TITLE PAGE

Structural and Functional Analysis of Conserved

Amino Acid Residues in Phosphodiesterase

Isoforms and Identifying Targets for Drug Discovery

Using Bioinformatics.

B. Sc. (Hons.), Biomedical Science, Anglia Ruskin University

Author: Sayeed (Rafi) Ali

Supervisor: Nick Pugh

ABSTRACT

Cyclic nucleotide phosphodiesterases (PDEs) are a large family of protein enzymes that regulate the cellular levels of the second messengers, cAMP and cGMP, by controlling their

rates of degradation. To date, 11 different PDE families have been recognized, with each family typically having several different isoforms and splice variants, which modulate

distinct regulatory pathways in cells. These properties therefore offer the opportunity for selectively targeting specific PDEs for treatment of specific disease states. This article

presents the structural and functional roles of conserved amino acid residues within the catalytic and GAF domains of PDEs. Particular residues and motifs conserved in the catalytic domains of PDE9 and PDE3 isoforms are aligned and discussed as possible allosteric cyclic nucleotide binding regions which may offer targets for drug development. This includes a divergent residue in PDE9 and a unique 44 amino-acid insert that is only found within the catalytic domain of PDE3 isoforms. The percentage sequence identity of several PDE-GAF domains are also presented which show a positive correlation between domains that are involved in cyclic nucleotide-binding. With the help of online genetic databases, sequence alignment tools, and research studies, a clearer understanding of the specific amino-acids

involved in substrate/inhibitor binding is conveyed.

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ACKNOWLEDGEMENTS

I would like to thank my mother and father for being a constant support throughout my three years at Anglia Ruskin University, without whom this would not have been possible.

I would also like to thank my supervisor Nicholas Pugh for his input and guidance in carrying out this project. I have thoroughly enjoyed working on this article and hope the work presented below is of benefit to the reader.

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TABLE OF CONTENTS

TITLE PAGE...............................................................................................................................1

ACKNOWLEDGEMENTS............................................................................................................3

TABLE OF CONTENTS............................................................................................................... 4

LIST OF FIGURES.......................................................................................................................6

ABBREVIATIONS.......................................................................................................................7

1.0 INTRODUCTION................................................................................................................8

1.1 PDE STRUCTURE AND REGULATION.........................................................................................11

1.12 GAF DOMAIN IN PDE.........................................................................................................13

1.13 GLUTAMIN SWITCH...........................................................................................................15

1.14 ENZYMATIC REGULATION.................................................................................................16

2.0 PDE INHIBITORS.............................................................................................................17

3.0 PHOSPHODIESTERASE IN PLATELETS - (PDE3 Specific)..................................................18

4.0 METHODS AND MATERIALS...........................................................................................20

4.1 SEQUENCE ALIGNMENT OF PDE FAMILIES...............................................................................20

4.2 IDENTIFACTION OF DOMAINS & 44 AMINO ACID INSERT IN PDE3.........................................22

4.3 AMINO ACID FREQUENCY PLOTS.............................................................................................23

5.0 RESULTS.........................................................................................................................24

5.1 SEQUENCE ALIGNMENT OF PDE FAMILIES..............................................................................24

5.2 UNIQUE FEATURE OF PDE9 CATALYTIC DOMAIN................................................................24

5.3 DIFFERENCE IN cAMP AND cGMP CATALYTIC DOMAIN STRUCTURE...................................25

5.4 ANALYSIS OF GAF DOMAINS................................................................................................26

5.5 44 AMINO ACID INSERT OF PDE3............................................................................................29

6.0 DISCUSSION...................................................................................................................32

6.1 DIVERGENT RESIDUES IN PDE9 CATALYTIC DOMAIN...............................................................33

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6.2 SUBSTRATE/INHIBITOR SPECIFICITY OF PDE4..........................................................................34

6.3 ANALYSIS OF PDE GAF DOMAINS.............................................................................................35

6.4 FUNCTIONAL ROLE OF THE CATALYTIC INSERTION IN PDE3....................................................37

7.0 CONCLUSION.................................................................................................................39

BIBLIOGRAPHY.......................................................................................................................41

APPENDIX – A........................................................................................................................ 46

Amino Acid Sequence of Human PDE1-11 Isoforms........................................................................46

APPENDIX - B......................................................................................................................... 52

Full PDE Family Sequence Alignment (1A-11A)................................................................................52

PDE3A – Sequence Alignment [Homo sapiens, Rattus norvegicus, Mus musculus, and Sus scrofa] 57

PDE3B – Sequence Alignment [Homo sapiens, Rattus norvegicus, Mus musculus].........................59

GAF DOMAIN SEQUENCE ALIGNMENT............................................................................................61

Raw Data - Amino Acid Frequency – PDE3A & PDE3B.....................................................................62

PDE3A..........................................................................................................................................62

PDE3B..........................................................................................................................................63

APPENDIX – C.........................................................................................................................64

Pairwise Sequence Alignment of PDE3 using Smith-Waterman Algorithm......................................64

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LIST OF FIGURES

Figure 1- Cyclic nucleotide hydrolysis.................................................................................................................10

Figure 2 –Stereo ribbon diagram of a phosphodiesterase structure (PDE5)......................................................13

Figure 3 – Imaging representation of PDE(5A) active site occupied by sildenafil. ………………………………………….14

Figure 4 - Domain organisation of mammalian PDEs.......................................................................................15

Figure 5 - catalytic domain structure of PDE5 showing the hypothesised hydrogen bond interaction between amide side chains and the guanine ring of 5'GMP (reaction product)..............................................................17

Figure 6 – NCBI Protein database.......................................................................................................................22

Figure 7 – Clustal Omega Multiple Sequence Alignment Tool..........................................................................23

Figure 8 – BLAST search and results...................................................................................................................24

Figure 9 - EMBOSS Water Tool...........................................................................................................................25

Figure 10 – Sequence of PDE9A and unique features of catalytic domain. …………………………………………………..26

Figure 11 – Alignment of PDE4 with consensus sequence for cAMP-specific PDEs and consensus sequence for cGMP-specific PDEs............................................................................................................................................27

Figure 12 – Clustal O sequence alignment of GAF1 and GAF2 domains of mammalian PDEs…………………………28

Figure 13 - The relatedness of human PDE GAF domains. A,.............................................................................29

Figure 14 – Mean percentage identities between all PDE-GAF domains...........................................................30

Figure 15 –PDE3A Protein sequence & identification 44 amin-acid insert........................................................31

Figure 16 – Sequence alignment of 44-amino acid insert in PDE3 gene family for human, Rattus norvegicus (Rat), Mus musculus (Mouse), and Sus scrofa (Pig)...........................................................................................32

Figure 17 – Shows a triplet of amino-acid comparisons between human and mouse PDE3. A, Amino Acid Frequency of HsPDE3 isoforms...........................................................................................................................33

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Figure 18 - Hypothesized interactions of cGMP with the putative NKXnD motif..............................................38

ABBREVIATIONS

PDE – Phosphodiesterase

PDEI – Phosphodiesterase inhibitor

cGMP – Cyclic guanosine monophosphate

cAMP – Cyclic adenosine monophosphate

cNMP – Cyclic nucleotide monophosphate

cN – Cyclic nucleotide

PKA – Protein Kinase A

PKB – Protein Kinase B

PKG – Protein Kinas G

CaM – Calmodium

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1.0 INTRODUCTION

Cyclic nucleotide phosphodiesterases (PDEs) are protein enzymes that can be found ubiquitously

across human tissue which control cellular levels of second messenger molecules cyclic adenosine

monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) by regulating their rates of

degradation (Bender, 2006). These cyclic nucleotides are of pivotal importance, and along with

intracellular calcium and IP3, they control many key cellular functions within the body such as

metabolic function, cell proliferation, and cell-cycle regulation. PDEs regulate the amplitude, duration

and compartmentation of cyclic nucleotide activity by selectively catalysing the hydrolysis of 3’-5’ –

cyclic nucleotides to the corresponding 5’ –monophosphate nucleoside (Fig.1), and by doing so make

them inactive (Authi, Bruchhausen and Walter,1997).The portrayal of PDE activity was initially

presented by Butcher & Sutherland in 1962, shortly after the discovery of 3’-5’ – cyclic

monophosphate by Dr. Earl Sutherland, during which time the focus was on the characterisation of

biochemical activity of PDE and the determination of their functional role (Beavo, 1995). Currently

there are 11 different but homologous PDE families recognised (PDE1 - PDE11), with each family

also having multiple isoforms and splice variants (Omori and Kotera, 2007). These unique PDE

families and isoforms differ in their three-dimensional structure, modes of regulation, cellular

expression, kinetic properties, intracellular localisation, and substrate selectivity. PDEs 1, 2, 3, 10, and

11 degrade both cAMP and cGMP to 5’ –monophosphate nucleoside, whereas PDEs 4, 7, and 8

selectively degrade only cAMP, and PDE 5, 6, and 9 selectively degrades cGMP (Lugnier, 2006)

(Table. 1)

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Figure 1- Cyclic nucleotide hydrolysis to 5’-monophosphate nucleoside by cyclic nucleotide phosphodiesterase

The classification of PDEs 1-11 was recognised according to their biochemical properties, position of

the genes of which they are products, sequence homology, regulation, and their sensitivity to

pharmacological agents (Lugnier, 2006). Within the C-terminal catalytic domain, which establishes the

core of PDE, there are approximately 270 conserved amino acids (AA), with up to a 50% sequence

identity among different PDE families (Omori and Kotera, 2007). Some PDE families are composed of

2 to 4 subfamily genes with more than 70% sequence similarity and identical organisation of protein

domains. The identification of multiple transcriptional products arising from alternative splicing and

promotor transcription of PDE genes now show that the PDE superfamily is composed of 21 genes,

engineering more than 50 PDE variants (M. Essayan, 2001).

PDE Family (no. of genes)

Substrate Property Primary Tissue Distribution Specific Inhibitors

PDE1 (3) cAMP, cGMP

Ca-calmodulin-activated

Heart, brain, lung, smooth muscle

Nimodipine

PDE2 (1) cAMP, cGMP

cGMP-activated, Adrenal gland, heart, lung, liver, platelets

EHNA

PDE3 (2) cAMP, cGMP

cGMP-inhibited Heart, lung, liver, platelets, adipose tissue, immunocytes

Cilostamide, milrinone

PDE4 (4) cAMP cGMP-insensitive Sertoli cells, kidney, brain, liver, lung, immunocytes

Rolipram, Ro 20-1724, roflumilast

PDE5 (1) cGMP PKA/PKG-phosphorylated

Lung, platelets, smooth muscle Zaprinast, DMPPO, E4021, Sildenafil

PDE6 (3) cGMP Transducin-activated Photoreceptors Zaprinast, DMPPO, E4021, Sildenafil

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PDE7 (2) cAMP Rolipram-insensitive Skeletal muscle, heart, kidney, brain, pancreas, T lymphocytes

BRL 50481, ICI242

PDE8 (1) cAMP Rolipram-insensitive IBMX-insensitive

Testes, eye, liver, skeletal muscle, heart, kidney, ovary, brain, T lymphocytes

Unknown

PDE9 (1) cGMP IBMX-insensitive Kidney, liver, lung, brain Unknown

PDE10 (1) cAMP, cGMP

Unknown Testes, brain Unknown

PDE11 (1) cAMP, cGMP

Unknown Skeletal muscle, prostate, kidney, liver, pituitary and salivary glands, testes

Unknown

Table 1 - Classification of PDE family showing substrate specificity, tissue distribution and selective inhibitors

To characterize each PDE isozyme (eg, PDE4A1), a specific nomenclature is used. The first number

after PDE indicates the gene family. The capital letter following the family number designates a

separate subfamily gene. The last number following the subtype letter represents the isoform. This

extensive range of PDE proteins allows specialised intracellular localisation of PDEs in the vicinity of

multiple protein effectors encouraging enhancement of compartmentalized regulation for cAMP and

cGMP (Lugnier, 2006).

PDEs are expressed in a number of different tissues (Table. 1) where they are responsible for

governing normal and pathological cell responses and influence a number of biological processes

including muscle contraction, production and action of pro-inflammatory mediators, ion channel

function, apoptosis, gluconeogenesis, and lipogenesis just to name a few (Perry and Higgs, 1998).

The importance of PDE in governing these essential reactions has led to PDEs becoming recognized

as important drug targets for treatment of various diseases, such as, depression, heart failure, asthma

and most famously erectile dysfunction. As intracellular levels of cyclic nucleotides increase, they

activate protein kinase A (PKA) and protein kinase G (PKG) by binding to these target enzymes (Jeon

et al., 2005). These protein kinases are liable for phosphorylating substrates such as contractile

proteins, ion channels, and transcription factors, which regulate crucial cellular functions.

Phosphorylation consequently changes the activity of these substrates and therefore alters cellular

activity (Krebs and Beavo, 1979). Evidently, altering the rate of cyclic nucleotide accumulation,

formation or degradation by PDE will change the activity of these pathways.

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This paper aims to analyse the amino acid sequence and coding regions of multiple PDE molecules

and splice variants, in order to study the function of conserved residues in various domains and

subdomains that characterizes the PDE structure and function. The primary source of data used in

this article are gathered from online biotechnology databases such as NCBI and UniProt, and

analysed using bioinformatics programmes such as Clustal and ExPASy. Hence, by using

bioinformatics, we hypothesize that it is possible to characterise the function(s) of specific amino-acids

of PDEs that may ultimately lead to pharmacological advances in producing PDE specific inhibitors to

treat various diseases without the risk of hindering the effects of other PDE families.

1.1 PDE STRUCTURE AND REGULATION

As mentioned above the catalytic domain of PDEs encompass a region of ∼ 270 AAs. There are also

other common shared structural elements among different PDE families. Including the catalytic core,

PDEs contain three functional domains, which comprise of a regulatory N-terminus and the C-terminal

(Bolger, 1994). The amino terminal varies quite extensively among the different classes of PDEs and

is bordered by the catalytic core. It contains regions that auto-inhibit the catalytic domains and targets

specific sequences that regulate subcellular localization (Houslay and Adams, 2003). The N-terminal

contains a calmodium binding domain (CaMBD) In PDE1, and cGMP binding sites in the same region

of PDE2. In PDE6 there is a transduction binding domain, and in PDEs 1-5, there are various

phosphorylation sites for numerous protein kinases (Degerman, Belfrage and Manganiello, 1997).

Several studies have manage to elucidate the structure of the catalytic domains of PDEs, which

encloses the active site that inhibitors bind to, in order manufacture inhibitors for selective PDEs as

therapeutic drugs. Crystal catalytic domain structures of PDE1B (Zhang et al., 2004), PDE3B (Scapin

et al., 2004), PDE4B (Xu, 2000 & Xu et al., 2004), PDE4D (Lee et al., 2002 & Huai et al., 2004),

PDE5A (Zhang et al., 2004), and PDE10A (Huai et al., 2004) have provided considerable evidence

that there are three helical sub-domains to the catalytic domain of PDE (Fig.2).

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Figure 2 –Stereo ribbon diagram of a phosphodiesterase structure (PDE5). The three subdomains of the catalytic core are identified: N-terminal cyclin fold domain (yellow), a linker helical domain (blue) and a C-terminal helical bundle domain (violet). The green spheres represent two metal ions.

At the interface of the three catalytic core subdomains lies a profound hydrophobic pocket consisting

of four locums: a metal-binding site (M site), hydrophobic region (H pocket), core pocket (Q pocket)

and lid region (L region) (Degerman, Belfrage and Manganiello, 1997) (Fig.3). Numerous metal atoms

are located at the bottom of the pocket proximate to the M site. These metal atoms bind to specific

residues that are entirely conserved in all PDE family members. Metal coordinate ligand geometry and

X-ray diffraction behaviour suggest that the ions are Zn2+ and Mg2+ with an octahedral coordination

geometry (Francis et al., 1994). Organization of the zinc sphere consists of three histidine molecules,

one aspartate and two water molecules, whereas the coordination of the magnesium sphere contains

one aspartate and five water molecules, one of which is shared with the zinc molecule. Although the

exact roles of these metal ions are not concrete, it is presumed that they are involved in stabilization

of the PDE structure and activation of hydroxide to mediate catalysis (Jeon et al., 2005).

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Figure 3 – Imaging representation of PDE(5A) active site occupied by sildenafil. The four sub-sites of the active site are presented: metal-binding site (M site), core pocket (Q pocket), hydrophobic pocket (H pocket) and lid region (L region).

1.12 GAF DOMAIN IN PDE

Five members of the PDE family have regulatory segments called the GAF domains. These GAF

domains are one of the most widespread and largest domain families found across the animal

kingdom (Heikaus et al., 2009 and Anantharaman et al., 2001), however, they are rare in human

proteins. The PDE family is the only protein family that have a GAF domain present in the humans, of

which there are ~14 GAF domain-comprising proteins (Schultz et al., 1998), all of which have an array

of functions which include protein to protein interactions and binding of small molecules. In the PDE

family, these GAF domains are present in PDE2, PDE5, PDE6, PDE10 and PDE11 and are therefore

labelled the GAFPDE subfamily (Fig. 4). The acronym, GAF, originates from the first three such

classes of proteins that were identified to harber this domain: mammalian cGMP-binding PDEs,

Anabaena adenylyl cyclases, and Escherichia coli FhIA (Zoraghi, 2004).

