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Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions
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Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions Scott H Soderling and Joseph A Beavo The past eighteen months have provided much progress in the cyclic nucleotide phosphodiesterase (PDE) field. Six new phosphodiesterase genes have been discovered and characterized. In addition, several new highly specific PDE inhibitors have been developed and approved for clinical use. Finally, new strategies have been employed to determine PDE function in model systems including the use of antisense oligonucleotide and disruption techniques. Address Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA Current Opinion in Cell Biology 2000, 12:174–179 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations IGF insulin-like growth factor PDE phosphodiesterase PKA protein kinase A PYP photoactive yellow protein TCR T-cell receptor
phosphodiesterase families, PDE8, PDE9 and PDE10 were identified, essentially simultaneously, by several independent laboratories [3•,4•,5,6•–11•]. PDE8A and PDE8B
PDE8A is specific for the hydrolysis of cAMP, with a low KM of approximately 70 nM [3•,4•]. Interestingly, PDE8 was the first example (PDE9 is now the second) of a PDE that is not inhibited effectively by the non-selective inhibitor, IBMX. Thus it should be emphasized that the lack of an effect of IBMX may not be a useful indicator that PDEs do not regulate a physiological function in all cases. In mouse, PDE8A expression is highest in testis followed by eye, liver, kidney, skeletal muscle, embryo, ovary and brain [3•]. In humans, PDE8A has a similar tissue distribution [4•]. In situ hybridization in mouse testis indicates that PDE8A expression is regulated both temporally and spatially in spermatocytes, with expression limited to midto-late pachytene spermatocytes [3•]. Although the function of PDE8 during these stages is uncertain, the stage-specific expression suggests that it probably plays a defined role(s) during germ-cell development.
Introduction Given that cyclic nucleotide signaling regulates a wide variety of cellular functions, it is not surprising that cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. From comparative, structural and functional studies, PDEs are now known to possess a modular architecture, with a conserved catalytic domain proximal to the carboxyl terminus and regulatory domains or motifs often near the amino terminus (see Figure 1). The PDE superfamily currently includes 19 different genes subgrouped into 10 different PDE families, and it is likely that more will be added in the coming years. Each family is distinguished functionally by its unique combination of enzymatic characteristics and pharmacological inhibitory profiles. Individual PDE families also exhibit regulation by distinct allosteric activators or inhibitors. Finally, each gene within a family also has specific tissue, cellular, and sometimes also subcellular distributions. Thus the precise cellular and subcellular profile of PDE expression determines how a tissue responds to first messengers. Here, we review recent work on the newly discovered PDE families (PDE8, PDE9 and PDE10) and also recent insights into the roles of other specific PDEs in the regulation of T-cell activation, insulin secretion, growth, fertility and penile erection.
New PDEs The past year has provided a number of surprises in the cyclic nucleotide signaling field. Not only was a new cAMP target discovered [1••,2••], but also three new
Only the description of a 5′ truncated human cDNA of PDE8B has been published to date [5]. Although only limited kinetic studies were done, it appears that PDE8B is also specific for cAMP hydrolysis and is also not inhibited by IBMX. Of the tissues examined so far, PDE8B expression appears highest in human thyroid gland, but is also expressed at lower levels in human brain, kidney, pancreas, spinal cord, placenta, prostate and uterus. An intriguing feature of PDE8 is the presence of a single PAS domain (for Per, ARNT, and Sim proteins for which this was originally identified) at the amino terminus [3•] (see Figures 1 and 2). In several other proteins containing PAS domains this domain mediates specific homomeric and heteromeric protein–protein interactions that often regulate subcellular distribution [12–16]. Crystallographic studies of proteins containing PAS domains demonstrate that this domain also can complex small ligands [17•,18]. For instance, the PAS domain of FixL contains a heme group, and that of photoactive yellow protein (PYP) to form a complex with a chromophore. In both PYP and FixL, these ligands serve in a sensory capacity, altering their activity state in response to light or oxygen, respectively [19••]. The PDE8 PAS domain sequence can be modeled very effectively on the crystal coordinates of the other PAS domain proteins, suggesting that it also has a similar structure and perhaps function (Figure 2). It will be interesting to see whether this domain in PDE8A or PDE8B also serves a protein interaction or regulatory/sensory function for these enzymes.
