic effects of PDE4 inhibitors, whereas the high affinity conformation correlates with undesirable central nervous system and gastric side effects (16, 21). Indeed, compounds that do not favor the high affinity state of the enzyme often are not emetic (21).

    PDE4 Regulation and Its Role in cAMP Signaling
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In the 1970s and 1980s, the synthesis of second generation inhibitors provided important tools to distinguish PDE4-specific functions from those of other PDEs. In general, treatment with PDE4-selective inhibitors causes an increase in intracellular cAMP with enhancement or suppression of distal responses depending on the cell context. However, it is unclear whether functional changes that follow PDE4 inhibition are exclusively due to changes in cAMP "steady state" or whether additional, more subtle, and local disruptions of cAMP signaling produce the myriad effects reported. Because they participate in feedback regulations involved in cell desensitization, adaptation, signaling cross-talk, and cAMP signal compartmentalization, PDE4s are considered to be important cAMP homeostatic regulators.

Even though compounds with lower potency for PDE4C have been described (21), the PDE4 inhibitors thus far developed are nonselective because they inhibit PDE4A, PDE4B, and PDE4D isoforms with comparable IC50 values. Thus, the question of distinct or overlapping functions of the PDE4s could not be addressed using a pharmacological approach. A genetic strategy of PDE4 gene inactivation has allowed the first insight into the function of different PDE4 proteins. Because clearly distinguishable phenotypes have been observed in the PDE4B- and PDE4D-null mice, one can conclude that each PDE4 gene subserves distinct functions (22, 23). To some extent, this specificity may reflect differences in the promoters of PDE4 genes, allowing expression of different proteins at different times during the life cycle of distinct cells. Because of the large array of splicing variants with distinct biochemical properties, it is more difficult to determine the extent of the overlap in the function of different PDE4 proteins. Certainly, differences in enzyme regulation or protein-protein interaction suggest specialized functions for each variant.

    PDE4 Regulation by PKA-mediated Phosphorylation
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The phosphorylation and activation of a PDE4 was demonstrated in a TSH-responsive thyroid cell line (24). In these cells, TSH produces a rapid, PKA-mediated phosphorylation of PDE4D3 at Ser-54 and an increase in activity (25). It is worth noting that the Ser and surrounding residues found in PDE4D3 are not only present in all mammalian PDE4 long splicing variants but are also conserved through evolution from C. elegans to human, thus implying a critical function for this domain. Phosphorylation of this site also causes a variable increase in activity in other long PDE4 variants in overexpression systems (26, 27). Activation of native PDE4D3 and PDE4D5 by phosphorylation has been demonstrated in vascular smooth muscle and lymphocytic cell lines (18, 28, 29).

Together with increased Vmax, phosphorylation causes an increase in affinity of PDE4D3 for Mg2+ (25). This finding has led to the proposal that an increased occupancy of Me2 of PDE4 causes changes in conformation and/or the increase in catalysis, as well as the observed changes in rolipram binding (18, 25). Regardless of the exact mechanism, these data underscore the idea that modifications at the amino terminus of PDE4 are reflected in conformational changes in the catalytic pocket.

    Presence of a PDE4D-PKA Complex in the Cell
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PDE4D3 phosphorylation contributes to cAMP homeostasis and to the shape of the cAMP transient because inhibition of PDE4D3 phosphorylation causes an increase in the intensity of the cAMP transient induced by TSH, whereas activation of PDE4D3 by cAMP analogs decreases cAMP levels (30). However, this feedback regulation must have additional functions that cannot be probed simply by measuring the overall intracellular cAMP concentration. A broader impact of PDE4 phosphorylation is implied by the observation that PDE4D3 and the PKA holoenzyme exist in a complex coordinated by the A kinase anchoring proteins (AKAPs) (31, 32). In rat Sertoli cells of the testis and cardiac myocytes, a PKA-PDE4D complex has been identified by co-immunoprecipitation and immunolocalization of PDE4D and RII (31, 32). (Fig. 2). The presence of AKAP450 and mAKAP in these complexes has been inferred by co-immunoprecipitation of these anchoring proteins in native or overexpressed complexes.