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Figure 4 - Domain organisation of mammalian PDEs. The conserved catalytic domain (shown in red) is located in the carboxyl-terminal portion of the phosphodiesterases (PDEs). The catalytic domain of PDE3 contains a unique 44-amino-acid

insert (shown in black). Many of the PDE families1, 2, 3 contain amino-terminal subdomains (such as GAF domains, transmembrane domains, targeting domains, upstream conserved regions (UCRs), PAS domains and REC domains) and N-

terminal hydrophobic regions that are important in subcellular localization, in the incorporation of PDEs into compartmentalized signalosomes, in interactions with signalling molecules and molecular scaffolds, and in the regulation of PDE activity. GAF domains regulate the allosteric binding of cGMP (to PDE2, PDE5, PDE6 and PDE11), the allosteric binding of cAMP (to PDE10) and the regulation of catalytic activity (in PDE2, PDE5 and PDE6). Phosphorylation sites are labelled on

the figure. CaMKII, calcium/calmodulin-dependent protein kinase II; ERK2, extracellular signal-regulated kinase 2; PKA, protein kinase A; Pat7, 7-residue nuclear localization signal.

GAF motifs in PDEs are composed of two segments of homologous sequences of ~110AAs arranged

in tandem, separated by approximately 70 AAs (Aravind and Ponting, 1997). The domain located

nearer the N-terminal is termed GAF 1 and the more C-terminal located domain is termed GAF 2

(Heikaus et al., 2009).

Although there are two GAF domains present in each GAFPDE-subfamily monomer, only one domain

in each has been shown to bind with cyclic-nucleotide monophosphates (cNMPs). Cyclic-GMP binds

to the GAF1 domains of PDE5, PDE6, and PDE11 and to the GAF2 domain of PDE2. Cyclic-AMP

selectively binds to the GAF2 domain of PDE10 (Zoraghi, 2004). It is known that the GAF domain is

responsible for cGMP binding-mediated allosteric regulation and dimerization of GAF PDEs (Bender,

2006). The catalytic activity of PDE2 and PDE5 are increased by the binding of cGMP to their

respected GAF domains (Rybalkin, 2003). In PDE5, allosteric cGMP binding enhances

phosphorylation through the cGMP-dependant protein kinase, which in turn increases PDE5 activity

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and cGMP binding affinity of GAF1 (Francis et al., 2002). Thus, elevation of intracellular cGMP

provides negative feedback control and enhances its own destruction via direct, cGMP-induced

allosteric activation of PDE5 and indirect activation due to phosphorylation by PKG. On the other

hand, inhibition of PDE5 by PDE5 inhibitors can increase cGMP, which binds to GAF domains; this, in

turn, increases binding of inhibitors to the catalytic site, thus providing positive feedback with respect

to the potentiation of cGMP accumulation by PDE5 inhibitors (Manganiello, 2004). Binding of cGMP to

the GAF1 domain of PDE6 increases affinity for the Pγ-subunit, an intrinsically disordered protein that

inhibits the catalytic activity of PDE6 when bound (Song et al., 2008), and alters the affinity for certain

catalytic site inhibitors (Zhang et al., 2008). Less is known about the GAF domain-dependent

regulatory mechanisms of PDE10 and PDE11. Binding of cAMP to the GAF B domain of full-length

PDE10A2 and binding of cGMP to the GAF A domain of full-length PDE11A4 has recently been

demonstrated (Matthiesen and Nielsen, 2009). PDEs1, 3, 4, 7, 8, and 9 belong to the non-GAF-PDE

subfamily. These PDEs have alternative binding sites and domains which regulate their activity and

function (see 1.14 Enzymatic Regulation).

An alignment of the GAF domains of all primary HsGAF-PDEs is carried out in this article to look for

highly conserved amino-acids and conserved motifs within the domains. Identification of detailed

residues can offer insight into the selective binding of specific cyclic nucleotides to the GAF domain

which is vital for cAMP and cGMP targeting.

1.13 GLUTAMIN SWITCH

The molecular mechanism for cyclic nucleotide specificity involves an invariant glutamine molecule

which stabilizes the binding of the purine ring in the binding pocket (Zhang et al., 2004) (Fig.4). In

order for correct hydrogen bonds to attach to both cAMP and cGMP, the glutamine molecule

essentially needs to rotate freely. It is hypothesized that in PDEs that are capable of hydrolyzing both

cyclic nucleotides, the ɣ-amino group of the conserved glutamine molecule in the active site of the

PDE is free to rotate and adopt two different orientations. For PDEs that selectively hydrolyze cAMP

at low substrate level, the glutamine is reserved by adjacent residues into the chosen orientation for

binding adenine, resulting in cAMP specificity. On the contrary, in cGMP hydrolyzing PDEs, the

glutamine is constrained in the position that favors guanine binding and consequently cGMP

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specificity. An exception to this hypothesis has recently been illustrated by Zoraghi et al, (2006) which

shows that PDE5 may have further interactions within the glutamine molecule that are important for

specificity. Ultimately, this “glutamine switch” occurs in PDEs that hydrolyze both cAMP and cGMP

and forms the molecular basis for much of the substrate selectivity eminent among different PDEs.

By using bioinformatics, this article aims to identify conserved amino acids within the PDE family

sequences that may be involved in preventing this glutamine switch which causes certain PDE

families to be cAMP or cGMP specific. Identification of such residues would allow for pharmacological

production of selective PDE inhibitors which bind specifically to the targeted PDE. This would enable

greater efficacy in the treatment of PDE related disease and improve the treatment by reducing side-

effects that arise from inhibitors targeting unspecified PDEs.

Figure 5 - catalytic domain structure of PDE5 showing the hypothesised hydrogen bond interaction between amide side chains and the guanine ring of 5'GMP (reaction product). The steric constraints does not allow the rotation of the glutamine

side chain and therefore PDE5A is selective for cGMP over cAMP. In PDE4A, the glutamine side chain is flipped, allowing

selectivity for cAMP, the prevention of the glutamine rotation is mediated by a different set of side chains. In both cAMP and cGMP hydrolysing PDEs, the glutamine is not fixed and is free

to rotate to bind to either cyclic nucleotide. (Figure edited from Jeon et al, (2005)).

1.14 ENZYMATIC REGULATION

[Table 2 displays the list of PDE regulators: kinases and association proteins]. PDE1 activity is

stimulated by the binding of Ca2+/CaM to the N-terminal binding site. This in vitro regulation has led to

speculation that all PDE1s function in cell signal pathways mediated by cAMP and cGMP with

pathways that regulate intracellular calcium levels (Bender, 2006). PKA-dependent and CaM kinase

II-dependent phosphorylation reduces the binding affinities of PDE1A and PDE1B, respectively

(Omori and Kotera, 2007). Geoffroy et al., (1999) reported the activation of PDE2A in the Golgi

endosomal fraction by Ca2/phospholipid-dependent protein kinase, there is however no evidence of

PDE2A phosphorylation. PDE2A2 on the other hand is phosphorylated by associated protein kinases

which inhibits activity (Bentley, 2005). The enzymatic alteration associated with PDE2 phosphorylation

have still not been reported. Activation of PDE3s are reported in the following section (see

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Phosphodiesterases in Platelets). Activity of PDE4 is regulated by PKA in the UCR1 region (Fig.5)

and also by extracellular signal-regulated kinase (ERK) in the C-terminal region however, regulation is

dependent on specific isoforms of PDE4 (Conti, 1996, Houslay and Adams, 2003).

It is thought that PKA phosphorylation of PDE4D3 and PDE4A4 (long forms) is caused by disruptions

of the UCR1-UCR2 interaction and/or by UCR2 interactions within the catalytic region (Lim, 1999,

Richter and Conti, 2002). A consensus motif, RXSP, situated within the catalytic domains of PDE4B,

PDE4C, and PDE4D is phosphorylated by ERK in assistance with a kinase interaction motif (KIM).

Cyclic AMP hydrolytic activity is reduced by up to 50% during ERK phosphorylation of PDE4D3 (long

form), which consequently leads to the activation of PKA. Accordingly, this leads to PKA

phosphorylation of the long form PDE4s which are emitted from ERK-mediated inhibition (MacKenzie,

2000, Omori and Kotera, 2007). PDE5 is also regulated by PKA/PKG phosphorylation. A report by

Corbin et al., (2000) showed that PKA and PKG phosphorylation of PDE5A1 at Ser102 increased

PDE5 activity. Copurification of PDE6γ with PDE5 have been reported (Lochhead et al., 1997),

however, the physiological implications of this association is uncertain, showing no inhibitory effect of

PDE6γ on PDE5 activity in vitro (Omori and Kotera, 2007). Very little is understood about the

enzymatic regulation of PDEs 8-11. PKA phosphorylates the N-terminal sequences of PDE7A&B,

PDE10A2, and PDE11A4, however, there is little evidence to suggest phosphorylation causes

enzymatic regulation of these PDEs (Bender, 2006). There are no studies reporting PDE9A

phosphorylation.

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Table 2 - Biochemical characteristics of human PDE families

2.0 PDE INHIBITORS

As the understanding of the role and function of the 11 different PDE families and isoforms at cellular

and molecular level increases, it provides a drive for many researchers to develop selective inhibitors

of selective PDEs for the treatment of multiple diseases. Today there are several drugs in production

which inhibit the action of phosphodiesterase (thus increasing the cellular levels of cAMP and cGMP)

which have a beneficial effect on the heart, lungs, and vasculatures and also on platelet function and

inflammatory mechanisms (Feneck, 2008).

Several of these PDE inhibiting drugs affect more than one isoform. Since many tissues have more

than one PDE family or isoform present, it results in phosphodiesterase inhibitors (PDEI) having an

array of effects on the body (Beavo, 1995). For example, theophylline, which is an inhibitor designed

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to have a positive effect on the lungs, also has cardiac and vascular effects; amrinone, a PDE3

inhibitor used to treat congestive heart failure, effects cardiac, vascular, and platelet function.

Sildenafil, more commonly known as Viagra, was initially considered as a possible antianginal agent

targeting PDE5 (Nicholson, Challiss and Shahid, 1991). With this multiplicity of effects comes various

side effects associated with PDEIs. This paper aims to identify whether the unique 44-amino acid

insert of PDE3A is associated with interacting with substrate or inhibitors (see 3.0

Phosphodiesterase in Platelets).

3.0 PHOSPHODIESTERASE IN PLATELETS - (PDE3 Specific)

Platelets are the major components in the human body responsible for thrombosis and haemostasis.

In order to carry out their function, platelets must undergo a range of different responses including

change of cell shape, aggregation, adhesion and release of granular content. An increase in cAMP

and cGMP is associated with potent inhibition of these agonist-evoked platelet responses including an

increase in intracellular Ca2+, the effects of phospholipase A2 and C and the response of platelets to

thrombin (Manns et al., 2002). In human platelets, there are 3 distinct forms of cyclic nucleotide PDEs

that exist: cGMP-stimulated phosphodiesterase type II (cGS-PDE), the cGMP-inhibited

phosphodiesterase type III (cGI-PDE), and the cGMP-specific PDE type V (Sheth and Colman, 1995)

(Table 1). In short, cAMP in platelets are hydrolysed by PDE2 and PDE3, and cGMP is hydrolysed by

PDE2 and PDE5 (Gresele, Momi and Falcinelli, 2011). Decrease in intracellular cAMP levels

ultimately potentiates platelet activation. PDE3A in particular hydrolyses cAMP more readily than any

other PDE3 and PDE2 isoforms within platelets. Activity of PDE3A is increased by ∼50% during

thrombosis which involves protein kinase C (PKC)-mediated and Protein kinase B (PKB)-mediated

phosphorylation (Feijge et al., 2004). PDE3A activation is also facilitated by cAMP-dependant protein

kinase A (PKA)-mediated phosphorylation which suggests a possible negative-feedback loop for

cAMP signalling (Smolenski, 2012). cGMP-mediated inhibition of PDE3A has shown an increase of

cAMP concentration leading to activation of PKA. This may have an implication on nitric oxide

inhibition of platelet activation, in particular, platelet shape change (Smolenski, 2012, Jensen, 2004).

Inhibition of PDE3 by cGMP increases platelet cAMP. This has been shown to account for the

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synergistic inhibition of platelet aggregation by activators of guanylate and adenylate cyclases

(Maurice and Haslam, 1990). Inhibitor drugs of PDE3 increase the levels of cAMP in platelets which in

turn increases the phosphorylation of proteins by cyclic nucleotide-dependant kinases (Hung et al.,

2006). Presently, there are two competitive PDE3 inhibitors, milrinone and cilostazol which are used

for the treatment of acute congestive heart failure and intermittent claudication respectively. Collagen

induced primary platelet aggregation and adrenaline induced secondary platelet aggregation has

been shown to be inhibited by cilostazol (Kimura et al., 1995, Kariyazono et al., 2001). Cilistazol is

also associated with suppressing the expression of P-selectin (Kariyazono et al., 2001) and

suppressing the release of platelet-derived growth factors (Igawa et al., 1990) which enhances the

anti-platelet effects (Gresele, Momi and Falcinelli, 2011). Milrinone is a PDE3A-specific inhibitor which

induces an increase in intra-platelet cAMP levels in a dose-dependent manner, which results in the

inhibition of platelet aggregation. ADP, collagen, and U46619 induced platelet aggregation in platelet

rich plasma and whole blood along with arachidonic acid-induced platelet shape is also inhibited by

milrinone (Barradas et al., 1993).

However, cilostazol is unfortunately contraindicated in patients suffering from congestive heart failure,

and milrinone is associated with cardiac arrhythmias as a prominent side effect (Levy, Bailey and

Deeb, 2002, and Smith, 2002). Exploring the mechanism behind the inhibition of PDE3A is essential

to exploit alternative ways of inhibiting this protein-enzyme to minimalize adverse side effects.

In PDE3, there is a 44 amino acid insert within the catalytic domain that is unique only to the PDE3

gene family (Fig.5). In PDE3A, the 44-amino acid residue 773-816 (refer to results) is located between

helices 6 and 7, whereas in PDE3B the insertion is comprised of residues 767-781 (refer to results).

This insertion however lacks any secondary structural organisation (Scapin et al., 2004). This paper

aims to address whether this 44 amino-acid insert has an influence in the reugulation of enzyme

activity or interactions with substrate and/or inhibitors by characterising specific nucleotides within the

sequence and comparing them with known sequences of alternative protein enzymes or sequences

from mutagenesis studies such as Tang et al, (1997) and Hung et al, (2006). This can offer an insight

for targeting pathological conditions by modulating PDE3.

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4.0 METHODS AND MATERIALS

Bioinformatics is at the heart of this research and was used throughout in order to attain vital data

regarding the sequencing, alignment and identification of PDE genes and nucleotides (SNPs). This

allowed for a greater understanding of the genetic basis of PDE related disease. Alignments of protein

sequences mapped the location of specific domains which represented organizational principles

within the nucleic acid and protein sequences.

4.1 SEQUENCE ALIGNMENT OF PDE FAMILIES

A multiple sequence alignment was assembled of all the cyclic nucleotide families. For this alignment

all phosphodiesterase families (1-11) were included including various isoforms where relevant. To

acquire the amino-acid sequence of each wild-type phosphodiesterase isoform, the protein database

of http://www.ncbi.nlm.nih.gov/ was used. This database holds the full protein sequence for all known

genes from several sources. When selecting proteins from multiple species, more specified searches

were required to narrow the search result. The protein sequences for all given isoforms were gathered

in an in silico, FASTA format which allowed for manipulation on other bioinformatics programs.

Figure 6 – NCBI Protein database. Search for Phosphodiesterase 3A shown with specific selection of Homo sapiens specific proteins. FASTA format shown on right hand side.

21

Comparisons off all PDE family protein sequence was carried out using a web tool called Clustal -

http://www.ebi.ac.uk/Tools/msa/clustalo/. This program helps identify similarities between DNA or

protein sequences using seeded guide trees and HMM profile-profile techniques which generates

alignments between three or more sequences. The similarities show up as a series of asterisks (*),

colons (:) or full stops (.). An asterisk indicates positions which have a single, fully conserved residue.

A colon indicates conservation between groups of strongly similar properties. A full stop indicates

conservation between groups of weakly similar properties (Appendix B). This was carried out multiple

times to compare several PDE sequences, primarily looking for conserved regions among different

PDE families in relation to specific domains.

Figure 7 – Clustal Omega Multiple Sequence Alignment Tool. Example shows sequence input of PDE1A with that of PDE2A.

(Results are shown in Appendix B).

To carry out position specific analysis of PDE nucleotides, a consensus sequence was created for

cAMP-specific (PDE1, PDE3, PDE4, and PDE7) and cGMP-specific (PDE2, PDE5, and PDE6) PDEs.

This was done by picking the amino acid at each position with the maximum BLOSUM62 score

against all the nucleotides detected in that position. BLOSUM (BLOcks SUbstitution Matrix), is often

used for sequence alignment of proteins based on local alignments. The BLOSUM database is

scanned for conserved regions of protein families and then the relative frequency of amino acids and

their substitution is counted. A conserved region is that which matches 90% of the consensus residue

at the position with a positive BLOSUM62 score.