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Figure 1 Diagram representing the domain and motif organization of each PDE family. PDE1A ,B,C PDE2A PDE3A ,B PDE4A ,B,C,D PDE5A PDE6A ,B,C PDE7A PDE8A ,B PDE9A PDE10A = Ca/CaM binding domain, CaM activates = GAF domain, binds cGMP, other molecules? = Probable transmembrane domain = Upstream conserved region, inhibitory? = Gamma subunit, inhibits PDE6 = Prenyl groups, anchor to membrane = Delta subunit, solubilizes PDE6 = Putative PDZ domain binding sequence = PAS domain, ligand binding? protein binding? = Catalytic domain Current Opinion in Cell Biology
PDE9A
Unlike PDE8, PDE9 is specific for the high-affinity hydrolysis of cGMP, with a KM of 0.07 µM [6•,7•]. Like PDE8, however, PDE9 is not effectively inhibited by IBMX. PDE9 is expressed in small intestinal smooth muscle (SH Soderling, JA Beavo, unpublished data), kidney, liver, lung, brain, testis, skeletal muscle, heart, thymus and spleen [6•,7•]. To date, four 5′ alternative splice variants have been identified for PDE9; however, the functional consequence of these variants are currently unknown [8•]. PDE10A
PDE10, the most recently described phosphodiesterase, was reported simultaneously by three independent groups [9•–11•]. PDE10 has the capacity to hydrolyze both cAMP and cGMP; however, the Km for cAMP is approximately 0.05 µM, whereas the KM for cGMP is 3 µM. In addition, the Vmax for cAMP hydrolysis is fivefold lower than for cGMP. Because of these kinetics, cGMP hydrolysis by PDE10 is potently inhibited by cAMP in vitro, suggesting that PDE10 may function as a cAMP-inhibited cGMP phosphodiesterase in vivo [9•,11•]. Unlike PDE8 or PDE9, PDE10 is inhibited by IBMX with an IC50 (50% inhibitory concentration) of 2.6 µM.
PDE10 contains two amino-terminal domains that are similar to the cGMP-binding domains of PDE2, PDE5 and PDE6. Profile-pattern-database analysis has now demonstrated that these domains are conserved across a wide variety of proteins [20]. Because of the wide conservation of this domain, it is now referred to as the GAF domain (for the GAF proteins: cGMP binding phosphodiesterases; the cynobacterial Anabaena adenylyl cyclase; and the Escherichia coli transcriptional regulator fhlA). Although in PDE2, PDE5 and PDE6 the GAF domains do bind cGMP, this is probably not the primary function of this domain in all cases (e.g., E. coli are not thought to synthesize cGMP). Interestingly, in vitro binding studies of PDE10 indicate the dissociation constant (Kd) for cGMP binding is well above 9 µM [11•]. As in vivo concentrations of cGMP are not thought to reach such high levels in most cells, it seems likely that either the affinity of PDE10 for cGMP is increased by regulation, or that the primary function of the GAF domain in PDE10 may be for something other than cGMP binding.
New functions for ‘old’ PDEs The past two years have seen an explosion in our understanding of the physiological functions that phospho-
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the membrane is also necessary for regulation of GLP-1 (glucagon-like peptide 1) stimulated insulin secretion [24,25].
Figure 2
Protein interaction region?
T-cell activation
Regulation of T-cell activation is a complex process, requiring the coordinated occupation of the T-cell receptor (TCR)–CD3 receptor and one or more co-stimulatory receptors such as CD28. Recent work reveals that PDE7 levels are elevated by co-stimulation of both the TCR and the CD28 receptor, but not by low stimulation of either receptor alone [26••]. As PKA signaling is thought to be at least one inhibitory pathway that must be overcome by co-stimulation to produce T-cell proliferation, this result suggests that induction of PDE7 may provide such a mechanism.
Ligand binding pocket?
Current Opinion in Cell Biology
Structural model of the PDE8A PAS domain based on the homology to and known structure of the FixL PAS domain. The Molecular Operating Environment (MOE) software package was used to align the PDE8 PAS domain to FixL and to thread the sequence of PDE8A into the atomic coordinates of FixL. Note the ‘hand like’ structure of the PAS domain with the view facing the back of the hand with the palm oriented away from the reader. The putative protein interaction area corresponds to a hydrophobic pocket on the surface of the antiparallel β sheets similar to that described for the HERG structure [40•]. The possible ligand binding region within the ‘palm’ of the hand structure is based on similarities to both PYP and FixL [17•,18].
