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Glick Jen Glickr
Postdoctoral Student - Current research interests: Cyclic GMP (cGMP) made in response to atrial natriuretic peptide (ANP) or nitric oxide (NO) is an important regulator of short-term changes in smooth muscle tone and longer- term responses to chronic drug treatment or proliferative signals. The ability of smooth muscle cells (SMCs) to utilize different combinations of phosphodiesterase (PDE) isozymes allows cGMP to mediate these multiple processes. For example, PDE5 as a major cGMP-hydrolyzing PDE effectively controls the development of smooth muscle relaxation. In order for contraction to occur, PDE5 is activated and cGMP falls. Conversely, blockade of PDE5 activity allows the relaxation cycle to be prolonged and enhanced. A recently shown direct activation of PDE5 by cGMP binding to the GAF A domain suggests that this regulatory site might be a target for new drug development. The calcium surge associated with vasoconstrictor initiated contraction also activates a calcium/calmodulin-dependent PDE (PDE1A). Together, PDE5 and PDE1A lower cGMP sufficiently to allow contraction. Longer term, both PDE5 and PDE1A mRNA are induced by chronic stimulation of guanylyl cyclase. This induction is a major cause of the tolerance that develops to NO-releasing drugs. Finally, high levels of cGMP or cAMP also act as a brake to attenuate the proliferative response of SMCs to many mitogens. After vessel damage, in order for SMC proliferation to occur, the levels of cGMP and cAMP must be decreased. In humans this decrease is caused in large part by induction of another Ca2+/calmodulin-dependent PDE (PDE1C) that allows the brake to be released and proliferation to start.

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Mol Intervent. 2002 Sep;2(5):317-23. GAF Domains: Two-Billion-Year-Old Molecular Switches that Bind Cyclic Nucleotides. Martinez SE, Beavo JA, Hol WG.

GAF domains represent one of the largest families of small-molecule binding units present in nature. The first mammalian GAF domains discovered were the cGMP-binding regulatory domains of several cyclic nucleotide phosphodiesterases (PDEs). The crystal structure of the PDE2A GAF domains has provided our first look at the architecture of the binding site for the second messenger cGMP. The topology of this site differs greatly from all other previously determined cyclic nucleotide binding sites. In PDE2A, cGMP binds to a well-defined pocket in one of the two GAF domains that is analogous to the ligand-binding pocket of the distantly related PAS domains of photoactive yellow protein and FixL. The consensus cGMP-binding motif suggests strongly that only certain GAF domains will bind cGMP. Although the detailed mechanism for how cGMP binding to the GAF domain regulates catalysis remains to be determined, recent data from a GAF domain-containing cAMP-stimulated adenylyl cyclase from Anabaena suggest a mechanism conserved across two billion years of evolution. Because of their unique ligand-binding topologies, the GAF domains of PDEs are likely to offer good new targets for rational drug design.


Nature Reviews Sept 2002 3:710 "Cyclic nucleotide research - still expanding after 40 years" Beavo JA, Brunton LL.

Since the discovery in 1957 that cyclic AMP acts as a second messenger for the hormone adrenaline, interest in this molecule and its companion, cyclic GMP, has grown. Over a period of nearly 50 years, research into second messengers has provided a framework for understanding transmembrane signal transduction, receptor-effector coupling, protein- kinase cascades and downregulation of drug responsiveness. The breadth and impact of this work is reflected by five different Nobel prizes.

Recent research articles:

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Crystal Structure of the Tandem GAF Domains from a Cyanobacterial Adenylyl Cyclase: Novel Modes of Ligand-Binding and Dimerization (Martinez et al PNAS Feb, 2005)

ABSTRACT In several species, GAF domains, which are widely expressed small molecule binding domains that regulate enzyme activity, are known to bind cyclic nucleotides. However, the molecular mechanism by which cyclic nucleotide binding affects enzyme activity is not known for any GAF domain. In the cyanobacterium, Anabaena, the cyaB1 and cyaB2 genes encode adenylyl cyclases that are stimulated by binding of cAMP to their N-terminal GAF domains. Replacement of the tandem GAF-A/B domains in cyaB1 with the mammalian phosphodiesterase 2A GAF-A/B tandem domains allows regulation of the chimeric protein by cGMP suggesting a highly conserved mechanism of activation. Here we describe the 1.9 ŕ crystal structure of the tandem GAF-A/B domains of cyaB2 with bound cAMP and compare it to the previously reported structure of the PDE2A GAF-A/B. Unexpectedly, the cyaB2 GAF-A/B dimer is antiparallel, unlike the parallel dimer of PDE2A. Moreover, there is clear electron density for cAMP in both GAF-A and GAF-B whereas in PDE2A, cGMP is found only in GAF-B. Phosphate and ribose group contacts are similar to those in PDE2A. However, the purine-binding pockets appear very different from that in PDE2A GAF-B. Differences in the ɿ2-ɿ3 loop suggest that this loop confers much of the ligand specificity in this and perhaps in many other GAF domains. Finally, a conserved asparagine appears to be a new addition to the signature NKFDE motif and a mechanism for this motif to stabilize the cNMP binding pocket is proposed.