22

4.2 IDENTIFACTION OF DOMAINS & 44 AMINO ACID INSERT IN PDE3

A multiple sequence alignment was carried for all PDE families in order to locate and identify the 44

amino acid insert that characterizes the catalytic domain of PDE3. Full protein sequence for all PDE

families (1-11) were obtained from the National Centre for Biotechnology Information and aligned

using Clustal Omega. http://www.uniprot.org/ was used to locate the conserved domains of each PDE

family . This website allows the identification of any conserved domains of a protein by searching for

the desired protein within their database. Once the insert was identified, a blast search using

http://blast.ncbi.nlm.nih.gov/Blast.cgi was carried out to find regions of similarity between the other

biological sequences that may have the same 44 amino acid insert to compare functional similarities.

The 44-amino acid sequence is entered into the “query” section of the website after selecting the

“human” and “blastp” tabs respectively. The search then presents proteins that have the same or a

similar sequence, listed in ascending order with the highest percentage similarity at the top.

The conserved catalytic domain nucleotide sequence of PDE3A and PDE3B for Rattus norvegicus

(Rat), Mus musculus (Mouse), and Sus scrofa (Pig) were also obtained and aligned with the human

PDE3A and PDE3B to look for shared conserved regions within the sequence in order to analyse the

function of individual conserved nucleotides. These were also attained from the National Centre for

Biotechnology Information.

23

Figure 8 – BLAST search and results – “blastp” search for the 44-amino acid insert of PDE3A (Left). Result, with colour key alignment score showing similarities in order. Scrolling over and clicking each line shows alignments associated with query (Right)

4.3 AMINO ACID FREQUENCY PLOTS

To carry out statistical analysis on the number of individual nucleotides that are present in PDE3A and

PDE3B and also within the 44-amino-acid insert of the catalytic domain The ExPASy ProtParam tool

accessed via http://web.expasy.org/protparam/ was used. This is a tool which allows the computation

of various physical and chemical parameters for a given protein stored in Swiss-Prot or TrEMBL or for

a user entered protein sequence. The computed parameters include the molecular weight, theoretical

pI, amino acid composition, atomic composition, extinction coefficient, estimated half-life, instability

index, aliphatic index and grand average of hydropathicity (GRAVY) (Web.expasy.org, 2015). Using

this data, an amino acid frequency plot for PDE3A and PDE3B in humans created using Microsoft

Excel.

The pairwise alignment tool accessed via: http://www.ebi.ac.uk/Tools/psa/emboss_water/ was used to

calculate the percentage similarity, identity and gaps. This was carried out to analyze relationships

between the GAF domains of PDE2, -5, -10, and -11 and for the two PDE3 isoforms of humans and

mouse. The program uses the Smith-Waterman algorithm to calculate the local alignment of two

sequences (Thompson, Higgins and Gibson, 1994). This data was used to present a graph

representing similarities between the two isoforms in both species for analysis of functional

differences.

24

Figure 9 - EMBOSS Water Tool. Presented are the HsPDE3A and MmPDE3A sequences entered into the pairwise sequence alignment tool. (Results are shown

in Appendix C)

5.0 RESULTS

5.1 SEQUENCE ALIGNMENT OF PDE FAMILIES

Sequence alignment of all PDE families 1-11 using Clustal allowed for an in depth analysis of the

amino acid make up of individual PDEs, including similarities and differences that characterize the

function and structure of different PDE families and isoforms, especially within the catalytic and GAF

domains. Specific findings are represented below. (For complete alignment refer to Appendix B).

5.2 UNIQUE FEATURE OF PDE9 CATALYTIC DOMAIN

B2A IYKEFFSQ GDLE-KAMGNRP-MEM· MDREKA-YIPELQISFMEHIAMPIYKLLQDLFPKA10A IYAEFWAQ GD-EMKKLGIQP-IPM· MDRDKKDEVPQGQLGFYNAVAIPCYTTLTQILPPT6A VAAEFWEQ GDLERTVLQQNP-IPM· MDRNKADELPKLQVGFIDFVCTFVYKEFSRFHEEI5A VATEFFDQ GDRERKELNIEP-TDL· MNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDC11A VTSEFFEQ GDRERLELKLTP-SAI· MNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDC3A IVNEFYEQ GDEE-ASLGLPI-SPF· MDRSAP-QLANLQESFISHIVGPLCNSYDSAGLM-8A ISEEYFSQ TDEE-KQQGLPVVMPV· FDRNTC-SIPKSQISFIDYFITDMFDAWDAFVDL-1A LMEEFFLQ GDKE-AELGLPF-SPL· CDRKST-MVAQSQIGFIDFIVEPTFSLLTDSTEKI4A IMAEFFQQ GDRE-RERGMEI-SPM· CDKHTA-SVEKSQVGFIDYIVHPLWETWADLVHPD7A VTEEFFHQ GDIE-KKYHLGV-SPL· CDRHTE-SIANIQIGFMTYLVEPLFTEWARFSNTR9A LLEEYFMQ SDRE-KSEGLPV-APF· MDRDKV-TKATAQIGFIKFVLIPMFETVTKLFPMV *:: : * . :: * : .

Figure 10 – Sequence of PDE9A and unique features of catalytic domain. A, showing amino acid sequence of PDE9A. The boxed amino acid sequence (in red) represents the catalytic domain based on conservation of this region to other PDEs. B,

unique feature of PDE9A (and PDE8) conserved domain. Amino acid alignment are represented of the 10 other PDE families to PDE9. Grey boxes represent either chemically conserved residues or residues conserved among all known mammalian

PDEs. 1, 2, and 3 mark where PDE9, and in some places PDE8, diverges from all previously known PDEs (highlighted in purple). Position 1 is a conservative change from a phenylalanine (F) to a tyrosine (Y) in both PDE8 and PDE9. Position 2

shows the switch from a glycine to a threonine (T) in in PDE8 and to a serine (S) in PDE9. Position 3 represents a switch from the chemically conserved isoleucine, leucine, and valine residues found in PDE families 1-8, 10 and 11, to a lysine (K) in

PDE9.

25

On comparison of the catalytic domains of all PDEs (1-11), there was a noticeable difference within

the catalytic domain of PDE9 (and PDE8). In this very highly conserved region, PDE9A had three

differing amino acids (two in PDE8) compared to the other PDE families, all found towards the C-

terminal end of the catalytic domain. Fig.10A shows the amino acid sequence of PDE9A, with the

sequence of the catalytic domain highlighted in red. Fig.10B highlights the different amino acids found

in PDE8A and PDE9A. The first divergent amino acid (1, Fig.10B) shows a change from a conserved

phenylalanine, which is present in all other PDE families, to a tyrosine in PDE8A and PDE9A. The

second variation presented (2, Fig.10B) displays the replacement of glycine, that is completely

conserved in all other human PDEs to a threonine at this position in PDE8A and a serine in PDE9A.

The final aberration found within the catalytic domain sequence of PDE9 (3, Fig.10B) was the

presence of a positively charged lysine residue, in the position where all other PDE families retained

the hydrophobic amino acids valine, isoleucine, or leucine.

5.3 DIFFERENCE IN cAMP AND cGMP CATALYTIC DOMAIN STRUCTURE

A consensus sequence comparison of the catalytic domains was carried out between cGMP specific

PDEs with that of cAMP specific PDEs. The results shows that there are particular sequence

difference between the two sub-families. One specific difference was noted in the N-terminus of the

PDE4D catalytic domain at residue 333 (Fig.11). The cAMP-specific PDEs have a conserved aspartic

acid at this residue, cGMP-specific PDEs on the other hand have a conserved asparagine. This

position of the catalytic domain has been proven to be within the binding site of PDEs as it effects the

binding of the PDE4 specific inhibitor rolipram (Pillai et al., 1993). Centered on these data, it is

plausible to hypothesize that the difference between aspartic acid and asparagine could be related to

substrate selectivity of cAMP and cGMP-specific PDEs.

26

Figure 11 – Alignment of PDE4 with consensus sequence for cAMP-specific PDEs and consensus sequence for cGMP-specific PDEs. The three sequences listed include the sequence for the catalytic domain of human PDE4D and cAMP-specific and cGMP-specific

consensus sequence constructed as described in Methods and Materials. The capital letters indicate conserved positions. The first two putative metal-binding histidines are boxed in dark grey. The light grey box show the amino acid residue at position 333,

responsible for substrate specificity. The aspartic acid in the PDE4D sequence corresponds to residue number 333.

5.4 ANALYSIS OF GAF DOMAINS

Sequence alignment of the GAF domains from all the primary GAF-PDEs is represented below.

PDE2_GAF1 DASSLQLKVLQYLQQETRASRCCLLLVSEDNLQLSC---KVFG-------D---------PDE5_GAF1 DVTALCHKIFLHIHGLISADRYSLFLVCEDSSNDKFLISRLFDVAE---GSTL-------PDE6_GAF1 QTEKCIFNVMKKLCFLLQADRMSLFMYRT-RNGIAELATRLFNVHK---DAVLE------PDE10_GAF1 DNQLLLYELSSIIKIATKADGFALYFLGECNNS-LC----IFT-------------PPGIPDE11_GAF1 DLTSLSYKILIFVCLMVDADRCSLFLVEGAAAGKKTLVSKFFDVHA---GTPL-------PDE2_GAF2 DVSVLLQEIITEARNLSNAEICSVFLLDQ---N--ELVAKVFDG----------------PDE5_GAF2 SLEVILKKIAATIISFMQVQKCTIFIVDEDCSD---SFSSVFHMECEELEKSS-------PDE6_GAF2 DIERQFHKALYTVRAFLNCDRYSVGLLDMTKQK------EFFDVWPVLMGEVPPYSGPRTPDE10_GAF2 AIDSLLEHIMIYAKNLVNADRCALFQVDH---KNKELYSDLFDIGE---EKEG-------PDE11_GAF2 DLEKIVKKIMHRAQTLLKCERCSVLLLEDIESPVVK-FTKSFELMS---PKCSA------ . . : :

PDE2_GAF1 ------------------------KVLGEEVSFPL-TGCLGQVVEDKKSIQLKDLTSEDVPDE5_GAF1 -------------------EEV----SNNCIRLEWNKGIVGHVAALGEPLNIKDAYEDPRPDE6_GAF1 -DC-L-------------------VMPDQEIVFPLDMGIVGHVAHSKKIANVPNTEEDEHPDE10_GAF1 KEG---------------KP-----RLIPAGPITQGTTVSAYVAKSRKTLLVEDILGDERPDE11_GAF1 -------------------LPCSSTENSNEVQVPWGKGIIGYVGEHGETVNIPDAYQDRRPDE2_GAF2 -------------------GVV--DDESYEIRIPADQGIAGHVATTGQILNIPDAYAHPLPDE5_GAF2 -----------------------DTLTREHDANKINYMYAQYVKNTMEPLNIPDVSKDKRPDE6_GAF2 PDGREINFYKVIDYILHGKEDIKVIPNPPPDHWALVSGLPAYVAQNGLICNIMNAPAEDFPDE10_GAF2 -------------------KPV--FKKTKEIRFSIEKGIAGQVARTGEVLNIPDAYADPRPDE11_GAF2 -DA-E-------------NSFKESMEKSSYSDWLINNSIAELVASTGLPVNISDAYQDPR * : : .

PDE2_GAF1 Q-----QLQSMLGCELQAMLCVPVISRATDQVVALACAFNKLE-----GDLFTDEDEHVIPDE5_GAF1 F--NAEVD-QITGYKTQSILCMPIKNHR-EEVVGVAQAINKKS---GNGGTFTEKDEKDFPDE6_GAF1 F--CDFVD-ILTEYKTKNILASPIMNG--KDVVAIIMAVNKVD-----GSHFTKRDEEILPDE10_GAF1 FPRG-QMVN-T-GLESGTRIQSVLCLPIVTAI-GDLIGINELYRHWG-KEAFCLSHQEVAPDE11_GAF1 F--NDEID-KLTGYKTKSLLCMPIRSSD-GEIIGVAQAINKI----PEGAPFTEDDEKVMPDE2_GAF2 F--YRGVD-DSTGFRTRNILCFPIKNEN-QEVIGVAELVNKIN-----GPWFSKFDEDLAPDE5_GAF2 FPWTTENTGNVNQQCIRSLLCTPIKNGKKNKVIGVCQLVNKMEENTGKVKPFNRNDEQFLPDE6_GAF2 FAFQKEPL-DESGWMIKNVLSMPIVNKK-EEIVGVATFYNRKD-----GKPFDEMDETLMPDE10_GAF2 F--NREVD-LYTGYTTRNILCMPIVSR--GSVIGVVQMVNKIS-----GSAFSKTDENNF PDE11_GAF2 F--DAEAD-QISGFHIRSVLCVPIWNSN-HQIIGVAQVLNRLD-----GKPFDDADQRLF

: :*. *: . .::.: .: * .: PDE2_GAF1 QHCFHYTSTVLPDE5_GAF1 AAYLAFCGIVLPDE6_GAF1 LKYLNFANLIMPDE10_GAF1 TANLAWASVAIPDE11_GAF1 QMYLPFCGIAIPDE2_GAF2 TAFSIYCGISIPDE5_GAF2 EAFVIFCGLGIPDE6_GAF2 ESLTQFLGWSVPDE10_GAF2 KMFAVFCALALPDE11_GAF2 EAFVIFCGLGI

The GAF domain alignment shows highly conserved regions across all mammalian GAF-PDEs. The

initial motif identified as a signature GAF-PDE domain by Aravind and Ponting (1997) is shown in the

grey box (Fig.12). Located towards the N-terminus, this motif (NKXnFX3DE) has been proposed to

create an alternative cGMP binding region within the GAF domain (Haik, 1996).

27

Figure 12 – Clustal O sequence alignment of GAF1 and GAF2 domains of mammalian PDEs: PDE2A, PDE5A, PDE6A, PDE10A, and PDE11A. The region containing the sequence NKXnFX3DE originally identified as a PDE GAF domain signature is indicated in the grey box. Identical residues are indicated in white (black box), and conserved residues are shown in red.

cGMP-binding GAF domains are highlighted in green and the cAMP-binding GAF2 domain of PDE10 is shown in dark yellow.

The cGMP-binding GAF domains (highlighted in green) and the cAMP-binding GAF domain

(Highlighted in yellow) (Fig. 12) do not show any significant differences in conserved regions when

compared to the GAF domains that do not bind cyclic nucleotides. The sequence identity between the

human GAF-PDE domains and the phylogenetic tree of origin are shown in Fig. 13.

Analysis of the sequence identity of all GAF domains from Primary PDEs indicates that there is no

significant increase in sequence identity percentage among GAF-domains from the same PDE-family

in comparison to GAF-domains from a different family. For example, the GAF1 domain of PDE2 has a

higher percentage identity with the GAF1 domain of PDE5 (28.3%) than it has with its own GAF2

domain (22.8%) (Fig.13B).

28

Figure 13 - The relatedness of human PDE GAF domains. A, the AA sequence conservation (percentage of identity) among GAF domains of human PDE families. The grey boxes represent the individual GAF

domains of each PDE. Percentage identities among cyclic nucleotide-binding GAF domains are boxed in red) – calculated using EMBOSS water pairwise sequence alignment. B (edited from Zoraghi, (2004)), the

phylogenic tree of the GAF domains of human PDE families. The domains mentioned in this article are boxed in green. The lower case letters “a” and “b” correspond to GAF1 and GAF2 respectively.

The GAF1 and GAF2 domains of PDE5 and PDE11 have relatively high sequence identities (51.6%

and 43.7% respectively) in comparison to domains from other PDEs (Fig.13A). This relatedness is

highlighted in the phylogenetic tree (Fig.13B), depicting a similar genetic origin of the two domains.

This is also the case when comparing the GAF2 domains of PDE2 and PDE10 which shows a 46.9%

identity. This enhances the idea that similarities among domains are a consequence of a shared

ancestral background. Cyclic nucleotide-binding GAF domains (Fig.13A-red boxes) certainly share a

higher proportion of sequence identities in relation to domains that do not bind do not bind cyclic

nucleotides.

PDE2_GAF1

PDE2_GAF2

PDE5_GAF1

PDE5_GAF2

PDE6_GAF1

PDE6_GAF2

PDE10_GAF1

PDE10_GAF2

PDE11_GAF1

PDE11_GAF2

0 5 10 15 20 25 30 35 40

23.8666666666667

33.6555555555556

37

28.0555555555556

26.8222222222222

27.8333333333333

22.3666666666667

34.7666666666667

34.6444444444444

33.2777777777778

Mean Percentage Identity Between All PDE-GAF Domains

Percentage Similarities

Dom

ains

Figure 14 – Mean percentage identities between all PDE-GAF domains – Graph shows that PDE5-GAF1 domain has the highest average percentage identity (37%) with all other GAF domains. The GAF1 domain of PDE10 has the lowest with

22.4%. The mean percentages identities of each GAF domain is shown at the end of each bar.

Comparison of the mean percentage identities among all GAF domains shows that PDE5-GAF1 has

the highest average sequence identity, whereas the GAF1 domain of PDE10 has the lowest identity

(Fig.14). There also seems to be no significantly stronger average in sequence identity of GAF1

domains over GAF2 domains and vice versa.

29

5.5 44 AMINO ACID INSERT OF PDE3

The location of the 44 amino acid insert of PDE3 was identified by Clustal alignment of PDE3

(Fig.15A) with the 10 other PDE families. Since the insert is specific to PDE3, it was noticeable as a

conserved region within the catalytic domain residue which was not present in other PDE families

(Fig.15B.