Because specific inhibitors for PDE7 were not available, antisense oligonucleotides specific to PDE7 were used to selectively inhibit PDE7 induction. Isolated peripheral T-cells treated with antisense, but not control, oligonucleotides showed an 80% reduction in proliferation and interleukin–2 production when co-stimulated [26••]. The antisense oligonucleotides reduced PDE7 activity, an effect that was reversed by an inhibitor of PKA, Rp-cAMP. Overall, the data suggest that PDE7 is necessary for the induction of T-cell proliferation and that PDE7 may be a useful drug target for the treatment of T-cell-mediated pathologies. Fertility and growth
diesterases regulate. One of the more satisfying aspects of these studies is that they have borne out the prevailing premise that distinct phosphodiesterases regulate specific cellular functions. We will describe several recent examples. Regulation of insulin secretion
It is thought that cAMP increases insulin secretion by activating protein kinase A (PKA), which inhibits an ATPsensitive potassium channel, and/or activates calcium channels [21]. Previous work has established that PDE3B mediates insulin-like growth factor (IGF)-1 control of insulin secretion from pancreatic B cells [22]. Regulation of insulin secretion by PDE3B, however, is not only limited to IGF-1, it is also mediated by leptin, thus establishing an alternative pathway to JAK/STAT (Janus kinase/signal transducer and activators of transcription) signaling for leptin in B cells [23••]. For example, leptin will inhibit insulin secretion stimulated by a cell-permeable cAMP analog that can be hydrolyzed by PDE3B but not by one that cannot be hydrolyzed, suggesting that PDE3B has a central role in mediating the effect. As with IGF-1, PDE3-specific inhibitors also blocked the effect of leptin. Although PDE4 inhibitors also raise cAMP levels in pancreatic B cells, they do not block the effect of leptin on insulin secretion. This suggests that PDE3B specifically modulates a discrete intracellular pool of cAMP that regulates insulin secretion. In support of this idea, AKAP (a kinase anchoring protein) targeting of a subset of PKA to
Although the above studies have relied on the acute inhibition of PDE activity by either pharmacological or antisense strategies, genetic manipulation of PDE activity has also proven to be a useful strategy for establishing new insights into PDE function. The function of PDE4D in mouse has now been examined by gene knockout studies [27••]. The resultant PDE4D-null mice display several interesting phenotypes including increased mortality, decreased size and body weight and reduced fertility. Increased mortality is most pronounced within the first four weeks postpartum, with approximately 40% of the null mice dying during this period. The underlying cause of the increased postnatal mortality is currently unknown, but is probably not due to genetic rearrangement during the recombination event, as an independently derived null strain also displayed a similar phenotype. Interestingly, surviving mice are growth retarded, with a 30–40% reduction in body weight at 2 weeks. This decreased body weight is caused by a decrease in bone, muscle and internal organ weight compared with wildtype littermates. Although it is not certain why the PDE4D-null mice fail to develop to a normal size, a possible cause may be the observed decrease in circulating IGF-1 in the null mice. Finally, female, but not male, mice exhibited a reduced fertility rate. Examination of oocytes recovered from the
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PDE4D-null mice revealed that more than 50% had overt signs of degeneration and fragmentation. Previous work has shown that PDE4D is expressed at high levels in murine granulosa cells [28] and PDE4D-null mice show a decrease in PDE4 activity in isolated granulosa cells. Somewhat surprisingly, when stimulated with FSH (follicle-stimulating hormone) the PDE4D-null granulosa cells do not display an increased cAMP accumulation as would be expected for cells with a lower cAMP PDE content. This was not due to a defect at the level of the cyclase as forskolin elicited a normal cAMP response. Although the reduced fertility observed in the PDE4D knockout mice may be due to a direct effect of PDE4D disruption in granulosa cells, it also seems plausible, on the basis of other studies [29], that this phenotype is related to the decreased circulating IGF-1.
regulate. Future work on both ‘new’ and ‘old’ phosphodiesterases should continue to provide additional exciting insights into the roles of PDEs in cAMP and cGMP signaling.
Penile erectile function
References and recommended reading
Upon sexual stimulation, penile nerve excitation releases nitric oxide (NO), relaxing vascular smooth muscle and allowing blood flow into the large corpora cavernosa and smaller corpus spongiosum leading to rigidity and erection. Erectile dysfunction may be caused by spinal injury, psychological issues or organic causes [30]. Although not life threatening, the psychological and social consequences of erectile dysfunction are serious, as previous labels have captured (‘impotence’ is from the Latin word meaning ‘without power’).
Papers of particular interest, published within the annual period of review, have been highlighted as:
Regardless of the cause of the dysfunction (including spinal injury), a number of studies have demonstrated the utility of stimulating the NO/cGMP pathway by inhibition of PDE5 for enhancing penile rigidity [31–33]. Inhibition of PDE5 can have dramatic effects on the cGMP content of the corpus cavernosum. Interestingly, sildenafil appears to have no direct effect on intracavernosal pressure in the absence of nerve stimulation, but instead only potentiates intracavernosal pressure that is induced by nerve stimulation [34•]. This corroborates clinical data suggesting that sildenafil enhances the natural male sexual response. Inhibition of PDE5 has thus changed the way in which erectile dysfunction is treated and has also established the importance of PDE5 in the regulation of a specific physiological process, penile erection.
Conclusions We have highlighted recent progress in the phosphodiesterase field related to the identification of previously unknown PDEs (see http://depts.washington.edu/pde/ for current PDE nomenclature updates), and to the characterization of some of the cellular functions that PDEs regulate. There has not been room to discuss other important studies in this burgeoning field, including PDE subcellular targeting (I Verde et al., unpublished data; [35,36••,37•]) and PDE regulation [38•,39•]. The past 18 months have expanded our appreciation of the number of distinct phosphodiesterases that exist and our understanding of the biology that previously identified PDEs
Update After submission of this manuscript the first report of a new PDE11 gene family was published [41••]. This isozyme hydrolyzes both cAMP and cGMP and contains a GAF domain. Little is yet known about its regulation.
Acknowledgements The authors would like to thank Elinor T Adman of the Department of Biological Structure at the University of Washington for her help in analyzing the PAS domain structure of PDEB. Work from the authors was supported by grants DK2173, HL60178 and HL4498 from the National Institutes of Health.