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Differentiation of human monocytes in vitro with granulocyte-macrophage colony- stimulating factor and macrophage colony-stimulating factor produces distinct changes in cGMP phosphodiesterase expression. Bender AT, Ostenson CL, Giordano D, Beavo JA. Cell Signal. 2004 Mar;16(3):365-74.

The cytokines macrophage colony-stimulating factor (M-CSF) and granulocyte- macrophage colony-stimulating factor (GM-CSF) promote differentiation of monocytes into macrophages with distinct phenotypes and unique functional abilities. In this report, we characterize how monocytes and macrophages differentiated from monocytes with M- CSF and GM-CSF regulate their cGMP levels by controlling which phosphodiesterases (PDEs) and guanylyl cyclases (GCs) are expressed. We find that PDE1B and PDE2A are expressed at low levels in monocytes, but are the major cGMP PDEs expressed in macrophages. M-CSF differentiation triggers increased expression of PDE1B and PDE2A, while GM-CSF causes a large increase only in PDE1B. Based on PDE expression, we identified THP-1 and U937 cell lines as possible models for studying the roles of PDE1B and PDE2A in macrophage function. We additionally characterized changes in expression of GCs upon differentiation. We found that GM-CSF differentiation triggers a small decrease in soluble guanylyl cyclase (sGC) and a large increase in GC-A, while M-CSF significantly decreases sGC.

J Immunol. 2003 Dec 15;171(12):6421-30. Cyclic nucleotides promote monocyte differentiation toward a DC-SIGN+ (CD209) intermediate cell and impair differentiation into dendritic cells. Giordano D, Magaletti DM, Clark EA, Beavo JA.

Recruitment of monocytes into tissues and their differentiation into macrophages or dendritic cells (DCs) depend on the microenvironment of the inflammatory site. Although many factors affecting this process have been identified, the intracellular signaling pathways implicated are poorly understood. We found that cyclic nucleotides regulate certain steps of monocyte differentiation into DCs. Increased levels of the cyclic nucleotides, cAMP or cGMP, inhibit differentiation of CD14(+)/CD1a(low) monocytes into CD14(-)/CD1a(high) DCs. However, DC-specific ICAM-3-grabbing nonintegrin (CD209) up-regulation was not affected by cyclic nucleotides, indicating that DC development was not blocked at the monocyte stage. Interestingly, Ag-presenting function was increased by cyclic nucleotides, as measured by the higher expression of MHC class II, CD86, and an increased ability to stimulate CD4(+) T cell proliferation in allogeneic MLRs. Although cyclic nucleotides do not completely block DC differentiation, they do block the ability of DCs to be induced to mature by LPS. Treatment during DC differentiation with either cAMP or cGMP analogues hampered LPS-induced expression of CD83, DC-LAMP, and CCR7 and the ability of DCs to migrate toward CCL19/macrophage-inflammatory protein 3beta. Interestingly, the induction of a CD16(+) subpopulation of cells was also observed. Thus, signals causing an increase in either cAMP or cGMP levels during monocyte recruitment to inflammatory sites may restrain the activation of acquired immunity by blocking DC development and migration to lymph nodes. At the same time, these signals promote development of an active intermediate cell type having properties between those of macrophages and DCs, which might contribute to the innate immune response in the periphery.

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The two GAF domains in phosphodiesterase 2A have distinct roles in dimerization and in cGMP binding. Proc Natl Acad Sci U S A. 2002 Oct 1;99(20):13260-5 Martinez SE, Wu AY, Glavas NA, Tang XB, Turley S, Hol WG, Beavo JA.

Cyclic nucleotide phosphodiesterases (PDEs) regulate all pathways that use cGMP or cAMP as a second messenger. Five of the 11 PDE families have regulatory segments containing GAF domains, 3 of which are known to bind cGMP. In PDE2 binding of cGMP to the GAF domain causes an activation of the catalytic activity by a mechanism that apparently is shared even in the adenylyl cyclase of Anabaena, an organism separated from mouse by 2 billion years of evolution. The 2.9-A crystal structure of the mouse PDE2A regulatory segment reported in this paper reveals that the GAF A domain functions as a dimerization locus. The GAF B domain shows a deeply buried cGMP displaying a new cGMP-binding motif and is the first atomic structure of a physiological cGMP receptor with bound cGMP. Moreover, this cGMP site is located well away from the region predicted by previous mutagenesis and structural genomic approaches.