MAVPGDAARV RDKPVHSGVS QAPTAGRDCH HRADPASPRD SGCRGCWGDL VLQPLRSSRK LSSALCAGSL SFLLALLVRL VRGEVGCDLE QCKEAAAAEE EEAAPGAEGG VFPGPRGGAP GGGARLSPWL QPSALLFSLL CAFFWMGLYL LRAGVRLPLA VALLAACCGG EALVQIGLGV GEDHLLSLPA AGVVLSCLAA ATWLVLRLRL GVLMIALTSA VRTVSLISLE RFKVAWRPYL AYLAGVLGIL LARYVEQILP QSAEAAPREH LGSQLIAGTK EDIPVFKRRR RSSSVVSAEM SGCSSKSHRR TSLPCIPREQ LMGHSEWDHK RGPRGSQSSG TSITVDIAVM GEATASLPTS WQTLLFHQTC ATSLRAVSNL LSTQLTFQAI HKPRVNPVTS LSENYTCSDS EESSEKDKLA IPKRLRRSLP PGLLRRVSST WTTTTSATGL PTLEPAPVRR DRSTSIKLQE APSSSPDSWN NPVMMTLTKS RSFTSSYAIS AANHVKAKKQ SRPGALAKIS PLSSPCSSPL QGTPASSLVS KISAVQFPES ADTTAKQSLG SHRALTYTQS APDLSPQILT PPVICSSCGR PYSQGNPADE PLERSGVATR TPSRTDDTAQ VTSDYETNNN SDSSDIVQNE DETECLREPL RKASACSTYA PETMMFLDKP ILAPEPLVMD NLDSIMEQLN TWNFPIFDLV ENIGRKCGRI LSQVSYRLFE DMGLFEAFKI PIREFMNYFH ALEIGYRDIP YHNRIHATDV LHAVWYLTTQ PIPGLSTVIN DHGSTSDSDS DSGFTHGHMG YVFSKTYNVT DDKYGCLSGN IPALELMALY VAAAMHDYDH PGRTNAFLVA TSAPQAVLYN DRSVLENHHA AAAWNLFMSR PEYNFLINLD HVEFKHFRFL VIEAILATDL KKHFDFVAKF NGKVNDDVGI DWTNENDRLL VCQMCIKLAD INGPAKCKEL HLQWTDGIVN EFYEQGDEEA SLGLPISPFM DRSAPQLANL QESFISHIVG PLCNSYDSAG LMPGKWVEDS DESGDTDDPE EEEEEAPAPN EEETCENNES PKKKTFKRRK IYCQITQHLL QNHKMWKKVI EEEQRLAGIE NQSLDQTPQS HSSEQIQAIK EEEEEKGKPR GEEIPTQKPD

Q

2A KNLE--------------------------------------------LTNYLEDIEIFA10A QNN----------------------------------------------HTLFTDLERKG6A VTGK--------------------------------------------LKRYFTDLEALA5A KAGK--------------------------------------------IQNKLTDLEILA11A TTAG--------------------------------------------FQDILTEVEILA3A TQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMA9A WLCS--------------------------------------------LQEKFSQTDILI8A SKER--------------------------------------------IKETLDPIDEVA1A MLHTG--------------------------------------------IMHWLTELEIL4A ATPA--------------------------------------------LDAVFTDLEILA7A KEPK--------------------------------------------LANSVTPWDILL

:

Figure 15 – A, Amino acid sequence of PDE3A highlighting the catalytic domain (Boxed/blue) based on conservation of this region to other PDEs. B, A section of the protein alignment of the full PDE family (1-11) carried out on Clustal showing

conserved 44 amino acid residue of PDE3 (highlighted in blue) within the Catalytic domain (residues 726–860). Refer to Appendix B for full sequence alignment.

30

The conserved amino acid insert of PDE3A and PDE3B were also identified in Rattus norvegicus

(Rat), Mus musculus (Mouse), and Sus scrofa (Pig) and showed similar conserved regions across all

four species (Fig.16). A BLAST search of the PDE3A insert did not show any significant correlations

with other non-PDE human proteins.

Figure 16 – Sequence alignment of 44-amino acid insert in PDE3 gene family for human, Rattus norvegicus (Rat), Mus musculus (Mouse), and Sus scrofa (Pig). The sequence of the 44-amino acid inserts from 4 different species including human PDE3A and PDE3B genes are represented. The numbers indicate residue positioning relative to each protein

sequence. The conserved sequence (highlighted in blue) shows conserved triplets at the N-terminus, C-terminus, and the middle of the insert.

Analysis of the sequences showed that there are specific conserved regions of the 44-amino acid

insert of PDE3A and PDE3B in all four species. The alignment of human PDE3A and PDE3B insert

showed a 38.6% identity including conserved triplets at the N-terminus, the middle of the insert, and

the C-terminus (Fig.16). To identify the potential structural roles of the conserved regions, Garnier et

al, (1978) was used. The conserved sequences PGL and YCG located at the N-terminus and C-

terminus respectively, indicates a possible β-turn at their respected sites which offers insight into the

functional role of the insert discussed later in the article.

To further analyze the structural difference between the two PDE3 isoforms, an amino acid frequency

plot was carried out to look for percentage similarities within the two amino acid sequences. This was

done for both human and mouse PDE3s to analyze whether the two PDE3 isoforms were more

closely related in humans or mice.

31

32

Ala (A)

Arg (R)

Asn (N)

Asp (D)

Cys (C)

Gln (Q)

Glu (E)

Gly (G)

His (H)

Ile (I)

Leu (L)

Lys (K)

Met (M)

Phe (F)

Pro (P)

Ser (S)

Thr (T)

Tyr (Y)

Val (V)

012345678

Amino-acid Frequency of the 44-aa Insert in HsPDE3A & HsPDE3B

PDE3A PDE3B

Amino Acids

Freq

uenc

y

Ala (A)

Arg (R)

Asn (N

)

Asp (D

)

Cys (C)

Gln (Q)

Glu (E)

Gly (G

)

His (H

)Ile

(I)

Leu (L

)

Lys (K

)

Met (M)

Phe (F)

Pro (P)

Ser (S)

Thr (T)

Trp (W)

Tyr (Y)

Val (V)

020406080

100120140

Amino-acid Frequency of PDE3 Isoforms

PDE3A PDE3B

Amino Acids

Freq

uenc

y

HsPDE3A v HsPDE3B HsPDE3A v MmPDE3A HsPDE3B v MmPDE3B0

102030405060708090

100

Comparison of Amino-Acid Sequence Similarity of PDE3 Isoforms in Humans and Mouse.

Identity Similarity Gaps

Perc

enta

ge

Figure 17 – Shows a triplet of amino-acid comparisons between human and mouse PDE3. A, Amino Acid Frequency of HsPDE3 isoforms – High frequency similarity of amino acids that make-up PDE3A (blue) and PDE3B (orange). B, Amino Acid Frequency of the 44-aa insert of HsPDE3A and HsPDE3B – comparable frequency similarity seen within the 44-aa insert of HsPDE3 isoforms showing trend in the number of specific amino-acids that are present. C, Comparison of Amino-acid Sequence Similarity of PDE3 Isoforms in Human and Mouse– Analysis of PDE3 sequence alignment showed that PDE3As (or PDE3B) from different species (Human Vs Mice) are more closely

related than PDE3A and PDE3B from the same species. (Percentage similarities calculated on Clustal).

A

B

C

Analysis of the amino-acid sequence of both HsPDE3A and HsPDE3B shows that there is a trend in

the frequency of specific nucleotides that are present in both isoforms (Fig.17A). Leucine (119x in

PDE3A and 133x in PDE3B) and serine (114x in PDE3A and 108x in PDE3B) are the two most

prominent amino acids found in the complete protein sequences of both PDE3A and PDE3B

(Fig.17A). This is mirrored in the 44-amino-acid insert of the catalytic domain for PDE3, only glycine

replaces leucine as the most prominent amino-acid within the region (Fig.17B). In fig.17C, the identity

bar represents an exact match of the same amino acid in the corresponding position. Similarity bar

represents all conserved regions found in both sequences including exact matches and the gaps bar

indicates the percentage of complete mismatches between the two sequences. Comparison of PDE3s

between humans and mice showed that there is a greater relation between PDE3As (or PDE3B) of

humans and mice than PDE3A and PDE3B from the same species (Fig.17C). HsPDE3A and

HsPDE3B had a 56.7% similarity (687/1212 aa’s), whereas HsPDE3A and MmPDE3A shared an

87.2% similarity (1006/1154) and HsPDE3B and MmPDE3B showed an 85.8% similarity (964/1124).

(Refer to Appendix B for raw data). This elucidates the individuality of each PDE3 isoform, and

implicates that both isoforms play a similar role in their respected species.

6.0 DISCUSSION

Members of the phosphodiesterase super-family are present in almost every cell of the human body,

with some cells expressing multiple PDE gene families and isoforms. It is vital, given the vast

distribution and molecular significance of PDEs, that the full repertoire of PDEs are identified and

extensively studied to gain an in depth understanding of the biochemical properties they each

possess. This will ultimately present greater opportunities for effective PDE drug targets for

pharmacological intervention. This paper aims to shown that with the help of online biotechnology

databases such as NCBI and UniProt, and alignment programs such as Clustal and EMBOSS, it is

possible to theorize and in some cases identify, with good confidence, the specific location of

individual amino acids within the sequence of various PDEs that are responsible for structure, binding

and substrate selectivity by analyzing protein sequences and comparative data from reliable sources.

33

6.1 DIVERGENT RESIDUES IN PDE9 CATALYTIC DOMAIN

The initial finding in this report presents a unique feature of the catalytic domain of PDE9A in

comparison to all other PDE families characterized to date. Many residues within the catalytic domain

of PDEs are either chemically or absolutely preserved across species, from yeast to humans, and

between PDE families (Fisher, 1998). Interestingly, the catalytic domain of PDE9A have three

divergent amino-acids in the position where all other PDEs have highly conserved residues. The

differing amino-acids recognized in this article in human PDE9 are also conserved in mouse PDE9

(Bloom, 2002). Fig.10B shows an alignment of the catalytic domain, in particular the C-terminal end,

from all the alpha representatives of PDE families. Two of the three divergent substitutions, Y424

(Y748 in PDE8) (1, Fig.10B) and S428 (T752 in PDE8) (2, Fig.10B) are present in both PDE8A and

PDE9A, however the third is unique to PDE9. In this position, PDE9 conserves positively charged

lysine residue, where all other members of the PDE family retain the hydrophobic amino acids valine,

isoleucine, or leucine (3, Fig.10B). It is difficult to identify the exact function of these divergent

residues in and PDE9A, however it can be hypothesized that some of these deviations play a possible

role in the unique kinetic characteristics of PDE9A. Kinetic analysis of PDE9 have shown that it has

the highest affinity for cGMP of all PDEs (Km = 170nM) which is a 1000-fold higher than its affinity for

cAMP (Km = 240nM) (Fisher, 1998). Comparative analysis of the consensus sequence of cAMP and

cGMP specific PDE putative regulatory domains (Fig. 11) with that of PDE9, showed that PDE9A did

not have sequence homologues to the ~80 amino-acid cGMP-binding regions, that can serve as

allosteric regulatory sites, which are present in cGMP-binding PDEs (PDE2, PDE5, and PDE6) (data

not shown).

Therefore, it can be assumed that all binding of cGMP to PDE9A occurs within the catalytic region.

This enhances the hypothesis that the three divergent amino-acids within the catalytic domain of

PDE9A are responsible for its high affinity towards cGMP. Pharmacological inhibitors to target these

residues could aid in reducing PDE9s high affinity to cGMP, consequently increasing cellular levels of

cGMP. Since PDE9 is highly expressed in the brain, it is thought that they are involved in regulating

neuronal cGMP levels (Garthwaite, 1991). Multiple studies have indicated that cGMP plays a

34

contributing factor in the process of learning and memory (Bernabeu et al., 1996). One such study

showed that intra-hippocampal injection of the cGMP analog 8-bromo-cGMP improves learning

(Prickaerts et al., 2002). Thus, inhibitors targeting the region identified in Fig.10B may possibly be

beneficial in the treatment of Alzheimer’s disease. However In order to have a more solid

understanding of the functional roles of these identified residues, high resolution crystal structure

solutions of PDE9 will be necessary. This will enable researchers to recognize exactly how separate

amino-acids bind to the cyclic nucleotides and the change in spatial arrangement this causes.

6.2 SUBSTRATE/INHIBITOR SPECIFICITY OF PDE4

As mentioned above, a consensus sequence of the catalytic domain of all cAMP-specific and cGMP-

specific PDEs was created (Fig. 11) in order to analyze similarities and differences within the

sequence of the two nucleotide specific sub-families. A particular finding in this section was the switch

in residue 333 in the N-terminus of PDE4D catalytic domain. Near the two putative metal-binding

histidines residues of both PDE sub-families is a conserved amino acid that differs between cAMP

and cGMP-specific PDEs. cAMP-specific PDEs have a highly conserved aspartic acid at this residue,

which differs to cGMP-specific PDEs that conserve asparagine. Mutagenesis study using yeast

expression system by Pillai et al, (1993) showed that the region presented above (Fig. 11) effects the

binding of rolipram, a PDE4 specific inhibitor and therefore is within the binding site of cAMP specific

PDEs (Pillai et al., 1993). Alternative mutagenesis studies have identified five residues (Y432, H588,

Y602, F613, and F645) (highlighted in Appendix A) in PDE4 as possible inhibitor binding partners

based on a hypothetical for the PDE4 binding site. In particular, residues Y432 (Y325 in PDE4D (Fig.

11)) and H558 (H527 in PDE4D) were shown to be conserved only in PDE4 isoform (Richter et al.,

2001). This indicates that conserved aspartic acid at residue 333 and the conserved tyrosine at

position 325 in PDE4D may determine inhibitor specificity and substrate specificity. It is known that

different inhibitors for PDE4 have different bind sites and parameters (Jacobitz et al., 1996). The

characterized PDE4 binding sequence presented (Fig. 11) may be used to calculate possible side-

effects when producing new drugs. This can be done by comparing the potency of side effects of

individual PDE4 inhibitors with their sensitivities against PDE4 mutants. Ultimately, this will improve

the effeteness of PDE4 inhibitors by minimalizing side effects associated with certain molecules.

35

6.3 ANALYSIS OF PDE GAF DOMAINS

Analysis of the GAF domain sequence alignment (Fig.12), sequence identity (Fig.13A) and branching

pattern in the phylogenetic tree (Fig.13B) show that the origin of the GAF domains and subtypes were

derived from a common ancestor. Of all the cGMP-binding GAF domains, GAF1 domain of PDE5 and

the GAF1 domain of PDE11 have the highest sequence identity of approximately 52% (Fig.13A).

When comparing this to the phylogenetic tree of GAF domains (Fig.13B), it is visible that PDE5 and

PDE11 have a common origin, indicating similar functioning properties in the domain besides cGMP-

binding. It is known that individual GAF domains in PDEs have multiple functional roles (Hofbauer,

Schultz and Schultz, 2008) which is highlighted by the relatively low sequence identity among GAF-

domains (Fig.13A). The sequence analysis presented above (Fig.12 and Fig.13A) illustrates that there

is a higher sequence homology among cNMP binding domains, indicating functional similarities in the

binding site. However, there is no substantial sequence homology between the PDE-GAF1 domains

and GAF2 domains to predict binding regions, nor is there a high similarity between GAF1 and GAF2

domains from the same PDE. This differs from the pattern found in the cyclic nucleotide (cN) binding

sites in the cAMP and cGMP-dependant protein kinases (PKA and PKG) (Shabb and Corbin, 1992).

These protein kinases have a pair of conserved homologous cyclic nucleotide-binding sites, each

composed of ~120 AAs which are different from the GAF motifs.

The conserved NKXnFX3DE motif identified in all GAF domains (Fig.12) is thought to be involved in

cGMP binding. This was identified by mutational analysis, where point mutations of the aspartate

residue ceased cGMP binding in GAF1 of PDE5 (McAllister-Lucas et al., 1995). Research by Haik,

(1996) went on to further identify the properties and potential roles of asparagine (Asn), lysine (Lys),

aspartic acid (Asp) and glutamic acid (Glu) in the NKXnFX3DE sequence of cGMP-binding PDEs. The

Asn residue at the head of the motif was shown to interact with the nitrogen at position 7 of the

guanine ring. Lys provided methylene carbons for van der Waals interactions with the guanine ring. It

is proposed that the aspartic acid residue in the recognized NKXnD motif (Fig.12) interacts with the

hydrogen of the N-1 endo-nitrogen guanine base (Beltman et al., 1995). Glu is unchanged in both

binding sites of cGMP-binding PDEs (Haik, 1996). However, mutagenesis of the Glu residue had no

36

noticeable effects on cGMP affinity, therefore it can be concluded that glutamic acid residue in the

NKXnFX3DE motif does not interact with cGMP.