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Guipponi M, Scott HS, Kudoh J, Kawasaki K, Shibuya K, Shintani A, Asakawa S, Chen H, Lalioti MD, Rossier C et al.: Identification and characterization of a novel cyclic nucleotide phosphodiesterase gene (PDE9A) that maps to 21q22.3: alternative splicing of mRNA transcripts, genomic structure and sequence. Hum Genet 1998, 103:386-392. These two papers [7•,8•] describe the PDE9 family in mouse [7•] and human [8•]. 9. •
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27. ••
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32. Moreland RB, Goldstein I, Traish A: Sildenafil, a novel inhibitor of phosphodiesterase type 5 in human corpus cavernosum smooth muscle cells. Life Sci 1998, 62:309-318.
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33. Ballard SA, Gingell CJ, Tang K, Turner LA, Price ME, Naylor AM: Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isozymes. J Urol 1998, 159:2164-2171.
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Gong W, Hao B, Mansy SS, Gonzalez G, Gilles-Gonzalez MA, Chan MK: Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction. Proc Natl Acad Sci USA 1998, 95:15177-15182. This paper describes the crystal structure for the PAS domain of FixL, which is remarkably similar to that of PYP [18], with different ligands complexed within the ‘palm’ of the PAS ‘hand’. 18. Borgstahl GE, Williams DR, Getzoff ED: 1.4 A structure of photoactive yellow protein, a cytosolic photoreceptor: unusual fold, active site, and chromophore. Biochemistry 1995, 34:6278-6287. 19. Pellequer JL, Brudler R, Getzoff ED: Biological sensors: more than •• one way to sense oxygen. Curr Biol 1999, 9:R416-R418. This paper reviews the structural features of the PAS domain. It contains an informative figure that aligns the structures of the FixL, PYP and HERG (human-ether-a-go-go potassium channel) PAS domains together to visually demonstrate that, in spite of limited sequence conservation, strong structural homology exists. 20. Aravind L, Ponting CP: The GAF domain: an evolutionary link between diverse phototransducing proteins. Trends Biochem Sci 1997, 22:458-459. 21. Holz GG, Habener JF: Signal transduction crosstalk in the endocrine system: pancreatic beta- cells and the glucose competence concept. Trends Biochem Sci 1992, 17:388-393. 22. Zhao AZ, Zhao H, Teague J, Fujimoto W, Beavo JA: Attenuation of insulin secretion by insulin-like growth factor 1 is mediated through activation of phosphodiesterase 3B. Proc Natl Acad Sci USA 1997, 94:3223-3228. 23. Zhao AZ, Bornfeldt KE, Beavo JA: Leptin inhibits insulin secretion •• by activation of phosphodiesterase 3B. J Clin Invest 1998, 102:869-873. This paper shows that both IGF-1 and leptin can reduce insulin secretion by activating PDE3B and thereby lowering cAMP levels. The authors also suggest that different cAMP pools regulated by localized PDEs regulate insulin secretion.
34. Carter AJ, Ballard SA, Naylor AM: Effect of the selective • phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog. J Urol 1998, 160:242-246. This paper demonstrates the effect of inhibiting PDE5 on penile erection. It suggests that PDE5 does not regulate basal cGMP, but rather cGMP in response to sexual stimulation. 35. O’Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD: The SH3 domain of Src tyrosyl protein kinase interacts with the N- terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J 1996, 318:255-261. 36. Yarwood SJ, Steele MR, Scotland G, Houslay MD, Bolger GB: The •• RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 1999, 274:14909-14917. This paper, along with [35], demonstrates the specific interaction of PDE4 isozymes with cellular scaffolding proteins. These studies help to define the emerging theme in the PDE field that that spatially distinct targeting of PDEs is likely to be important for regulation of cellular function. 37. •
Marzesco AM, Galli T, Louvard D, Zahraoui A: The rod cGMP phosphodiesterase delta subunit dissociates the small GTPase Rab13 from membranes. J Biol Chem 1998, 273:22340-22345. This study suggests that the function of the PDE6 delta subunit may not be limited to the solubilization of PDE6 in photoreceptors. It also suggests that the delta subunit may be targeted to subcellular compartments by a PDZ binding motif at the C-terminus. 38. Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD: The • MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J 1999, 18:893-903. The authors demonstrate that PDE4D3 can be regulated by ERK2. This in turn suggests that the MAP kinase and cAMP signaling systems may crosstalk at the level of PDE4.
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molecular protein interaction, is presented. This structure is similar to the structures of the PAS domains of PYP and FixL. Additionally, mutagenesis suggests the HERG PAS domain can bind to other regions of the potassium channel by a hydrophobic patch located on the surface of the antiparallel β sheets. An analogous hydrophobic patch is found on the PDE8A PAS domain structure model (see Figure 2), suggesting that this may be a common protein interaction region.
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41. Fawcett L, Baxendale R, Stacey P, McGrouther C, Harrow I, •• Soderling SA, Helman J, Beavo JA, Phillips SC: Molecular cloning and characterization of a novel human phosphodiesterase gene family: PDE11A. Proc Natl Acad Sci USA 2000, in press. This isozyme hydrolyzes both cAMP and cGMP and contains a GAF domain. Little is known yet about its regulation.