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Fig. 2.   Regulation and subcellular compartmentalization of PDE4s. The known PDE4 macromolecular complexes and their subcellular localization are reported in a hypothetical cell. Anchoring proteins are represented as yellow barrels, receptors as seven transmembrane domain bundles, and adenylyl cyclase as two connected bundles of transmembrane helices. The modified cyclic nucleotide-gated channel used as a cyclic AMP biosensor is represented as a bundle of blue transmembrane domains. The location of the PDE4 in proximity to the EP2 receptors is inferred by the studies with this biosensor. beta ar, beta -arrestin; beta 2, adrenergic receptor; Gs, heterotrimeric guanine nucleotide binding protein; SH3, Src homology 3 binding domain.

The functional consequences of the presence of these AKAP-PKA-PDE4D complexes are inferred by the fact that PDE4D3 is phosphorylated by PKA. Thus, a local feedback regulation serves to control cAMP access to an anchored PKA (31, 32). An increase in cAMP causes activation of the PKA that in turn phosphorylates accessible substrates including the closely positioned PDE4D3. This phosphorylation causes PDE activation, a local decrease in cAMP, and a return of the PKA to a basal activity state.

Additional components of signaling are most likely present in the AKAP-PKA-PDE complex because AKAP450 and its splice variant Yotiao also bind PP1 and PP2A (33). Recently, it has been suggested that PDE4D3 is part of the complex that includes the Ca2+ channel RyR, PKA, mAKAP, and PP1 in cardiomyocytes (34, 35) (Fig. 2).

    PDE4s and Regulation of cAMP Diffusion
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Although cAMP is a small molecule that potentially equilibrates throughout the cell in milliseconds (calculated diffusion rate 780 µm2 s-1), the diffusion of this second messenger may be prevented either by physical barriers or rapid decay due to degradation by PDEs.

Twenty years of investigation have indicated compartmentalization of cAMP in cardiac myocytes (36) as well as other cells. Although they all promote cAMP accumulation, beta 1- and beta 2-adrenergic agonists and PGE1 produce distinct patterns of protein phosphorylation in these cells and distinct effects on contractility (36, 37). In addition, elegant work by Jurevicius and Fishmeister (38) measuring L-type channel activation by beta -adrenergic agonists has suggested that cAMP diffusion occurs only in a limited fashion and that PDEs, particularly PDE4s, may contribute to the prevention of this diffusion.

By modifying a cyclic nucleotide-gated channel to bind cAMP in the micromolar range, Karpen and coworkers (39) have developed a cAMP biosensor to study local cAMP concentration and diffusion. Use of these channels expressed in heterologous systems to measure cAMP-regulated Ca2+ currents has provided evidence that a pool of cAMP below the plasma membrane does not equilibrate rapidly with the bulk of the cytoplasm. More importantly, they have been able to show that a PDE4 is important for regulating the cAMP concentration in this "microdomain" and have suggested that PDE4s are rapidly activated when a GPCR is occupied by its ligand (39). Thus, a PDE4 regulation is likely involved in controlling cAMP access to its effectors close to the plasma membrane, the region most critical to signaling (Fig. 2).

A similar cAMP microdomain may be functioning in cardiac myocytes where it has been shown that cAMP accumulation in response to beta -adrenergic agonists occurs preferentially in a region overlapping with the Z band and the T tubules (40). Even more significant, this preferential accumulation is obliterated by the inhibition of PDE4s. Myomegalin is a PDE4-interacting protein that may serve to anchor the long forms of PDE4 close to the Z band in the vicinity of the L-type channels, RyR and PKA, as indicated by immunolocalization data (34, 41-43).

    Other PDE4 Anchoring Proteins and Subcellular Targeting
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The interaction of PDE4s with different anchoring proteins, their inclusion in macromolecular signaling complexes, and localization in discrete compartments in the cell are probably the rule rather than the exception.

Among the different PDE4 variants, the short form PDE4A1 is recovered mostly in the particulate fraction of brain homogenates. Studies from Houslay and collaborators (44) have shown that this targeting is due to a unique amino terminus present in this variant. This domain has been characterized extensively and found to be the prototype of a domain (TAPAS-1) that binds lipids in the membrane bilayer (44). PDE4A1 binds to phosphatidic acid via this domain, thus anchoring this protein in the Golgi region in proximity to phospholipase A. Phosphatidic acid binding has been reported for other PDE4s suggesting a localization close to the membrane (45). Although phosphatidic acid binding increases the activity of PDE4 long forms, the exact physiological significance of the interaction with PDE4A1, in addition to targeting, remains to be elucidated.