Furthermore, Studies carried out on the GAF1 and GAF2 domains of bovine PDE2A and PDE5A

respectively, demonstrated that all residues in the GAF2 domain of PDE that are involved in cGMP-

binding are conserved or conservatively substituted in the GAF1 domain of HsPDE5A. The residues

identified were D206 in PDE5A and D439 in PDE2A which were shown to engage with the guanine

base of cGMP in both back-bone and side chain interactions (Sopory et al., 2003). Additionally, the

adjacent phenylalanine (Phe) residue, F205 (F438 in PDE2A), is positioned in an obscure manner

which causes an interaction with the ring of the guanine base. F205 in GAF1 of PDE is positioned as

one of the highly conserved phenylalanine’s found in all PDE GAF-domains (Fig.12). In the GAF2

domain of PDE5, phenylalanine and aspartic acid are replaced by histidine and methionine, and

isoleucine and glycine are the replacements in the GAF2 domain of PDE2A (Fig.12). These could be

substitutions which compromise cGMP binding to these domains.

The medical importance of these cGMP-GAF sequence repeats for normal function of PDEs is

highlighted by the naturally occurring mutation H258N in human PDE6B, which leads to congenital

stationary night blindness in humans (Gal et al., 1994). PDEs are the only protein family in humans to

possess GAF motifs. These motifs play an important role in a number of PDEs by regulating activity,

thus making them a key target for pharmacological intervention of cAMP and cGMP signalling

(Zoraghi, 2004).

37

Figure 18 - Hypothesized interactions of cGMP with the putative NKXnD motif. Showing

interactions of Asn276, Lys227, and Asp289 of PDE5-GAF1 with cGMP

6.4 FUNCTIONAL ROLE OF THE CATALYTIC INSERTION IN PDE3

The catalytic domain of PDE3 is unique from all other PDEs in that it has an extra 44-amino acid

insert (Fig.15B) which shares no sequence homology with any other member of the PDE-superfamily

or with any other sequences currently in the protein databases. The position of the insertion intersects

the first (of two) recognized Zn2+ binding domains present in the catalytic domains of all PDEs (Francis

et al., 1994). There is however conserved residues within the insert that are expressed in both

isoforms of human PDE3. These unvaried amino acids were also present when aligned with PDE3

isoforms from Rattus norvegicus, Mus musculus, and Sus scrofa (Fig. 16), indicating that the

conservation of amino acids is associated with a particular function in all four species. Amino-acids

with potential structural roles were recognized by consulting Garnier et al, (1978), which identified the

PGL and YCG residues located near the N-terminus and C-terminus respectively (Fig.16) that may be

structurally involved in a β-turn at either terminal. β-turns cause the amino-acid chain to fold back on

itself to produce a compact shape and connect regions of more formal structure, forming simple motifs

(Fort, 2015). Mutagenesis of these β-turns in PDE3 completely abolished enzyme activity, which

demonstrates the significance of the insert for function.

Amino acid frequency plots of the HsPDE3A and HsPDE3B sequences and the 44-amino acid insert

sequences identified a tendency of particular amino acids present in both isoforms (Fig.17A&B). In

both isoforms, the hydrophobic AA leucine and the polar uncharged AA serine were noticeably more

ubiquitous than any other amino acid. While the significance of frequent amino acids within a

sequence is yet to be determined, the resemblance of the two PDE3 proteins establishes that both

isoforms were derived from the same gene may indicate similar modes of function and regulation.

This was reflected in 44-amino acid insert (Fig.17B). However, larger resemblances were noted from

the sequence identity/similarity of the PDE3 insert between different species than with isoforms of the

same species. For example, the PDE3 insert in HsPDE3A and MmPDE3A shared a greater

38

percentage identity (81.9%) compared to HsPDE3A and HsPDE3B (43.4%) (Fig.17C) which

exemplifies the unique nature of each isoform and suggests that each PDE3 isoform is localized in a

similar positions and performs similar functions in their respected species.

Mutagenesis studies by Tang et al, (1997), carried out on platelet derived PDE3B molecules explored

the unique properties of the 44-amino acid insert. Mutations were made in the conserved putative β-

turn residues residing near the N-terminus and C-terminus (Fig.16) to analyze whether these

sequences altered enzyme activity. Mutations P1A G2A at the N-terminus and Y42A and G43A at the

C-terminus were shown to markedly reduce cAMP and cGMP hydrolysis in platelets in comparison to

the wild-type PDE3B sequence, however the ratio of cAMP to cGMP hydrolysis at low concentrations

remained unchanged by the alanine mutations (Tang et al., 1997). Conserved negatively charged

residues found at the center of the PDE3 insert (Fig.16) have shown no effect on the biochemical

properties of PDE3 (He et al., 1998). These results imply that the 44-amino acid insert of PDE3 may

have be involved in the hydrolysis of cNMPs in platelets, which in turn help to regulate platelet

activation by reducing cNMP levels.

A recent study has further elaborated on the functional roles of individual amino acids within the

insert, and have identified a new cAMP-binding residue (Tyr807 – position 35 of the insertion) in

HsPDE3A which forms a unique flexible loop only found in the PDE3 gene family (Hung et al., 2006).

The residue in the corresponding position of PDE3B shows a cysteine which forms a comparable loop

to that found in PDE3A. This suggests that cN-binding in both PDE isoforms have a similar

mechanism, corresponding to similar positions within the catalytic insert. A further eight amino-acids

(His782, His796, His798, Ser804, Lys805, Thr810, Tyr814, and Gly815) within the catalytic insert of

PDE3A were also defined as amino acids involved in catalysis and/or metal binding (Hung et al.,

2006). This novel insight into the functional role of the PDE3 insert offers a new approach for the

development of structure-based inhibitors to be

designed that can target specific cN-binding sites in

platelets.

Figure – 19 Molecular model of PDE3A. The 44-amino acid insert is depicted as a solid red ribbon. The hypothesized cAMP-binding pocket of Tyr807 (green) and Asn845, Glu866, Glu971, Phe972, and Phe1004 labelled in black. Mutants in the insert that affect the kcat are labelled in blue. (This figure was edited from Hung et al, (2006)

39

7.0 CONCLUSION

Over the last 50 years, our understanding of the regulation, structure, and function of PDEs have

evolved considerably, shedding new light on the importance of PDEs as key regulators of numerous

different cellular functions. Identification of novel PDE isoforms have significantly aided our

understanding of PDEs and their involvement in pathological diseases. In particular, the use of

bioinformatics has made it possible to overcome complications that have plagued the field of PDE

investigation. A great deal of development has been seen in targeting the active site of single PDEs

with selective inhibitors which have proved to be difficult in the past due to the high degree of

similarity within the binding sites of PDEs. Sequence alignments of functional domains, involved in

cyclic nucleotide selectivity and PDE regulation have highlighted conserved regions unique to specific

PDEs which have assisted pharmacological advances in targeting PDE. This paper has highlighted

several regions within the catalytic domains of PDE9, PDE4, and PDE3, as well as conserved regions

within the GAF domains of several PDEs that are unique to specific PDEs and PDE-subfamilies. PDE9

has been shown to have divergent residues within a highly conserved region among other PDEs and

is thought to be responsible for its high affinity towards cGMP. The variance between cAMP-binding

and cGMP-binding PDEs have also be portrayed in this article through the use of consensus

alignments of cN-specific PDEs. An aspartic acid residue in cAMP-specific PDEs is replaced by

asparagine in the corresponding position of cGMP-specific PDEs. Based on the fact that the identified

sequence is a known inhibitor binding region for rolipram, it can be assumed that the switch in

amino acid is cN-specific PDEs is responsible for substrate selectivity. The unique 44 amino-acid

insert of PDE3 was also identified using FASTA protein sequences from biotechnology databases and

40

aligning it with all the primary PDEs that are currently known. Further Alignment of the catalytic

insert with those of PDE3s of mice, rats, and pig found conserved triplets of amino acids at each end

of the insertion present across all four species. Amino acid frequency plots showed no significant

correlations between PDE3A and PDEB inserts, however references from mutagenesis studies gave

evidence to show that the insert is partially responsible for cNMP-binding especially in platelet

derived PDE3A. Further research into the signaling network in platelets have the potential to identify

new indicators of platelet function and possibly new targets for therapeutic drugs.

Analysis of the GAF motifs identified a known cGMP binding region conserved in all GAF-PDEs.

Percentage identity correlations showed an increased sequence identity among cN-binding GAF

domains in comparison to non-binding domains. This was mirrored in the phylogenetic tree showing

domains with higher sequence identities were more closely related from a common origin.

The gathering of basic knowledge shown in this article sets the foundation for targeting these PDEs

by specific inhibitors for clinical treatment. The impact of PDE targeting drugs has been

demonstrated by the global success of the PDE5 inhibitor, sildenafil, both commercially and

clinically. This has attracted great interest for research into PDE isoforms both academically and

commercially which is beneficial for progressing drug discovery.

.

41

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Zoraghi, R. (2004). Properties and Functions of GAF Domains in Cyclic Nucleotide Phosphodiesterases and Other Proteins. Molecular Pharmacology, 65(2), pp.267-278.

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APPENDIX – AAmino Acid Sequence of Human PDE1-11 Isoforms (Including sequences for animal PDE3A & PDE3B used in text).

46

>gi|21361262|ref|NP_005010.2| calcium/calmodulin-dependent 3',5'-cyclic nucleotide phosphodiesterase 1A [Homo sapiens] MGSSATEIEELENTTFKYLTGEQTEKMWQRLKGILRCLVKQLERGDVNVVDLKKNIEYAASVLEAVYIDETRRLLDTEDELSDIQTDSVPSEVRDWLASTFTRKMGMTKKKPEEKPKFRSIVHAVQAGIFVERMYRKTYHMVGLAYPAAVIVTLKDVDKWSFDVFALNEASGEHSLKFMIYELFTRYDLINRFKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIMLHTGIMHWLTELEILAMVFAAAIHDYEHTGTTNNFHIQTRSDVAILYNDRSVLENHHVSAAYRLMQEEEMNILINLSKDDWRDLRNLVIEMVLSTDMSGHFQQIKNIRNSLQQPEGIDRAKTMSLILHAADISHPAKSWKLHYRWTMALMEEFFLQGDKEAELGLPFSPLCDRKSTMVAQSQIGFIDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIVGLHIADALRRSNTKGSMSDGSYSPDYSLAAVDLKSFKNNLVDIIQQNKERWKELAAQGESDLHKNSEDLVNAEEKHDETHS

>gi|26454702|gb|AAH40974.1| Phosphodiesterase 2A, cGMP-stimulated [Homo sapiens] MGQACGHSILCRSQQYPAARPAEPRGQQVFLKPDEPPPPPQQCADSLQDALLSLGSVIDISGLQRAVKEALSAVLPRVETVYTYLLDGESQLVCEDPPHELPQEGKVREAIISQKRLGCNGLGFSDLPGKPLARLVAPLAPDTQVLVMPLADKEAGAVAAVILVHCGQLSDNEEWSLQAVEKHTLVALRRVQVLQQRGPREAPRAVQNPPEGTAEDQKGGAAYTDRDRKILQLCGELYDLDASSLQLKVLQYLQQETRASRCCLLLVSEDNLQLSCKVIGDKVLGEEVSFPLTGCLGQVVEDKKSIQLKDLTSEDVQQLQSMLGCELQAMLCVPVISRATDQVVALACAFNKLEGDLFTDEDEHVIQHCFHYTSTVLTSTLAFQKEQKLKCECQALLQVAKNLFTHLDDVSVLLQEIITEARNLSNAEICSVFLLDQNELVAKVFDGGVVDDESYEIRIPADQGIAGHVATTGQILNIPDAYAHPLFYRGVDDSTGFRTRNILCFPIKNENQEVIGVAELVNKINGPWFSKFDEDLATAFSIYCGISIAHSLLYKKVNEAQYRSHLANEMMMYHMKVSDDEYTKLLHDGIQPVAAIDSNFASFTYTPRSLPEDDTSMAILSMLQDMNFINNYKIDCPTLARFCLMVKKGYRDPPYHNWMHAFSVSHFCYLLYKNLELTNYLEDIEIFALFISCMCHDLDHRGTNNSFQVASKSVLAALYSSEGSVMERHHFAQAIAILNTHGCNIFDHFSRKDYQRMLDLMRDIILATDLAHHLRIFKDLQKMAEVGYDRNNKQHHRLLLCLLMTSCDLSDQTKGWKTTRKIAELIYKEFFSQGDLEKAMGNRPMEMMDREKAYIPELQISFMEHIAMPIYKLLQDLFPKAAELYERVASNGEHWTKVSHKFTIRGIPSNNSLDFLDEEYEVPDLDGTRAPINGCCSLDAE

>gi|3059109|emb|CAA06304.1| phosphodiesterase 3A [Homo sapiens]MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGCRGCWGDLVLQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGAPGGGARLSPWLQPSALLFSLLCAFFWMGLYLLRAGVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAAGVVLSCLAAATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYVEQILPQSAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEATASLPTSWQTLLFHQTCATSLRAVSNLLSTQLTFQAIHKPRVNPVTSLSENYTCSDSEESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSTSIKLQEAPSSSPDSWNNPVMMTLTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPASSLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVICSSCGRPYSQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECLREPLRKASACSTYAPETMMFLDKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEAFKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQITQHLLQNHKMWKKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPTQKPDQ

>gi|149049096|gb|EDM01550.1| phosphodiesterase 3A [Rattus norvegicus]MAVRGEAAQDWAKPGLRGPSPAPVARGDHRCRGGSPSSPRGSGCCWRALALQPLRRSPQLSSALCAGSLSVLLALLVRLVGGEVGGELESSQEAAAEEEEEEGARGGVFPGPRGGAPGGGAQLSPWLQPAALLFSLLCAFFWMGLCLLRAGVRLPLAVALLAACCAGEALVQLSLGVGDGRLLSLPAAGVLLSCLGGATWLVLRLRLGVLMVALTSALRTVALVSLERFKVAWRPYLAYLAAVLGLLLARYAEQLLPQCSGPAPPRERFGSQSSARTKEEIPGWKRRRRSSSVVAGEMSGCGGKSHRRTSLPCIPREQLMGHSEWDHKRGSRGSQSGTSVTVDIAVMGEA

47

HGLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPTVTFSENYTCSDSEEGLEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSASIKPHEAPSPSAVNPDSWNAPVLMTLTKSRSFTSSYAVSAANHVKAKKQNRPGGLDKISPVPSPSSSPPQGSPTSSPVSGIASVQFPESPEVTTKRGPGSHRALTYTQSAPDLSPQIPPSPVICSSCGRPYSQGNPADGPSERSGPAMQKPNRTDDTSQVTSDYETNNNSDSSDILQNDEEAECQREPLRKASACGTYTPQTMIFLDKPILAPEPLVMDNLDSIMDQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEAFKIPVREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLPSVIGDHGSASDSDSDSGFTHGHMGYVFSKAYHVPDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLVNLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNAKVNDDVGIDWTNENDRLLVCQMCIKLADINGPAKCKDLHLRWTEGIASEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCHSYDSAGLMPGKWVDDSDDSGDTDDPEEEEEEAETPHEEETCENSEAPRKKSFKRRRIYCQITQHLLQNHMMWKKVIEEEQCLSGTENQAPDQAPLQHSSEQIQAIKEEEEEKGKPRAEETLAPQPDL

>gi|148678674|gb|EDL10621.1| phosphodiesterase 3A, cGMP inhibited [Mus musculus]MAVRGEAAQDLAKPGLGGASPARVARGNHRHRGESSPSPRGSGCCWRALALQPLRRSPQLSSALCAGSLSVLLALLVRLVGGEVGGELEKSQEAAAEEEEEEGARGGVFPGPRGGAPGGGAQLSPWLQPAALLFSLLCAFFWMGLCLLRAGVRLPLAVALLAACCAGEALVQLSLGVGDGRLLSLPAAGVLLSCLGGATWLVLRLRLGVLMVAWTSVLRTVALVSLERFKVAWRPYLAYLAAVLGLLLARYAEQILPQCSGPAPPRERFGSQLSARTKEEIPGWKRRRRSSSVVAGEMSGCSGKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSGTSITVDIAVMGEAHGLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPTVTFSENYTCSDSEEGLEKDKQAISKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSASIKPHEAPSPSAVNPDSWNAPGLTTLTKSRSFTSSYAVSAANHVKAKKQNRPGGLAKISPVPSPSSSPPQGSPASSPVSNSASQQFPESPEVTIKRGPGSHRALTYTQSAPDLSPQIPPPSVICSSCGRPYSQGNPADGPSERSGPAMLKPNRTDDTSQVTSDYETNNNSDSSDILQNEEEAECQREPQRKASACGTYTSQTMIFLDKPILAPEPLVMDNLDSIMDQLNTWNFPIFDLMENIGRKCGRILSQVSYRLFEDMGLFEAFKIPVREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLPSVIGDHGSASDSDSDSGFTHGHMGYVFSKMYHVPDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLVNLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNAKVNDDVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLRWTEGIASEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCHSYDSAGLMPGKWVDDSDDSGDTDDPEEEEEEAETPHEDEACESSIAPRKKSFKRRRIYCQITQHLLQNHMMWKKVIEEEQCLSGTENQSLDQVPLQHPSEQIQAIKEEEEEKGKPRAEETLAPQPDL