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Regulation of cAMP and cGMP signaling: new phosphodiesterases and new functions Scott H Soderling and Joseph A Beavo The past eighteen months have provided much progress in the cyclic nucleotide phosphodiesterase (PDE) field. Six new phosphodiesterase genes have been discovered and characterized. In addition, several new highly specific PDE inhibitors have been developed and approved for clinical use. Finally, new strategies have been employed to determine PDE function in model systems including the use of antisense oligonucleotide and disruption techniques. Address Department of Pharmacology, Box 357280, University of Washington, Seattle, Washington 98195, USA Current Opinion in Cell Biology 2000, 12:174–179 0955-0674/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations IGF insulin-like growth factor PDE phosphodiesterase PKA protein kinase A PYP photoactive yellow protein TCR T-cell receptor
phosphodiesterase families, PDE8, PDE9 and PDE10 were identified, essentially simultaneously, by several independent laboratories [3•,4•,5,6•–11•]. PDE8A and PDE8B
PDE8A is specific for the hydrolysis of cAMP, with a low KM of approximately 70 nM [3•,4•]. Interestingly, PDE8 was the first example (PDE9 is now the second) of a PDE that is not inhibited effectively by the non-selective inhibitor, IBMX. Thus it should be emphasized that the lack of an effect of IBMX may not be a useful indicator that PDEs do not regulate a physiological function in all cases. In mouse, PDE8A expression is highest in testis followed by eye, liver, kidney, skeletal muscle, embryo, ovary and brain [3•]. In humans, PDE8A has a similar tissue distribution [4•]. In situ hybridization in mouse testis indicates that PDE8A expression is regulated both temporally and spatially in spermatocytes, with expression limited to midto-late pachytene spermatocytes [3•]. Although the function of PDE8 during these stages is uncertain, the stage-specific expression suggests that it probably plays a defined role(s) during germ-cell development.
Introduction Given that cyclic nucleotide signaling regulates a wide variety of cellular functions, it is not surprising that cyclic nucleotide phosphodiesterases (PDEs) are represented by a large superfamily of enzymes. From comparative, structural and functional studies, PDEs are now known to possess a modular architecture, with a conserved catalytic domain proximal to the carboxyl terminus and regulatory domains or motifs often near the amino terminus (see Figure 1). The PDE superfamily currently includes 19 different genes subgrouped into 10 different PDE families, and it is likely that more will be added in the coming years. Each family is distinguished functionally by its unique combination of enzymatic characteristics and pharmacological inhibitory profiles. Individual PDE families also exhibit regulation by distinct allosteric activators or inhibitors. Finally, each gene within a family also has specific tissue, cellular, and sometimes also subcellular distributions. Thus the precise cellular and subcellular profile of PDE expression determines how a tissue responds to first messengers. Here, we review recent work on the newly discovered PDE families (PDE8, PDE9 and PDE10) and also recent insights into the roles of other specific PDEs in the regulation of T-cell activation, insulin secretion, growth, fertility and penile erection.
New PDEs The past year has provided a number of surprises in the cyclic nucleotide signaling field. Not only was a new cAMP target discovered [1••,2••], but also three new
Only the description of a 5′ truncated human cDNA of PDE8B has been published to date [5]. Although only limited kinetic studies were done, it appears that PDE8B is also specific for cAMP hydrolysis and is also not inhibited by IBMX. Of the tissues examined so far, PDE8B expression appears highest in human thyroid gland, but is also expressed at lower levels in human brain, kidney, pancreas, spinal cord, placenta, prostate and uterus. An intriguing feature of PDE8 is the presence of a single PAS domain (for Per, ARNT, and Sim proteins for which this was originally identified) at the amino terminus [3•] (see Figures 1 and 2). In several other proteins containing PAS domains this domain mediates specific homomeric and heteromeric protein–protein interactions that often regulate subcellular distribution [12–16]. Crystallographic studies of proteins containing PAS domains demonstrate that this domain also can complex small ligands [17•,18]. For instance, the PAS domain of FixL contains a heme group, and that of photoactive yellow protein (PYP) to form a complex with a chromophore. In both PYP and FixL, these ligands serve in a sensory capacity, altering their activity state in response to light or oxygen, respectively [19••]. The PDE8 PAS domain sequence can be modeled very effectively on the crystal coordinates of the other PAS domain proteins, suggesting that it also has a similar structure and perhaps function (Figure 2). It will be interesting to see whether this domain in PDE8A or PDE8B also serves a protein interaction or regulatory/sensory function for these enzymes.