A more dynamic localization of PDE4 to the plasma membrane in proximity to GPCRs is suggested by the discovery of an interaction between PDE4 and beta -arrestin, a scaffold protein with the dual function of coordinating signaling molecules and receptor trafficking (46). In cells overexpressing the beta -adrenergic receptor, beta -arrestin and PDE4 are translocated to the membrane whereas this translocation is absent in MEF cells deficient in beta -arrestin 1 and 2 (46). The presence of a GPCR-arrestin-PDE4 complex obviously has important implications in receptor signaling and desensitization. Thus, beta -arrestin recruitment to the phosphorylated receptor brings a PDE to the complex, contributing to the termination of the signal and preventing the complex from further signaling through cAMP (46).

Several PDE4 long isoforms contain a polyproline motif that interacts with SH3 domains of Fyn/Lyn kinases (7). Such a domain has been mapped in human PDE4A4 and the orthologous rat PDE4A5 at the amino terminus as well as in the linker domain between UCR2 and the catalytic domain. Although it is unclear to what extent these PDEs are in complex with the kinases in the intact cell, it is likely that other SH3 domain-containing proteins interact with PDE4s. It has been reported that these polyprolines are required for targeting to the particulate fraction in membrane ruffles and in a perinuclear region (7). Finally, PDE4D5 has been shown to interact with RACK1 through its unique amino-terminal domain (47).

    Other PDE4 Regulation by Phosphorylation: MAPK Feedback
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During studies on the expression of PDE4s, it was observed that the recombinant PDE4B2 expressed in insect cells is phosphorylated by MAPK at a serine in a carboxyl-terminal SPS motif (48). The implications of this finding came into focus when it was demonstrated that PDE4D3 overexpressed in mammalian cells is phosphorylated in a similar motif by MAPK leading to an inhibition of the catalytic activity (49). It has been proposed that this regulation of PDE4D and other PDE4 long forms by MAPK constitutes feedback regulation. Inhibition of the PDE4 activity induces an increase in cAMP, and it has been speculated that the local activation of PKA in turn suppresses the MAPK cascade via phosphorylation of RAF (50). Pharmacological manipulation of the aortic smooth muscle cells shows that PKA phosphorylation and activation of PDE4D5 overcomes or obliterates the initial inhibition due to MAPK phosphorylation (51). Further work is required to understand the physiological significance of the intricate positive and negative regulations. An increase in PDE4A activity has also been observed after activation of the phosphatidylinositol 3-kinase signaling pathway (52).

    cAMP Gating and Long Term Feedback Regulations Involving PDE4 Expression
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In addition to short term regulation of PDE4 due to post-translational modifications, the expression of PDE4 is regulated at the transcriptional level. In both the PDE4B and PDE4D genes, a cAMP-regulated intronic promoter has been identified (53, 54). This promoter includes several potential cAMP regulatory elements as well as other elements involved in transcription. Hormonal stimulation in vitro or in vivo as well as pharmacological manipulation of intracellular cAMP causes large increases in PDE4D and PDE4B mRNA and the corresponding short form proteins (55, 56). In addition, mRNA stability may contribute to the overall increase in PDE4D and PDE4B mRNA (56). Other promoters present in the PDE4 genes also may be regulated by cAMP as an increase in PDE4D3 and PDE4D5 mRNA were recently reported after a sustained increase in cAMP (57, 58).

In vitro studies have implicated this long term induction of PDE4 in desensitization and long term cell adaptation as treatment with protein synthesis or PDE4 inhibitors to some extent reverses the desensitization state (2). In addition, mutations that chronically activate the cAMP signaling in vivo are associated with increased PDE4 expression (59). A better understanding of the function of this feedback has come from the analysis of two phenotypes in the PDE4-null mice. In the ovary, the growth and maturation of the follicle is dependent on the concerted action of the pituitary gonadotropin follicle-stimulating hormone and luteinizing hormone (60). Both gonadotropins signal through cAMP and produce large increases in the mRNAs coding for the short forms of PDE4D. Ablation of the PDE4D gene causes a 75% decrease in the rate of ovulation and a consequent reduced fertility (22). More importantly, the pattern of gene expression required for follicle maturation/ovulation is disrupted, demonstrating that the PDE4D feedback regulation is critical in cAMP signaling and cell differentiation.