>gi|47523360|ref|NP_998901.1| cGMP-inhibited 3',5'-cyclic phosphodiesterase 3A [Sus scrofa]MGLYLLRAGVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYVEQILPQSAGAAPREHFGSQLLAGTKEDIPEFKRRRRSSSVVSAEMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEAHGLITDLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPAVSFSENYTCSDSEESAEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPSPVRRDRSASIKLHEAPSSSAINPDSWKNPVMMTLTKSRSFTSSYAVSASNHVKAKKQSRPGSLVKISPLSSPCSSALQGTPASSPVSKISTVQFPEPADATAKQGLSSHKALTYTQSAPDLSPHILTPPVICSSCGRPYSQGNPADGPLERSGPAIQAQSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECSREPLRKASACSAYTPDTMMFLDKPILAPEPLVMDNLDSIMEHLNTWNFPIFDLVEKIGRKCGRILSQVSYRLFEDMGLFEAFKIPIREFMNYFHALEIGYREIPYHNRIHATDVLHAVWYLTTQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKMYNVPDDKYGCLSGNIPALELMALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRTEYNFLVNLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNAKVNDEVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLQWTEGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDTDDPEEEEAPKEEETCENNDSPRKKTFKRRKIYCQITQHLLQNHKMWKKVIEEEQRLAGIESQSLDQAPQQHSSEQIQAIKEEDEDKGKPRGEETPTPKPNQ

>gi|1731706|dbj|BAA09306.1| phosphodiesterase 3B [Homo sapiens]MRRDERDAKAMRSLQPPDGAGSPPESLRNGYVKSCVSPLRQDPPRGFFFHLCRFCNVELRPPPASPQQPRRCSPFCRARLSLGDLAAFVLALLLGAEPESWAAGAAWLRTLLSVCSHSLSPLFSIACAFFFLTCFLTRTKRGPGPGRSCGSWWLLALPACCYLGDFLVWQWWSWPWGDGDAGSAAPHTPPEAAAGRLLLVLSCVGLLLTLAHPLRLRHCVLVLLLASFVWWVSFTSLGSLPSALRPLLSGLVGGAGCLLALGLDHFFQIREAPLHPRLSSAAEEKVPVIRPRRRSSCVSLGETAASYYGSCKIFRRPSLPCISREQMILWDWDLKQWYKPHYQNSGGGNGVDLSVLNEARNMVSDLLTDPSLPPQVISSLRSISSLMGAFSGSCRPKINPLTPFPGFYPCSEIEDPAEKGDRKLNKGLNRNSLPTPQLRRSSGTSGLLPVEQSSRWDRNNGKRPHQEFGISSQGCYLNGPFNSNLLTIPKQRSSSVSLTHHVGLRRAGVLSSLSPVNSSNHGPVS

48

TGSLTNRSPIEFPDTADFLNKPSVILQRSLGNAPNTPDFYQQLRNSDSNLCNSCGHQMLKYVSTSESDGTDCCSGKSGEEENIFSKESFKLMETQQEEETEKKDSRKLFQEGDKWLTEEAQSEQQTNIEQEVSLDLILVEEYDSLIEKMSNWNFPIFELVEKMGEKSGRILSQVMYTLFQDTGLLEIFKIPTQQFMNYFRALENGYRDIPYHNRIHATDVLHAVWYLTTRPVPGLQQIHNGCGTGNETDSDGRINHGRIAYISSKSCSNPDESYGCLSSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLHLDHVEFKRFRFLVIEAILATDLKKHFDFLAEFNAKANDVNSNGIEWSNENDRLLVCQVCIKLADINGPAKVRDLHLKWTEGIVNEFYEQGDEEANLGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQWLEAEEDNDTESGDDEDGEELDTEDEEMENNLNPKPPRRKSRRRIFCQLMHHLTENHKIWKEIVEEEEKCKADGNKLQVENSSLPQADEIQVIEEADEEE

>gi|149068220|gb|EDM17772.1| phosphodiesterase 3B, [Rattus norvegicus]MRKDERERDTPAMRSPYAAAARPATATAASPPESLRNGYVKSCVSPLRQDPPRSFFFHLCRFCNVEPPAASLRAGARLSLAALAAFVLAALLGAGPERWAAAATGLRTLLSACSLSLSPLFSIACAFFFLTCFLTRAQRGPDRGAGSWWLLALPACCYLGDFAAWQWWSWLRGEPAAAAAGRLCLVLSCVGLLTLAPRVRLRHGVLVLLFAGLVWWVSFSGLGALPPALRPLLSCLVGGAGCLLALGLDHFFHVRGASPPPRSASTADEKVPVIRPRRRSSCVSLGESAAGYYGSGKMFRRPSLPCISREQMILWDWDLKQWCKPHYQNSGGGNGVDLSVLNEARNMVSDLLIDPSLPPQVISSLRSISSLMGAFSGSCRPKINSFTPFPGFYPCSEVEDPVEKGDRKLHKGLSSKPSFPTAQLRRSSGASGLLTSEHHSRWDRSGGKRPYQELSVSSHGCHLNGPFSSNLMTIPKQRSSSVSLTHHAGLRRAGALPSPSLLNSSSHVPVSAGCLTNRSPVGFLDTSDFLTKPSVTLHRSLGSVSSAADFHQYLRNSDSSLCSSCGHQILKYVSTCEPDGTDHHNEKSGEEDSTVFSKERLNIVETQEEETVKEDCRELFLEGDDHLMEEAQQPNIDQEVLLDPMLVEDYDSLIEKMSNWNFQIFELVEKMGEKSGRILSQVMYTLFQDTGLLETFKIPTQEFMNYFRALENGYRDIPYHNRVHATDVLHAVWYLTTRPIPGLQQLHNNHETETKADSDARLSSGQIAYLSSKSCCIPDKSYGCLSSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLNLDHMEFKRFRFLVIEAILATDLKKHFEFLAEFNAKANDVNSNGIEWSSENDRLLVCQVCIKLADINGPAKDRDLHLRWTEGIVNEFYEQGDEEATLGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQWIEAEEGDDTESDDDDDDDDDDDDDDDDDDEELDSDDEETEDNLNPKPQRRKGRRRIFCQLMHHLTENHKIWKEIIEEEEKCKAEGNKLQVDNASLPQADEIQVIEEADEEEEQMFE

>gi|24210984|gb|AAN52086.1| phosphodiesterase 3B [Mus musculus]MRKDERERDAPAMRSPPPPPASAASPPESLRNGYVKSCVSPLRQDPPRSFFFHLCRFCNVEPPAASLRAGARLSLGVLAAFVLAALLGARPERWAAAAAGLRTLLSACSLSLSPLFSIACAFFFLTCFLTRAQRGPGRGAGSWWLLALPACCYLGDFAAWQWWSWLRGEPAAAGRLCLVLSCVGLLTLAPRVRLRHGVLVLLFAGLVWWVSFSGLGALPPALRPLLSCLVGGAGCLLALGLDHFFHVRGASPPPRSASTAEEKVPVIRPRRRSSCVSLGESAAGYYGSGKMFRRPSLPCISREQMILWDWDLKQWCKPHYQNSGGGNGVDLSVLNEARNMVSDLLIDPSLPPQVISSLRSISSLMGAFSGSCRPKINSFTPFPGFYPCSEVEDPVEKGDRKLHKGLSGRTSFPTPQLRRSSGASSLLTNEHCSRWDRSSGKRSYQELSVSSHGCHLNGPFSSNLFTIPKQRSSSVSLTHHAGLRRAGALPSHSLLNSSSHVPVSAGSLTNRSPIGFPDTTDFLTKPNIILHRSLGSVSSAADFHQYLRNSDSNLCSSCGHQILKYVSTCEPDGTDHPSEKSGEEDSSVFSKEPLNIVETQEEETMKKACRELFLEGDSHLMEEAQQPNIDQEVSLDPMLVEDYDSLIEKMNNWNFQIFELVEKMGEKSGRILSQVMYTLFQDTGLLETFKIPTQEFMNYFRALENGYRDIPYHNRVHATDVLHAVWYLTTRPIPGLPQIHNNHETETKADSDGRLGSGQIAYISSKSCCIPDMSYGCLSSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLNLDHMEFKRFRFLVIEAILATDLKKHFDFLAEFNAKANDVNSNGIEWSSENDRLLVCQVCIKLADINGPAKDRDLHLRWTEGIVNEFYEQGDEEAALGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQWIETEEGDDTESDDDDDDDDGDGGEELDSDDEETEDNLNPKPQRRKGRRRIFCQLMHHLTENHKIWKEIIEEEEEKCKAEGNKLQVDNASLPQADEIQVIEEADEEEEQMFE

>gi|162329608|ref|NP_001104777.1| cAMP-specific 3',5'-cyclic phosphodiesterase 4A [Homo sapiens]EPPTVPSERSLSLSLPGPREGQATLKPPPQHLWRQPRTPIRIQQRGYSDSAERAERERQPHRPIERADAMDTSDRPGLRTTRMSWPSSFHGTGTGSGGAGGGSSRRFEAENGPTPSPGRSPLDSQASPGLVLHAGAATSQRRESFLYRSDSDYDMSPKTMSRNSSVTSEAHAEDLIVTPFAQVLASLRSVRSNFSLLTNVPVPSNKRSPLGGPTPVCKATLSEETCQQLARETLEELDWCLEQLETMQTYRSVSEMASHKFKRMLNRELTHLSEMSRSGNQVSEYISTTFLDKQNEVEIPSPTMKEREKQQAPRPRPSQPPPPPVPHLQPMSQITGLKKLMHSNSLNNSNIPRFGVKTDQEELLAQELENLNKWGLNIFCVSDYAGGRSLTCIMYMIFQERDLLKKFRIPVDTMVTYMLTLEDHYHADVAYHNSLHAADVLQSTHVLLAT

49

PALDAVFTDLEILAALFAAAIHDVDHPGVSNQFLINTNSELALMYNDESVLENHHLAVGFKLLQEDNCDIFQNLSKRQRQSLRKMVIDMVLATDMSKHMTLLADLKTMVETKKVTSSGVLLLDNYSDRIQVLRNMVHCADLSNPTKPLELYRQWTDRIMAEFFQQGDRERERGMEISPMCDKHTASVEKSQVGFIDYIVHPLWETWADLVHPDAQEILDTLEDNRDWYYSAIRQSPSPPPEEESRGPGHPPLPDKFQFELTLEEEEEEEISMAQIPCTAQEALTAQGLSGVEEALDATIAWEASPAQESLEVMAQEASLEAELEAVYLTQQAQSTGSAPVAPDEFSSREEFVVAVSHSSPSALALQSPLLPAWRTLSVSEHAPGLPGLPTAAEVEAQREHQAAKRACSACAGTFGEDTSALPAPGGGGSGGDPT

>gi|32306513|ref|NP_006194.2| cAMP-specific 3',5'-cyclic phosphodiesterase 4D isoform PDE4D3 [Homo sapiens]MMHVNNFPFRRHSWICFDVDNGTSAGRSPLDPMTSPGSGLILQANFVHSQRRESFLYRSDSDYDLSPKSMSRNSSIASDIHGDDLIVTPFAQVLASLRTVRNNFAALTNLQDRAPSKRSPMCNQPSINKATITEEAYQKLASETLEELDWCLDQLETLQTRHSVSEMASNKFKRMLNRELTHLSEMSRSGNQVSEFISNTFLDKQHEVEIPSPTQKEKEKKKRPMSQISGVKKLMHSSSLTNSSIPRFGVKTEQEDVLAKELEDVNKWGLHVFRIAELSGNRPLTVIMHTIFQERDLLKTFKIPVDTLITYLMTLEDHYHADVAYHNNIHAADVVQSTHVLLSTPALEAVFTDLEILAAIFASAIHDVDHPGVSNQFLINTNSELALMYNDSSVLENHHLAVGFKLLQEENCDIFQNLTKKQRQSLRKMVIDIVLATDMSKHMNLLADLKTMVETKKVTSSGVLLLDNYSDRIQVLQNMVHCADLSNPTKPLQLYRQWTDRIMEEFFRQGDRERERGMEISPMCDKHNASVEKSQVGFIDYIVHPLWETWADLVHPDAQDILDTLEDNREWYQSTIPQSPSPAPDDPEEGRQGQTEKFQFELTLEEDGESDTEKDSGSQVEEDTSCSDSKTLCTQDSESTEIPLDEQVEEEAVGEEEESQPEACVIDDRSPDT

>gi|365776143|gb|AEW91484.1| phosphodiesterase 5A [Homo sapiens]MERAGPSFGQQRQQQQPQQQKQQQRDQDSVEAWLDDHWDFTFSYFVRKATREMVNAWFAERVHTIPVCKEGIRGHTESCSCPLQQSPRADNSVPGTPTRKISASEFDRPLRPIVVKDSEGTVSFLSDSEKKEQMPLTPPRFDHDEGDQCSRLLELVKDISSHLDVTALCHKIFLHIHGLISADRYSLFLVCEDSSNDKFLISRLFDVAEGSTLEEVSNNCIRLEWNKGIVGHVAALGEPLNIKDAYEDPRFNAEVDQITGYKTQSILCMPIKNHREEVVGVAQAINKKSGNGGTFTEKDEKDFAAYLAFCGIVLHNAQLYETSLLENKRNQVLLDLASLIFEEQQSLEVILKKIAATIISFMQVQKCTIFIVDEDCSDSFSSVFHMECEELEKSSDTLTREHDANKINYMYAQYVKNTMEPLNIPDVSKDKRFPWTTENTGNVNQQCIRSLLCTPIKNGKKNKVIGVCQLVNKMEENTGKVKPFNRNDEQFLEAFVIFCGLGIQNTQMYEAVERAMAKQMVTLEVLSYHASAAEEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLETALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYRKNVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDLEILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHSIMEHHHFDQCLMILNSPGNQILSGLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDCFPLLDGCRKNRQKWQALAEQQEKMLINGESGQAKRN

>gi|112180437|gb|AAH35909.1| Phosphodiesterase 6A, cGMP-specific, rod, alpha [Homo sapiens]MGEVTAEEVEKFLDSNIGFAKQYYNLHYRAKLISDLLGAKEAAVDFSNYHSPSSMEESEIIFDLLRDFQENLQTEKCIFNVMKKLCFLLQADRMSLFMYRTRNGIAELATRLFNVHKDAVLEDCLVMPDQEIVFPLDMGIVGHVAHSKKIANVPNTEEDEHFCDFVDILTEYKTKNILASPIMNGKDVVAIIMAVNKVDGSHFTKRDEEILLKYLNFANLIMKVYHLSYLHNCETRRGQILLWSGSKVFEELTDIERQFHKALYTVRAFLNCDRYSVGLLDMTKQKEFFDVWPVLMGEVPPYSGPRTPDGREINFYKVIDYILHGKEDIKVIPNPPPDHWALVSGLPAYVAQNGLICNIMNAPAEDFFAFQKEPLDESGWMIKNVLSMPIVNKKEEIVGVATFYNRKDGKPFDEMDETLMESLTQFLGWSVLNPDTYESMNKLENRKDIFQDIVKYHVKCDNEEIQKILKTREVYGKEPWECEEEELAEILQAELPDADKYEINKFHFSDLPLTELELVKCGIQMYYELKVVDKFHIPQEALVRFMYSLSKGYRKITYHNWRHGFNVGQTMFSLLVTGKLKRYFTDLEALAMVTAAFCHDIDHRGTNNLYQMKSQNPLAKLHGSSILERHHLEFGKTLLRDESLNIFQNLNRRQHEHAIHMMDIAIIATDLALYFKKRTMFQKIVDQSKTYESEQEWTQYMMLEQTRKEIVMAMMMTACDLSAITKPWEVQSQVALLVAAEFWEQGDLERTVLQQNPIPMMDRNKADELPKLQVGFIDFVCTFVYKEFSRFHEEITPMLDGITNNRKEWKALADEYDAKMKVQEEKKQKQQSAKSAAAGNQPGGNPSPGGATTSKSCCIQ

>gi|12652977|gb|AAH00249.1| Phosphodiesterase 6B, cGMP-specific, rod, beta [Homo sapiens]MSLSEEQARSFLDQNPDFARQYFGKKLSPENVAAACEDGCPPDCDSLRDLCQVEESTALLELVQDMQESINMERVVFKVLRRLCTLLQADRCSLFMYRQRNGVAELATRLFSVQPDSVLEDCLVPPDSEIVFPLDIGVVGHVAQTKKMVNVEDVAECPHFSSFADELTDYKTKNMLATPIMNGKDVVAVIMAVNKLNGPFFTSEDEDVFLKYLNFATLYLKIYHLSYLHNCETRRGQVLLWSANKVFEELTDIERQFHKAFYTVRAYLNCERYSVGLLDMTKEKEFFDVWSVLMGESQPYSGPRTPDGREIVFYKVIDYVLHGKEEIKVIPTPSADHWALASGLPSYVAESGFICNIMNASADEMFKFQEGALDD

50

SGWLIKNVLSMPIVNKKEEIVGVATFYNRKDGKPFDEQDEVLMESLTQFLGWSVMNTDTYDKMNKLENRKDIAQDMVLYHVKCDRDEIQLILPTRARLGKEPADCDEDELGEILKEELPGPTTFDIYEFHFSDLECTELDLVKCGIQMYYELGVVRKFQIPQEVLVRFLFSISKGYRRITYHNWRHGFNVAQTMFTLLMTGKLKSYYTDLEAFAMVTAGLCHDIDHRGTNNLYQMKSQNPLAKLHGSSILERHHLEFGKFLLSEETLNIYQNLNRRQHEHVIHLMDIAIIATDLALYFKKRAMFQKIVDESKNYQDKKSWVEYLSLETTRKEIVMAMMMTACDLSAITKPWEVQSKVALLVAAEFWEQGDLERTVLDQQPIPMMDRNKAAELPKLQVGFIDFVCTFVYKEFSRFHEEILPMFDRLQNNRKEWKALADEYEAKVKALEEKEEEERVAAKKVGTEICNGGPAPKSSTCCIL