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Figure 1 Diagram representing the domain and motif organization of each PDE family. PDE1A ,B,C PDE2A PDE3A ,B PDE4A ,B,C,D PDE5A PDE6A ,B,C PDE7A PDE8A ,B PDE9A PDE10A = Ca/CaM binding domain, CaM activates = GAF domain, binds cGMP, other molecules? = Probable transmembrane domain = Upstream conserved region, inhibitory? = Gamma subunit, inhibits PDE6 = Prenyl groups, anchor to membrane = Delta subunit, solubilizes PDE6 = Putative PDZ domain binding sequence = PAS domain, ligand binding? protein binding? = Catalytic domain Current Opinion in Cell Biology
PDE9A
Unlike PDE8, PDE9 is specific for the high-affinity hydrolysis of cGMP, with a KM of 0.07 µM [6•,7•]. Like PDE8, however, PDE9 is not effectively inhibited by IBMX. PDE9 is expressed in small intestinal smooth muscle (SH Soderling, JA Beavo, unpublished data), kidney, liver, lung, brain, testis, skeletal muscle, heart, thymus and spleen [6•,7•]. To date, four 5′ alternative splice variants have been identified for PDE9; however, the functional consequence of these variants are currently unknown [8•]. PDE10A
PDE10, the most recently described phosphodiesterase, was reported simultaneously by three independent groups [9•–11•]. PDE10 has the capacity to hydrolyze both cAMP and cGMP; however, the Km for cAMP is approximately 0.05 µM, whereas the KM for cGMP is 3 µM. In addition, the Vmax for cAMP hydrolysis is fivefold lower than for cGMP. Because of these kinetics, cGMP hydrolysis by PDE10 is potently inhibited by cAMP in vitro, suggesting that PDE10 may function as a cAMP-inhibited cGMP phosphodiesterase in vivo [9•,11•]. Unlike PDE8 or PDE9, PDE10 is inhibited by IBMX with an IC50 (50% inhibitory concentration) of 2.6 µM.
PDE10 contains two amino-terminal domains that are similar to the cGMP-binding domains of PDE2, PDE5 and PDE6. Profile-pattern-database analysis has now demonstrated that these domains are conserved across a wide variety of proteins [20]. Because of the wide conservation of this domain, it is now referred to as the GAF domain (for the GAF proteins: cGMP binding phosphodiesterases; the cynobacterial Anabaena adenylyl cyclase; and the Escherichia coli transcriptional regulator fhlA). Although in PDE2, PDE5 and PDE6 the GAF domains do bind cGMP, this is probably not the primary function of this domain in all cases (e.g., E. coli are not thought to synthesize cGMP). Interestingly, in vitro binding studies of PDE10 indicate the dissociation constant (Kd) for cGMP binding is well above 9 µM [11•]. As in vivo concentrations of cGMP are not thought to reach such high levels in most cells, it seems likely that either the affinity of PDE10 for cGMP is increased by regulation, or that the primary function of the GAF domain in PDE10 may be for something other than cGMP binding.
New functions for ‘old’ PDEs The past two years have seen an explosion in our understanding of the physiological functions that phospho-
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the membrane is also necessary for regulation of GLP-1 (glucagon-like peptide 1) stimulated insulin secretion [24,25].
Figure 2
Protein interaction region?
T-cell activation
Regulation of T-cell activation is a complex process, requiring the coordinated occupation of the T-cell receptor (TCR)–CD3 receptor and one or more co-stimulatory receptors such as CD28. Recent work reveals that PDE7 levels are elevated by co-stimulation of both the TCR and the CD28 receptor, but not by low stimulation of either receptor alone [26••]. As PKA signaling is thought to be at least one inhibitory pathway that must be overcome by co-stimulation to produce T-cell proliferation, this result suggests that induction of PDE7 may provide such a mechanism.
Ligand binding pocket?
Current Opinion in Cell Biology
Structural model of the PDE8A PAS domain based on the homology to and known structure of the FixL PAS domain. The Molecular Operating Environment (MOE) software package was used to align the PDE8 PAS domain to FixL and to thread the sequence of PDE8A into the atomic coordinates of FixL. Note the ‘hand like’ structure of the PAS domain with the view facing the back of the hand with the palm oriented away from the reader. The putative protein interaction area corresponds to a hydrophobic pocket on the surface of the antiparallel β sheets similar to that described for the HERG structure [40•]. The possible ligand binding region within the ‘palm’ of the hand structure is based on similarities to both PYP and FixL [17•,18].