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Cloning and characterization of a cAMP-specific phosphodiesterase (TbPDE2B) from Trypanosoma brucei. Rascon A, Soderling SH, Schaefer JB, Beavo JA. Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4714-9.br>

Here we report the cloning, expression, and characterization of a cAMP-specific phosphodiesterase (PDE) from Trypanosoma brucei (TbPDE2B). Using a bioinformatic approach, two different expressed sequence tag clones were identified and used to isolate the complete sequence of two identical PDE genes arranged in tandem. Each gene consists of 2,793 bases that predict a protein of 930 aa with a molecular mass of 103.2 kDa. Two GAF (for cGMP binding and stimulated PDEs, Anabaena adenylyl cyclases, and Escherichia coli FhlA) domains, similar to those contained in many signaling molecules including mammalian PDE2, PDE5, PDE6, PDE10, and PDE11, were located N-terminal to a consensus PDE catalytic domain. The catalytic domain is homologous to the catalytic domain of all 11 mammalian PDEs, the Dictyostelium discoideum RegA, and a probable PDE from Caenorhabditis elegans. It is most similar to the T. brucei PDE2A (89% identity). TbPDE2B has substrate specificity for cAMP with a K(m) of 2.4 microM. cGMP is not hydrolyzed by TbPDE2B nor does this cyclic nucleotide modulate cAMP PDE activity. The nonselective PDE inhibitors 3-isobutyl-1-methylxanthine, papaverine and pentoxifyline are poor inhibitors of TbPDE2B. Similarly, PDE inhibitors selective for the mammalian PDE families 2, 3, 5, and 6 (erythro-9-[3-(2-hydroxynonyl)]- adenine, enoximone, zaprinast, and sildenafil) were also unable to inhibit this enzyme. However, dipyridamole was a reasonably good inhibitor of this enzyme with an IC50 of 27 microM. cAMP plays key roles in cell growth and differentiation in this parasite, and PDEs are responsible for the hydrolysis of this important second messenger. Therefore, parasite PDEs, including this one, have the potential to be attractive targets for selective drug design

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Science 1999 Feb 5;283(5403):848-51 CD3- and CD28-dependent induction of PDE7 required for T cell activation. Li L, Yee C, Beavo JA

Costimulation of both the CD3 and CD28 receptors is essential for T cell activation. Induction of adenosine 3',5'-monophosphate (cAMP)-specific phosphodiesterase-7 (PDE7) was found to be a consequence of such costimulation. Increased PDE7 in T cells correlated with decreased cAMP, increased interleukin-2 expression, and increased proliferation. Selectively reducing PDE7 expression with a PDE7 antisense oligonucleotide inhibited T cell proliferation; inhibition was reversed by blocking the cAMP signaling pathways that operate through cAMP-dependent protein kinase (PKA). Thus, PDE7 induction and consequent suppression of PKA activity is required for T cell activation, and inhibition of PDE7 could be an approach to treating T cell-dependent disorders.

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Individual cerebellar Purkinje cells express different cGMP phosphodiesterases (PDEs): in vivo phosphorylation of cGMP-specific PDE (PDE5) as an indicator of cGMP- dependent protein kinase (PKG) activation. Shimizu-Albergine M, Rybalkin SD, Rybalkina IG, Feil R, Wolfsgruber W, Hofmann F, Beavo JA. J Neurosci. 2003 Jul 23;23(16):6452-9.

The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed. Although expression of guanylate cyclase and/or cGMP- dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cells in vivo has been demonstrated. To determine if changes in PKG activation and PDE5 regulation occur in vivo we have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylation in vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as an in vivo indicator for PKG activation.

Some cool pictures from one of Dawn Juilfs papers. Notice the different localization of different PDEs in various parts of the olfactory neurons.
Juilfs et al. (PNAS 94:3388-3395)


The data in this manuscript illustrate that different PDEs are localized to different regions of single olfactory neurons and also that a new subset of neurons expressing PDE2 and GCD are present in olfactory epithelia. These latter neurons do not appear to innervate a single glomerulus as is the case for most others that have been described to date. Rather they project to a ring like set of "necklace glomeruli" that surround the accessory olfactory bulb.

PDE1,2, & 4 Triple Stain Photo

Section of Olfactory Epithelium
This photo shows a immunocytochemical staining of the caudal portion of an olfactory turbinate triple stained for PDE1C2 (blue), PDE2A (red), and PDE4A (green). Note that the cilia of most neurons contain PDE1C2 while the dendrites, cell bodies, and axon bundles contain PDE4A. The red olfactory neurons do not contain either PDE1C2 or PDE4A. They do, however, contain guanylyl cyclase D (see later photo)

PDE1C2 and PDE4 photo

Olfactory Neurons
This photo shows a blow-up of an olfactory neuron stained for PDE1C2 (red) and PDE4A (green). Note the cilia containing PDE1C arising from the dendrites of individual neurons expressing PDE4 .

Co-expression of PDE2 and Guanylyl Cyclase D (GCD) in the same neurons

GCD & PDE2 Coexpression
Merged image of PDE2A staining (red) and guanylyl cyclase D (GCD) staining (green) of a subset of neurons located in the posterior part of the nasal epithelium. Note that PDE2 is present in the cell bodies of all neurons that are positive for GCD and that it is present in the cilia of most of them. Yellow color indicates co-expression.

 

 

PDE2 Neurons Project to Bulb

PDE2 expressing neurons projecting to olfactory bulb
Note the axon bundles projecting to multiple areas of the olfactory bulb. These axon bundles appear to terminate on one of a dozen or so glomerular like structures called the necklace glomeruli that encircle the accessory olfactory bulb.

 



 

 

 


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