>gi|116496881|gb|AAI26361.1| Phosphodiesterase 7A [Homo sapiens]MGITLIWCLALVLIKWITSKRRGAISYDSSDQTALYIRMLGDVRVRSRAGFESERRGSHPYIDFRIFHSQSEIEVSVSARNIRRLLSFQRYLRSSRFFRGTAVSNSLNILDDDYNGQAKCMLEKVGNWNFDIFLFDRLTNGNSLVSLTFHLFSLHGLIEYFHLDMMKLRRFLVMIQEDYHSQNPYHNAVHAADVTQAMHCYLKEPKLANSVTPWDILLSLIAAATHDLDHPGVNQPFLIKTNHYLATLYKNTSVLENHHWRSAVGLLRESGLFSHLPLESRQQMETQIGALILATDISRQNEYLSLFRSHLDRGDLCLEDTRHRHLVLQMALKCADICNPCRTWELSKQWSEKVTEEFFHQGDIEKKYHLGVSPLCDRHTESIANIQIGFMTYLVEPLFTEWARFSNTRLSQTMLGHVGLNKASWKGLQREQSSSEDTDAAFELNSQLLPQENRLS

>gi|49904180|gb|AAH75822.1| Phosphodiesterase 8A [Homo sapiens]MGCAPSIHISERLVAEDAPSPAAPPLSSGGPRLPQGQKTAALPRTRGAGLLESELRDGSGKKVAVADVQFGPMRFHQDQLQVLLVFTKEDNQCNGFCRACEKAGFKCTVTKEAQAVLACFLDKHHDIIIIDHRNPRQLDAEALCRSIRSSKLSENTVIVGVVRRVDREELSVMPFISAGFTRRYVENPNIMACYNELLQLEFGEVRSQLKLRACNSVFTALENSEDAIEITSEDRFIQYANPAFETTMGYQSGELIGKELGEVPINEKKADLLDTINSCIRIGKEWQGIYYAKKKNGDNIQQNVKIIPVIGQGGKIRHYVSIIRVCNGNNKAEKISECVQSDTHTDNQTGKHKDRRKGSLDVKAVASRATEVSSQRRHSSMARIHSMTIEAPITKVINIINAAQESSPMPVTEALDRVLEILRTTELYSPQFGAKDDDPHANDLVGGLMSDGLRRLSGNEYVLSTKNTQMVSSNIITPISLDDVPPRIARAMENEEYWDFDIFELEAATHNRPLIYLGLKMFARFGICEFLHCSESTLRSWLQIIEANYHSSNPYHNSTHSADVLHATAYFLSKERIKETLDPIDEVAALIAATIHDVDHPGRTNSFLCNAGSELAILYNDTAVLESHHAALAFQLTTGDDKCNIFKNMERNDYRTLRQGIIDMVLATEMTKHFEHVNKFVNSINKPLATLEENGETDKNQEVINTMLRTPENRTLIKRMLIKCADVSNPCRPLQYCIEWAARISEEYFSQTDEEKQQGLPVVMPVFDRNTCSIPKSQISFIDYFITDMFDAWDAFVDLPDLMQHLDNNFKYWKGLDEMKLRNLRPPPE

>gi|14290551|gb|AAH09047.1| Phosphodiesterase 9A [Homo sapiens]MGSGSSSYRPKAIYLDIDGRIQKVIFSKYCNSSDIMDLFCIATGLPRNTTISLLTTDDAMVSIDPTMPANSERTPYKVRPVAIKQLSEREELIQSVLAQVAEQFSRAFKINELKAEVANHLAVLEKRVELEGLKVVEIEKCKSDIKKMREELAARSSRTNCPCKYSFLDNHKKLTPRRDVPTYPKYLLSPETIEALRKPTFDVWLWEPNEMLSCLEHMYHDLGLVRDFSINPVTLRRWLFCVHDNYRNNPFHNFRHCFCVAQMMYSMVWLCSLQEKFSQTDILILMTAAICHDLDHPGYNNTYQINARTELAVRYNDISPLENHHCAVAFQILAEPECNIFSNIPPDGFKQIRQGMITLILATDMARHAEIMDSFKEKMENFDYSNEEHMTLLKMILIKCCDISNEVRPMEVAEPWVDCLLEEYFMQSDREKSEGLPVAPFMDRDKVTKATAQIGFIKFVLIPMFETVTKLFPMVEEIMLQPLWESRDRYEELKRIDDAMKELQKKTDSLTSGATEKSRERSRDVKNSEGDCA

>gi|119567931|gb|EAW47546.1| phosphodiesterase 10A [Homo sapiens]MRIEERKSQHLTGLTDEKVKAYLSLHPQVLDEFVSESVSAETVEKWLKRKNNKSEDESAPKEVSRYQDTNMQGVVYELNSYIEQRLDTGGDNQLLLYELSSIIKIATKADGFALYFLGECNNSLCIFTPPGIKEGKPRLIPAGPITQGTTVSAYVAKSRKTLLVEDILGDERFPRGTGLESGTRIQSVLCLPIVTAIGDLIGILELYRHWGKEAFCLSHQEVATANLAWASVAIHQVQVCRGLAKQTELNDFLLDVSKTYFDNIVAIDSLLEHIMIYAKNLVNADRCALFQVDHKNKELYSDLFDIGEEKEGKPVFKKTKEIRFSIEKGIAGQVARTGEVLNIPDAYADPRFNREVDLYTGYTTRNILCMPIVSRGSVIGVVQMVNKISGSAFSKTDENNFKMFAVFCALALHCANMYHRIRHSECIYRVTMEKLSYHSICTSEEWQGLM

51

QFTLPVRLCKEIELFHFDIGPFENMWPGIFVYMVHRSCGTSCFELEKLCRFIMSVKKNYRRVPYHNWKHAVTVAHCMYAILQNNHTLFTDLERKGLLIACLCHDLDHRGFSNSYLQKFDHPLAALYSTSTMEQHHFSQTVSILQLEGHNIFSTLSSSEYEQVLEIIRKAIIATDLALYFGNRKQLEEMYQTGSLNLNNQSHRDRVIGLMMTACDLCSVTKLWPVTKLTANDIYAEFWAEGDEMKKLGIQPIPMMDRDKKDEVPQGQLGFYNAVAIPCYTTLTQILPPTEPLLKACRDNLSQWEKVIRGEETATWISSPSVAQKAAASED

>gi|10716052|dbj|BAB16371.1| phosphodiesterase 11A [Homo sapiens]MAASRLDFGEVETFLDRHPELFEDYLMRKGKQEMVEKWLQRHSQGQGALGPRPSLAGTSSLAHSTCRGGSSVGGGTGPNGSAHSQPLPGGGDCGGVPLSPSWAGGSRGDGNLQRRASQKELRKSFARSKAIHVNRTYDEQVTSRAQEPLSSVRRRALLRKASSLPPTTAHILSALLESRVNLPQYPPTAIDYKCHLKKHNERQFFLELVKDISNDLDLTSLSYKILIFVCLMVDADRCSLFLVEGAAAGKKTLVSKFFDVHAGTPLLPCSSTENSNEVQVPWGKGIIGYVGEHGETVNIPDAYQDRRFNDEIDKLTGYKTKSLLCMPIRSSDGEIIGVAQAINKIPEGAPFTEDDEKVMQMYLPFCGIAISNAQLFAASRKEYERSRALLEVVNDLFEEQTDLEKIVKKIMHRAQTLLKCERCSVLLLEDIESPVVKFTKSFELMSPKCSADAENSFKESMEKSSYSDWLINNSIAELVASTGLPVNISDAYQDPRFDAEADQISGFHIRSVLCVPIWNSNHQIIGVAQVLNRLDGKPFDDADQRLFEAFVIFCGLGINNTIMYDQVKKSWAKQSVALDVLSYHATCSKAEVDKFKAANIPLVSELAIDDIHFDDFSLDVDAMITAALRMFMELGMVQKFKIDYETLCRWLLTVRKNYRMVLYHNWRHAFNVCQLMFAMLTTAGFQDILTEVEILAVIVGCLCHDLDHRGTNNAFQAKSGSALAQLYGTSATLEHHHFNHAVMILQSEGHNIFANLSSKEYSDLMQLLKQSILATDLTLYFERRTEFFELVSKGEYDWNIKNHRDIFRSMLMTACDLGAVTKPWEISRQVAELVTSEFFEQGDRERLELKLTPSAIFDRNRKDELPRLQLEWIDSICMPLYQALVKVNVKLKPMLDSVATNRSKWEELHQKRLLASTASSSSPASVMVAKEDRN

APPENDIX - BFull PDE Family Sequence Alignment (1A-11A)(Significant data highlighted in red)

2A MGQACGHSILCRSQQ-YPAARPAEPRGQQVFLKPDEPPPPPQQCADSLQDALLS----LG10A -----------------------------M-----RIEERKSQHLTG-----LT----DE6A ------------------------------------------------------------5A ------------------MERAGPSFGQQR-----QQQQPQQQKQQQ-----RD----QD

52

11A ------------------------------------------MAASR-----LD----FG3A MAVP-GDAARVRDKPVHSGVSQAPTAGRDCHHRADPASPRDSGCRGCWGDLVLQPLRSSR9A ------------------------------------------------------------8A ------------------------------------------------------------1A ------------------------------------------------------------4A ------------------------------------------------------------7A ------------------------------------------------------------ 2A SV-IDISGLQRAVKEALSAVLPRVETVYTYLLDGESQLVC--------------------10A KV-KAYLSLHPQVLDEFVSESVSAETVEKWLKRKNNKSE---------------------6A ------------------------------------------------------------5A SV-EAWLDDHWDFTFSYFVRKATREMVNAWFAERVHTIP---------------------11A EV-ETFLDRHPELFEDYLMRKGKQEMVEKWLQRHSQGQGA--------------------3A KLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGA9A ------------------------------------------------------------8A -------------------------MGCAPSIHISERLVAEDAPSPAAPPLSSGGPRLPQ1A ------------------------------------------------------------4A ---------------------------MEPP----------TVPSERSLSLSLPGPRE--7A ------------------------------------------------------------ 2A ----------------------------------------------------------ED10A ------------------------------------------------------------6A ------------------------------------------------------------5A ------------------------------------------------------------11A ----------------------------------------------------------LG3A ---PGGGARLSPWLQPSALLFSLLCAFFWMG------------------------LYLLR9A ------------------------------------------------------------8A GQKTAALP--------R----TRGAGLLESELRDGSGK----KVAVADVQFG--PMRFHQ1A ------------------------------------------------------------4A GQATLKPPPQHLWRQPRTPIRIQQRGYSDSAERAERERQPHRPIERADAMDTSDRPGLRT7A ------------------------------------------------------------ 2A PPHELPQEGK-----VREAIISQKRLGCNGLGFSDLPGKPLARLVAPLAPDTQVLVMPLA10A ------------------------------------------------------------6A ------------------------------------------------------------5A --------------VCKEGIR--GHT--------------------------ESCSCPLQ11A PRPSLAGTSSLAHSTCRGGSSVGGGTGPNGSAH-----------SQPLPGGGDCGGVPLS3A AGVRLPLAVALLAACCGGE-----ALVQIGLGVGE-------DHLLSLPAAGVVLSCLAA9A ------------------------------------------------------------8A DQLQVLLVFTKEDNQCNGF-----CRACEKAGFK----------CTVTKEAQAVLACFLD1A ------------------------------------------------------------4A TRMSWPSSFHGTGTGSGGA-----G-GGSSRRFE----------AENGPTPSP-GRSPLD7A ------------------------------------------------------------ 2A DKEAGAVAAVI---------------LVHCGQLSDNEEWSLQAV--EKHTLVALRRVQVL10A ------------------------------------------------------------6A ---------MGE-----VTAEEVE-KFLDSNIGFAKQYYNL--------HYRA-------5A QSPRADNSVPGTPTR-KISASEFDR------------------------PLRPIVV----11A PSWAGGSRGDGNLQR-RASQKELRKSFARSKAIHVNRTYDEQVTSRAQEPLSSVRRRALL3A ATWLVLRLRLGVLMIALTSA--V-------------------------------------9A ------------------------------------------------------------8A K-----HH--DIIIIDHRNPRQLDAEALCRSIRS----S---------------------1A ------------------------------------------------------------4A S-----QASPGLVLHAGAATSQRRESFLYRSDSD----Y---------------------7A ------------------------------------------------------------2A QQRGPREAPR--------------AVQNPPEGTAEDQKGGAAYTDRDRKILQLCGEL-YD10A ------------------------DESAPKEVSR--YQD----TNMQGVVYELNSYIEQR6A ------------KLISDLL----GAKEAAVDFSN--YH-SPSSMEESEIIFDLLRDFQEN5A -KDS----EGTVSFLS-DSEKKEQMPLTPPRF-------DHDEGDQCSRLLELVKDISSH11A RKAS-SLPPTTAHILSALLESRVNLPQYPPTAID--YKCHLKKHNERQFFLELVKDISND3A ---------RTVSLI---------------------------------------------9A ------------------------------------------------------------8A -KLSENTV--IVGVV---------------------------------------------1A ------------------------------------------------------------4A -DMSPKTMSRNSSVT---------------------------------------------7A ------------------------------------------------------------ 2A LDA----SSLQLKVLQYLQQETRASRCCLLLVSEDNLQLSCK------VIGDKVL-----

53

10A LDTGGDNQLLLYELSSIIKIATKADGFALYFLGECNNSLCIFTPP--GIKEGKPR---LI6A LQT----EKCIFNVMKKLCFLLQADRMSLFMYRT-RNGIAELATRLFNVHKDAVLEDCLV5A LDV----TALCHKIFLHIHGLISADRYSLFLVCEDSSNDKFLISRLFDVAEGSTLEEV--11A LDL----TSLSYKILIFVCLMVDADRCSLFLVEGAAAGKKTLVSKFFDVHAGTPLLPCSS3A ----------------------SLERFKVAWRP--------YLAYLAGVLGILLARYVEQ9A ------------------------------------------------------------8A ---------------------RRVDREELSVMP--------FIS------AGFTRRYVEN1A ------------------------------------------------------------4A ---------------------SEAHAEDLIVTP--------FA-----------------7A ------------------------------------------------------------ 2A ---GEEVSFPL-TGCLGQVVEDKKSIQLKDLTSEDV--Q-QLQSMLGCELQ-----AMLC10A --P--AGPITQGTTVSAYVAKSRKTLLVEDILGDERFPR-GTGLESGTRIQ-----SVLC6A M-PDQEIVFPLDMGIVGHVAHSKKIANVPNTEEDEHFCD-FVDILTEYKTK-----NILA5A --SNNCIRLEWNKGIVGHVAALGEPLNIKDAYEDPRFNA-EVDQITGYKTQ-----SILC11A TENSNEVQVPWGKGIIGYVGEHGETVNIPDAYQDRRFND-EIDKLTGYKTK-----SLLC3A ILPQSAEAAPREHLGSQLIAGTKEDIPVF--KRRRRSSSVVSAEMSGCSSKSHRRTSLPC9A ------------------------------------------------------------8A PN---------------IMACYNELLQLE--FGEVR----SQLKLRACNSVFTALENS--1A ------------------------------------------------------------4A ---------------------------------QVL----AS--LRSVRSNFSLLTNV--7A ------------------------------------------------------------ 2A VPVISRATDQVVALACAFNKL--EGDLFTDEDEHVIQH---CF--------------HYT10A LPIVTAI-GDLIGILELYRHW--GKEAFCLSHQEVATA---NL--------------AWA6A SPIMNGK--DVVAIIMAVNKV--DGSHFTKRDEEILLK---YL--------------NFA5A MPIKNHR-EEVVGVAQAINKKSGNGGTFTEKDEKDFAA---YL--------------AFC11A MPIRSSD-GEIIGVAQAINKI-PEGAPFTEDDEKVMQM---YL--------------PFC3A IPR-----EQLMGHSEWDHKRGPRGSQSS--------GTSITVDIAVMGEATASLPTSWQ9A -----------------------MGSGSS-----SYRPKAIYLDID---G-------RIQ8A -ED-----AI----EITSEDRF-IQYANP-----AFETTMGYQSGELIGK-------ELG1A -----------------------MGSSAT-EIEELENTTFKYLTGEQT------------4A -P-------------VPSNKRSPLGGPTPVCKATLSEETCQQLARE--------------7A ------------------------------------------------------------ 2A STVLTSTLAFQKEQKLKCECQALLQVAKNLFTHLDDVSVLLQEIITEARNLSNAEICS--10A SVAIHQVQVCRGLAKQTELNDFLLDVSKTYFDNIVAIDSLLEHIMIYAKNLVNADRCA--6A NLIMKVYHLSYLHNCETRRGQILLWSGSKVFEELTDIERQFHKALYTVRAFLNCDRYS--5A GIVLHNAQLYETSLLENKRNQVLLDLASLIFEEQQSLEVILKKIAATIISFMQVQKCT--11A GIAISNAQLFAASRKEYERSRALLEVVNDLFEEQTDLEKIVKKIMHRAQTLLKCERCS--3A TLLFHQTCAT--------SLRAVSNL----LSTQLTFQAIHKPRVNPVTSLSENYTCSDS9A KVIFSKYCNS--------SDIMDLFC----------------------------------8A EVPINEKKAD--------LLDTINSC----------------------------------1A -----EKMWQ--------RLKGILRC----------------------------------4A ------------------TLEELDWC----------------------------------7A ------------------MGITLIWC----------------------------------