Because specific inhibitors for PDE7 were not available, antisense oligonucleotides specific to PDE7 were used to selectively inhibit PDE7 induction. Isolated peripheral T-cells treated with antisense, but not control, oligonucleotides showed an 80% reduction in proliferation and interleukin–2 production when co-stimulated [26••]. The antisense oligonucleotides reduced PDE7 activity, an effect that was reversed by an inhibitor of PKA, Rp-cAMP. Overall, the data suggest that PDE7 is necessary for the induction of T-cell proliferation and that PDE7 may be a useful drug target for the treatment of T-cell-mediated pathologies. Fertility and growth
diesterases regulate. One of the more satisfying aspects of these studies is that they have borne out the prevailing premise that distinct phosphodiesterases regulate specific cellular functions. We will describe several recent examples. Regulation of insulin secretion
It is thought that cAMP increases insulin secretion by activating protein kinase A (PKA), which inhibits an ATPsensitive potassium channel, and/or activates calcium channels [21]. Previous work has established that PDE3B mediates insulin-like growth factor (IGF)-1 control of insulin secretion from pancreatic B cells [22]. Regulation of insulin secretion by PDE3B, however, is not only limited to IGF-1, it is also mediated by leptin, thus establishing an alternative pathway to JAK/STAT (Janus kinase/signal transducer and activators of transcription) signaling for leptin in B cells [23••]. For example, leptin will inhibit insulin secretion stimulated by a cell-permeable cAMP analog that can be hydrolyzed by PDE3B but not by one that cannot be hydrolyzed, suggesting that PDE3B has a central role in mediating the effect. As with IGF-1, PDE3-specific inhibitors also blocked the effect of leptin. Although PDE4 inhibitors also raise cAMP levels in pancreatic B cells, they do not block the effect of leptin on insulin secretion. This suggests that PDE3B specifically modulates a discrete intracellular pool of cAMP that regulates insulin secretion. In support of this idea, AKAP (a kinase anchoring protein) targeting of a subset of PKA to
Although the above studies have relied on the acute inhibition of PDE activity by either pharmacological or antisense strategies, genetic manipulation of PDE activity has also proven to be a useful strategy for establishing new insights into PDE function. The function of PDE4D in mouse has now been examined by gene knockout studies [27••]. The resultant PDE4D-null mice display several interesting phenotypes including increased mortality, decreased size and body weight and reduced fertility. Increased mortality is most pronounced within the first four weeks postpartum, with approximately 40% of the null mice dying during this period. The underlying cause of the increased postnatal mortality is currently unknown, but is probably not due to genetic rearrangement during the recombination event, as an independently derived null strain also displayed a similar phenotype. Interestingly, surviving mice are growth retarded, with a 30–40% reduction in body weight at 2 weeks. This decreased body weight is caused by a decrease in bone, muscle and internal organ weight compared with wildtype littermates. Although it is not certain why the PDE4D-null mice fail to develop to a normal size, a possible cause may be the observed decrease in circulating IGF-1 in the null mice. Finally, female, but not male, mice exhibited a reduced fertility rate. Examination of oocytes recovered from the
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PDE4D-null mice revealed that more than 50% had overt signs of degeneration and fragmentation. Previous work has shown that PDE4D is expressed at high levels in murine granulosa cells [28] and PDE4D-null mice show a decrease in PDE4 activity in isolated granulosa cells. Somewhat surprisingly, when stimulated with FSH (follicle-stimulating hormone) the PDE4D-null granulosa cells do not display an increased cAMP accumulation as would be expected for cells with a lower cAMP PDE content. This was not due to a defect at the level of the cyclase as forskolin elicited a normal cAMP response. Although the reduced fertility observed in the PDE4D knockout mice may be due to a direct effect of PDE4D disruption in granulosa cells, it also seems plausible, on the basis of other studies [29], that this phenotype is related to the decreased circulating IGF-1.
regulate. Future work on both ‘new’ and ‘old’ phosphodiesterases should continue to provide additional exciting insights into the roles of PDEs in cAMP and cGMP signaling.
Penile erectile function
References and recommended reading
Upon sexual stimulation, penile nerve excitation releases nitric oxide (NO), relaxing vascular smooth muscle and allowing blood flow into the large corpora cavernosa and smaller corpus spongiosum leading to rigidity and erection. Erectile dysfunction may be caused by spinal injury, psychological issues or organic causes [30]. Although not life threatening, the psychological and social consequences of erectile dysfunction are serious, as previous labels have captured (‘impotence’ is from the Latin word meaning ‘without power’).
Papers of particular interest, published within the annual period of review, have been highlighted as:
Regardless of the cause of the dysfunction (including spinal injury), a number of studies have demonstrated the utility of stimulating the NO/cGMP pathway by inhibition of PDE5 for enhancing penile rigidity [31–33]. Inhibition of PDE5 can have dramatic effects on the cGMP content of the corpus cavernosum. Interestingly, sildenafil appears to have no direct effect on intracavernosal pressure in the absence of nerve stimulation, but instead only potentiates intracavernosal pressure that is induced by nerve stimulation [34•]. This corroborates clinical data suggesting that sildenafil enhances the natural male sexual response. Inhibition of PDE5 has thus changed the way in which erectile dysfunction is treated and has also established the importance of PDE5 in the regulation of a specific physiological process, penile erection.
Conclusions We have highlighted recent progress in the phosphodiesterase field related to the identification of previously unknown PDEs (see http://depts.washington.edu/pde/ for current PDE nomenclature updates), and to the characterization of some of the cellular functions that PDEs regulate. There has not been room to discuss other important studies in this burgeoning field, including PDE subcellular targeting (I Verde et al., unpublished data; [35,36••,37•]) and PDE regulation [38•,39•]. The past 18 months have expanded our appreciation of the number of distinct phosphodiesterases that exist and our understanding of the biology that previously identified PDEs
Update After submission of this manuscript the first report of a new PDE11 gene family was published [41••]. This isozyme hydrolyzes both cAMP and cGMP and contains a GAF domain. Little is yet known about its regulation.
Acknowledgements The authors would like to thank Elinor T Adman of the Department of Biological Structure at the University of Washington for her help in analyzing the PAS domain structure of PDEB. Work from the authors was supported by grants DK2173, HL60178 and HL4498 from the National Institutes of Health.