Secondly, expression of the PDE4B2 short form is induced by activation of the Toll receptor-related signaling pathway. In macrophages or monocytic cell lines as well as in circulating monocytes, LPS stimulation causes a large increase in PDE4B2 mRNA and protein (23, 61). In the PDE4B-null mice, where this regulation is absent, LPS stimulation of tumor necrosis factor-alpha production is reduced 90% (23). This finding indicates that the induction of PDE4B2 by LPS is a positive feedback regulation required to remove a negative cAMP constraint. Indeed, it is well established that cytokine production and activation of inflammatory cells is under negative regulation by cAMP, which functions as a gating pathway (62). This finding is consistent with the pharmacological tenet that PDE4 inhibition blocks inflammatory responses and therefore is a promising therapeutic strategy for inflammatory disorders (21).

    Additional Functions of PDE4 Inferred by the Phenotype of the PDE4D Knock-out Mouse
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The analysis of the phenotype of the PDE4D-null mouse has provided additional insight into the critical role of PDE4 in cAMP homeostasis. This function is particularly evident in those cells where cAMP is involved in balancing the effect of several signaling pathways. Through binding to muscarinic cholinergic receptors, acetylcholine released from the parasympathetic nerves induces contraction of the smooth muscle of the airway. This parasympathetic control of the smooth muscle tone involves M1/M3 receptors coupled to phospholipase C activation and Ca2+ signaling, as well as M2 receptors coupled to adenylyl cyclase inhibition (63). Activation of cAMP signaling by epinephrine or other ligands that activate adenylyl cyclase counteracts the cholinergic responses by inducing relaxation. In mice deficient in PDE4D, cholinergic agonists do not produce airway contraction as assessed in vivo by whole body plethysmography or ex vivo by measuring tracheal ring contractility (64).2 The absence of a cholinergic-mediated contraction is likely due to an increased cAMP tone that follows ablation of the PDE4D in the airway. Indeed, cAMP levels are increased in the lungs of PDE4D knock-out mice. In addition, an increased sensitivity to agonists that activate adenylyl cyclase is present in this organ together with a decreased signaling through M2 cholinergic receptors that inhibit cyclase (64). Therefore, PDE4D is critical for the control of cAMP levels in the airway and the fine tuning of the sensitivity to contracting and relaxing inputs.

In a different paradigm, anesthesia, monitored in mice as obliteration of the righting reflex, is induced by administration of alpha 2-adrenergic agonists, and its duration is greatly reduced by the specific alpha 2 antagonist MK-912 (65). In mice deficient in PDE4D but not PDE4B, the duration of anesthesia is reduced by more than 50%. The alpha 2 antagonists have an additional minor effect whereas PDE4 inhibitors that shorten the anesthesia in wild type mice have no effect in the PDE4D knock-out mice. These findings indicate that the decrease in cAMP induced by the alpha 2 agonist is disrupted by removal of a PDE4, re-emphasizing the critical role of PDE4D in the control of the balance between the positive and negative stimuli of the cAMP signaling pathway. Furthermore, this observation is important from a pharmacological standpoint as it has been proposed that the inhibition of anesthesia in mice is a correlate of emesis in ferrets (65), a major side effect produced by PDE4 inhibition.

    ACKNOWLEDGEMENTS

We are indebted to many colleagues for their discussions and comments regarding this review and to Caren Spencer for editorial assistance.

    FOOTNOTES

* This minireview will be reprinted in the 2003 Minireview Compendium, which will be available in January, 2004. The work done in the authors' laboratory is supported by National Institutes of Health Grants HD20788 and HL67674 and a grant from the Sandler Foundation.

Dagger To whom correspondence should be addressed: Division of Reproductive Biology, Dept. of Obstetrics and Gynecology, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5317. Tel.: 650-725-2452; Fax: 650-725-7102; E-mail: marco.conti@stanford.edu.

Published, JBC Papers in Press, December 18, 2002, DOI 10.1074/jbc.R200029200

2 C. Mehats, submitted for publication.

    ABBREVIATIONS

The abbreviations used are: GPCR, G protein-coupled receptor; PDE, phosphodiesterase; UCR, upstream conserved region; TSH, thyroid-stimulating hormone; AKAP, A kinase anchoring protein; MAPK, mitogen-activated protein kinase; PKA, protein kinase A; LPS, lipopolysaccharide.

    REFERENCES
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