• of special interest •• of outstanding interest 1. ••
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6. •
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Soderling SH, Bayuga SJ, Beavo JA: Identification and characterization of a novel family of cyclic nucleotide phosphodiesterases. J Biol Chem 1998, 273:15553-15558. See annotation [8•]. 8. •
Guipponi M, Scott HS, Kudoh J, Kawasaki K, Shibuya K, Shintani A, Asakawa S, Chen H, Lalioti MD, Rossier C et al.: Identification and characterization of a novel cyclic nucleotide phosphodiesterase gene (PDE9A) that maps to 21q22.3: alternative splicing of mRNA transcripts, genomic structure and sequence. Hum Genet 1998, 103:386-392. These two papers [7•,8•] describe the PDE9 family in mouse [7•] and human [8•]. 9. •
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27. ••
Jin SL, Richard FJ, Kuo W, D’Ercole AJ, Conti M: Impaired growth and fertility of cAMP-specific phosphodiesterase PDE4D-deficient mice. Proc Natl Acad Sci USA 1999, 96:11998-12003. This study is the first publication of a PDE4 gene knockout in mouse. Although PDE4D is one of four similar cAMP PDEs (4A, 4B, 4C and 4D), these knockout mice do not appear to compensate for the loss of PDE4D by upregulation of other PDE4 members. Additionally, these mice show many interesting phenotypes including growth and fertility impairments. 28. Tsafriri A, Chun SY, Zhang R, Hsueh AJW, Conti M: Oocyte maturation involves compartmentalization and opposing changes of cAMP levels in follicular somatic and germ cells: studies using selective phosphodiesterase inhibitors. Dev Biol 1996, 178:393-402.
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17. •
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34. Carter AJ, Ballard SA, Naylor AM: Effect of the selective • phosphodiesterase type 5 inhibitor sildenafil on erectile dysfunction in the anesthetized dog. J Urol 1998, 160:242-246. This paper demonstrates the effect of inhibiting PDE5 on penile erection. It suggests that PDE5 does not regulate basal cGMP, but rather cGMP in response to sexual stimulation. 35. O’Connell JC, McCallum JF, McPhee I, Wakefield J, Houslay ES, Wishart W, Bolger G, Frame M, Houslay MD: The SH3 domain of Src tyrosyl protein kinase interacts with the N- terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). Biochem J 1996, 318:255-261. 36. Yarwood SJ, Steele MR, Scotland G, Houslay MD, Bolger GB: The •• RACK1 signaling scaffold protein selectively interacts with the cAMP-specific phosphodiesterase PDE4D5 isoform. J Biol Chem 1999, 274:14909-14917. This paper, along with [35], demonstrates the specific interaction of PDE4 isozymes with cellular scaffolding proteins. These studies help to define the emerging theme in the PDE field that that spatially distinct targeting of PDEs is likely to be important for regulation of cellular function. 37. •
Marzesco AM, Galli T, Louvard D, Zahraoui A: The rod cGMP phosphodiesterase delta subunit dissociates the small GTPase Rab13 from membranes. J Biol Chem 1998, 273:22340-22345. This study suggests that the function of the PDE6 delta subunit may not be limited to the solubilization of PDE6 in photoreceptors. It also suggests that the delta subunit may be targeted to subcellular compartments by a PDZ binding motif at the C-terminus. 38. Hoffmann R, Baillie GS, MacKenzie SJ, Yarwood SJ, Houslay MD: The • MAP kinase ERK2 inhibits the cyclic AMP-specific phosphodiesterase HSPDE4D3 by phosphorylating it at Ser579. EMBO J 1999, 18:893-903. The authors demonstrate that PDE4D3 can be regulated by ERK2. This in turn suggests that the MAP kinase and cAMP signaling systems may crosstalk at the level of PDE4.
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40. Morais JH, Lee A, Cohen SL, Chait BT, Li M, Mackinnon R: Crystal • structure and functional analysis of the HERG potassium channel N terminus: a eukaryotic PAS domain. Cell 1998, 95:649-655. The crystal structure of the amino-terminal HERG PAS domain, which probably regulates the gating properties of this potassium channel by an intra-
24. Lester LB, Langeberg LK, Scott JD: Anchoring of protein kinase A facilitates hormone-mediated insulin secretion. Proc Natl Acad Sci USA 1997, 94:14942-14947.
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molecular protein interaction, is presented. This structure is similar to the structures of the PAS domains of PYP and FixL. Additionally, mutagenesis suggests the HERG PAS domain can bind to other regions of the potassium channel by a hydrophobic patch located on the surface of the antiparallel β sheets. An analogous hydrophobic patch is found on the PDE8A PAS domain structure model (see Figure 2), suggesting that this may be a common protein interaction region.
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41. Fawcett L, Baxendale R, Stacey P, McGrouther C, Harrow I, •• Soderling SA, Helman J, Beavo JA, Phillips SC: Molecular cloning and characterization of a novel human phosphodiesterase gene family: PDE11A. Proc Natl Acad Sci USA 2000, in press. This isozyme hydrolyzes both cAMP and cGMP and contains a GAF domain. Little is known yet about its regulation.