2A --------------------VFLLDQNEL------------------------VA-----10A --------------------LFQVDHKNK------------------------EL-----6A --------------------VGLLDMTKQ------------------------KE-----5A --------------------IFIVDEDCS------------------------DS-----11A --------------------VLLLEDIES------------------------PVV----3A EESSEKDKLAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSTSIKLQE9A ------------------IATGL-----PRNTTISLLTTD--------------------8A ------------------IRIGK------EWQGIYYAKKK-----------NGDNI--QQ1A ------------------LVKQL-----ERGDVNV-------------------------4A ------------------LEQLE-----TM------------------------------7A ------------------LALVL-----IKWIT--------------------------- 2A ------KVFDGGVVD----------------------------------DESYEIRIPAD

54

10A ----YSDLFDIGE----------------------------EKEGKPVFKKTKEIRFSIE6A ----FFDVWPVLMGEVPPYSGPRTPDGREINFYKVIDYILHGKEDIKVIPNPPPDHWALV5A ----FSSVFHM---E--------CEELE-------------KSSD--TLT-REHDANKIN11A ---KFTKSFELMSPK--------CSADA-------------ENSFKESMEKSSYSDWLIN3A APSSSPDSWNNPV----------MMTLTKSRSFTSSYAISAAN-----------------9A ---------------------------D---AMVSIDPTMPA-----------------N8A ------NVKIIPV----------IGQGGKIRHYVSIIRVCNGNNKAEKISECVQSDTHTD1A -----------------------VDLKKN-IEYAAS--VL--------------------4A ------------------------------QTYRSVSEMA--------------------7A -----------------------SKRRGA-ISYDSSDQ---------------------- 2A QGIAGHVATTGQILNIPDAYAHPL----FYRGV---DDSTGFRTRNILCFPIKNEN----10A KGIAGQVARTGEVLNIPDAYADPR----FNREV---DLYTGYTTRNILCMPIVSRG----6A SGLPAYVAQNGLICNIMNAPAEDF----FAFQKEPL-DESGWMIKNVLSMPIVNKK----5A YMYAQYVKNTMEPLNIPDVSKDKR----FPWTTENTGNVNQQCIRSLLCTPIKNGKK---11A NSIAELVASTGLPVNISDAYQDPR----FDAEA---DQISGFHIRSVLCVPIWNSN----3A HVKAKKQSRPGALAKISPLSSPC----SSPLQGTP--ASSLVSKISAVQFPESADTTAKQ9A SERTPYKVRP---VAIKQLSEREELIQSVLAQVAEQFS--RAFKINELKAEVANH-----8A NQTGKHKDRRKGSLDVKAVASRAT-EVSSQRRHS---SMARIHSM-TIEAPITKV-----1A --EAVYIDETRRLLDTEDELSDIQ-TDSVPSEVRDWLASTFTRKMG--------------4A --SHKFKRMLNRELTHLSEMSRSG-N-----QVSEYISTTFLDKQNEVEIPSPTM-----7A --TALYIRMLG----DVRVRSRAG-FESERRGSHPYIDFRIFHSQSEIEVSVSAR----- 2A -----------------------------QEVIGVAELVNKIN-----GPWFSKFDEDLA10A ------------------------------SVIGVVQMVNKIS-----GSAFSKTDENNF6A -----------------------------EEIVGVATFYNRKD-----GKPFDEMDETLM5A -----------------------------NKVIGVCQLVNKMEENTGKVKPFNRNDEQFL11A -----------------------------HQIIGVAQVLNRLD-----GKPFDDADQRLF3A SLGSHRALTYTQSAPDLSPQIL----TPPVICSSCGRPYS--Q-----GNPADE---PLE9A -----LAVLEK--RVELEGLKV-------VEIE---------------KCKSD------I8A -----INIINA--AQESSPMPVTEALDRVLEILRTTELYSPQF-----GAKDDD---PHA1A ---------M------TK------------------------------KKPEEK---PKF4A -----KE-REK--QQAPRPRPS--------------------Q-----PPPPPV---PHL7A -----N------------------------------------------------------ 2A TAFSIY-----CGISIAHSLLYKKVNEAQYRSHLANEMMMYHMKVSDDEYTKLLHD----10A KMFAVF-----CALALHCANMYHRIRHSECIYRVTMEKLSYHSICTSEEWQ---------6A ESLTQF-----LGWSVLNPDTYESMNKLENRKDIFQDIVKYHVKCDNEEIQKILKTREVY5A EAFVIF-----CGLGIQNTQMYEAVERAMAKQMVTLEVLSYHASAAEEETR---------11A EAFVIF-----CGLGINNTIMYDQVKKSWAKQSVALDVLSYHATCSKAEVD---------3A RSGVATRTPSRTDDTAQVTSDYETNNNSDSSDIVQNE---DETECLREPLRKASACSTY-9A KK------------------------------MRE--------ELAARSSRTNC-PCKY-8A ND------------------------------LVG--------GLMSDGLRRLS-GNEY-1A RS------------------------------IVH-------------AVQA-----GI-4A QP------------------------------MSQ--------I---TGLKKLMHSNSL-7A --------------------------------IRR--------L---LSFQRYLRSSRF-

2A ------------------------GIQP---VAAIDSNFASFTYTPRSLPE-------DD10A -------------------GLMQFTLPV---R--LCKEIELFHFDIGP----------FE6A GKEPWECEEEE------LAEILQAELPD---A--DKYEINKFHFSDLPLTE-------LE5A ----------E------LQSLAAAVVPS---A--QTLKITDFSFSDFELSD-------LE11A -------------------KFKAANIPL---V--SELAIDDIHFDDFSLDV-------DA3A --AP----ETMMFL---DKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRI9A --SFLDNHKKLTPRR------DVPTYPKYLLSPETIEALRKPTFDVWLWEP-------NE8A ---VLSTKNTQMVSSNIITPISLDDVPPR--IARAMENEEYWDFDIFELEAATH---NRP1A ---F----VERMYR-K-TYHMVGLAYPAA--VIVTLKDVDKWSFDVFALNEASG---EHS4A ---N----NSNIP----RFG-VKTDQEEL--LAQELENLNKWGLNIFCVSDYAG---GRS7A ---F----RGTAVS-N-SLNILDDDYNGQ--AKCMLEKVGNWNFDIFLFDRLTN---GNS 2A TSMAILSMLQDMNFINNYKIDCPTLARFCLMVKKGYR-D-PPYHNWMHAFSVSHFCYLLY

55

10A NMWPGIFVYMVHRSCGTSCFELEKLCRFIMSVKKNYR-R-VPYHNWKHAVTVAHCMYAIL6A LVKCGIQMYYELKVVDKFHIPQEALVRFMYSLSKGYR-K-ITYHNWRHGFNVGQTMFSLL5A TALCTIRMFTDLNLVQNFQMKHEVLCRWILSVKKNYR-KNVAYHNWRHAFNTAQCMFAAL11A MITAALRMFMELGMVQKFKIDYETLCRWLLTVRKNYR-M-VLYHNWRHAFNVCQLMFAML3A LSQVSYRLFEDMGLFEAFKIPIREFMNYFHALEIGYR--DIPYHNRIHATDVLHAVWYLT9A MLSCLEHMYHDLGLVRDFSINPVTLRRWLFCVHDNYR--NNPFHNFRHCFCVAQMMYSMV8A LIYLGLKMFARFGICEFLHCSESTLRSWLQIIEANYH-SSNPYHNSTHSADVLHATAYFL1A LKFMIYELFTRYDLINRFKIPVSCLITFAEALEVGYSKYKNPYHNLIHAADVTQTVHYIM4A LTCIMYMIFQERDLLKKFRIPVDTMVTYMLTLEDHYH-ADVAYHNSLHAADVLQSTHVLL7A LVSLTFHLFSLHGLIEYFHLDMMKLRRFLVMIQEDYH-SQNPYHNAVHAADVTQAMHCYL : : : : * :** * . :

2A KNLE--------------------------------------------LTNYLEDIEIFA10A QNN----------------------------------------------HTLFTDLERKG6A VTGK--------------------------------------------LKRYFTDLEALA5A KAGK--------------------------------------------IQNKLTDLEILA11A TTAG--------------------------------------------FQDILTEVEILA3A TQPIPGLSTVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMA9A WLCS--------------------------------------------LQEKFSQTDILI8A SKER--------------------------------------------IKETLDPIDEVA1A LHTG--------------------------------------------IMHWLTELEILA4A ATPA--------------------------------------------LDAVFTDLEILA7A KEPK--------------------------------------------LANSVTPWDILL :

2A LFISCMCHDLDHRGTNNSFQVASKSVLAALYSSEGSVMERHHFAQAIAILN-THGCNIFD10A LLIACLCHDLDHRGFSNSYLQKFDHPLAALYSTS--TMEQHHFSQTVSILQ-LEGHNIFS6A MVTAAFCHDIDHRGTNNLYQMKSQNPLAKLHGSS--ILERHHLEFGKTLLR-DESLNIFQ5A LLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCHS--IMEHHHFDQCLMILN-SPGNQILS11A VIVGCLCHDLDHRGTNNAFQAKSGSALAQLYGTS-ATLEHHHFNHAVMILQ-SEGHNIFA3A LYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDR-SVLENHHAAAAWNLFMSRPEYNFLI9A LMTAAICHDLDHPGYNNTYQINARTELAVRYNDI-SPLENHHCAVAFQILA-EPECNIFS8A ALIAATIHDVDHPGRTNSFLCNAGSELAILYNDT-AVLESHHAALAFQLTTGDDKCNIFK1A MVFAAAIHDYEHTGTTNNFHIQTRSDVAILYNDR-SVLENHHVSAAYRLMQ-EEEMNILI4A ALFAAAIHDVDHPGVSNQFLINTNSELALMYNDE-SVLENHHLAVGFKLLQ-EDNCDIFQ7A SLIAAATHDLDHPGVNQPFLIKTNHYLATLYKNT-SVLENHHWRSAVGLLR-ES--GLFS . ** :* * .: : * : :* ** : :

2A HFSRKDYQRMLDLMRDIILATDLAHHLRIFKDLQKMAEVG------------------YD10A TLSSSEYEQVLEIIRKAIIATDLALYFGNRKQLEEMYQTG-----------------SLN6A NLNRRQHEHAIHMMDIAIIATDLALYFKKRTMFQKIVDQSKT-------YESEQEWTQYM5A GLSIEEYKTTLKIIKQAILATDLALYIKRRGEFFELIRKN-----------------QFN11A NLSSKEYSDLMQLLKQSILATDLTLYFERRTEFFELVSKG-----------------EYD3A NLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNGKVNDDVG----------------ID9A NIPPDGFKQIRQGMITLILATDMARHAEIMDSFKEKMENF-------------------D8A NMERNDYRTLRQGIIDMVLATEMTKHFEHVNKFVNSINKPLATLEENGETDKNQEVINTM1A NLSKDDWRDLRNLVIEMVLSTDMSGHFQQIKNIRNSLQQ---------------------4A NLSKRQRQSLRKMVIDMVLATDMSKHMTLLADLKTMVETKKVT-----------SSGVLL7A HLPLESRQQMETQIGALILATDISRQNEYLSLFRSH------L-----------DRGDLC : : :::*:: :

2A RNNKQHHRLLLCLLMTSCDLSDQTKGWKTTRKIAELIYKEFFSQGDLE-KAMGNRP-MEM10A LNNQSHRDRVIGLMMTACDLCSVTKLWPVTKLTANDIYAEFWAEGD-EMKKLGIQP-IPM6A MLEQTRKEIVMAMMMTACDLSAITKPWEVQSQVALLVAAEFWEQGDLERTVLQQNP-IPM5A LEDPHQKELFLAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDRERKELNIEP-TDL11A WNIKNHRDIFRSMLMTACDLGAVTKPWEISRQVAELVTSEFFEQGDRERLELKLTP-SAI3A WTNENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEE-ASLGLPI-SPF9A YSNEEHMTLLKMILIKCCDISNEVRPMEVAEPWVDCLLEEYFMQSDRE-KSEGLPV-APF8A LRTPENRTLIKRMLIKCADVSNPCRPLQYCIEWAARISEEYFSQTDEE-KQQGLPVVMPV1A -PEGIDRAKTMSLILHAADISHPAKSWKLHYRWTMALMEEFFLQGDKE-AELGLPF-SPL4A LDNYSDRIQVLRNMVHCADLSNPTKPLELYRQWTDRIMAEFFQQGDRE-RERGMEI-SPM7A LEDTRHRHLVLQMALKCADICNPCRTWELSKQWSEKVTEEFFHQGDIE-KKYHLGV-SPL : .*: : : *:: : * .

56

2A MDREKA-YIPELQISFMEHIAMPIYKLLQDLFPKA--AELYERVASNGEHWTKVSHKFTI10A MDRDKKDEVPQGQLGFYNAVAIPCYTTLTQILPPT--EPLLKACRDNLSQWEKVIRGEET6A MDRNKADELPKLQVGFIDFVCTFVYKEFSRFHEEI--TPMLDGITNNRKEWKALADEYDA5A MNREKKNKIPSMQVGFIDAICLQLYEALTHVSEDC--FPLLDGCRKNRQKWQALAEQQEK11A FDRNRKDELPRLQLEWIDSICMPLYQALVKVNVKL--KPMLDSVATNRSKWEELHQKRLL3A MDRSAP-QLANLQESFISHIVGPLCNSYDSAGLM-------P------GKWVEDSDES--9A MDRDKV-TKATAQIGFIKFVLIPMFETVTKLFPMV-EEIMLQPLWESRDRYE--------8A FDRNTC-SIPKSQISFIDYFITDMFDAWDAFVDL---PDLMQHLDNNFKYWK--------1A CDRKST-MVAQSQIGFIDFIVEPTFSLLTDSTEKIVIPLIEEASKAETSSYVASSSTTIV4A CDKHTA-SVEKSQVGFIDYIVHPLWETWADLVHPDAQEI-LDTLEDN-RDWY--------7A CDRHTE-SIANIQIGFMTYLVEPLFTEWARFSNTRLSQTMLGHVGLNKASWK-------- :: * : . :

2A RGIPSNNSLDFL---------DEEYE-----VPDLDGTRAPIN-----GCCSLDAE----10A ATWISSPSVAQK------AAASED------------------------------------6A KMKVQEEKKQKQ------QSAKSAAAGNQPGGNPSPGGATTSK-----SCCIQ-------5A MLINGESGQAK------------------RN-----------------------------11A ASTASSSSPASV------MVAKED-----RN-----------------------------3A G--DTDDPEEEE----EEAPAPNEEETCENNESPKKKTFKRRKIY----CQITQHLLQNH9A ELKRIDDAMKELQKKTDSLTSGATEKSRERSR-----------DVKN--------SEGDC8A GLDEM----KLR-----NLRPPPE------------------------------------1A GLHIA-DALRRS-----NTKGSMSDGSYSPDYSLA------AVDLKSFKNNLVDIIQQNK4A YSAIR-----QS------PSPPPEEESRGPGHPPLPDKFQFELTLEEEEEEEISMAQIPC7A GLQRE-----QS------SSED-TDAAFELNSQLLPQENRLS------------------

2A ------------------------------------------------------------10A ------------------------------------------------------------6A ------------------------------------------------------------5A ------------------------------------------------------------11A ------------------------------------------------------------3A KMWKKVIEEEQRLAGIENQSLDQTPQSH--SSEQIQAIKEEEEEKGKPRG---------E9A A-----------------------------------------------------------8A ------------------------------------------------------------1A ERWKELAA--QGESDLHKNSEDLVNAEEK---------------------------HDET4A TAQEALTA--QGLSGVEEALDATIAWEASPAQESLEVMAQEASLEAELEAVYLTQQAQST7A ------------------------------------------------------------

2A ------------------------------------------------------------10A ------------------------------------------------------------6A ------------------------------------------------------------5A ------------------------------------------------------------11A ------------------------------------------------------------3A EIPTQKPDQ---------------------------------------------------9A ------------------------------------------------------------8A ------------------------------------------------------------1A HS----------------------------------------------------------4A GSAPVAPDEFSSREEFVVAVSHSSPSALALQSPLLPAWRTLSVSEHAPGLPGLPTAAEVE7A ------------------------------------------------------------

PDE3A – Sequence Alignment [Homo sapiens, Rattus norvegicus, Mus musculus, and Sus scrofa](44 amino-acid insert of catalytic domain is highlighted)

HsPDE3A MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHR-ADPASPRDSGCRGCWGDLVLQPLRSSRSsPDE3A ------------------------------------------------------------RnPDE3A MAVRGEAAQDWAKPGLRGPSPAPVARGDHRCRGGSPSSPRGSGC--CWRALALQPLRRSPMmPDE3A MAVRGEAAQDLAKPGLGGASPARVARGNHRHRGESSPSPRGSGC--CWRALALQPLRRSP

57

HsPDE3A KLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGASsPDE3A ------------------------------------------------------------RnPDE3A QLSSALCAGSLSVLLALLVRLVGGEVGGELESSQEAAAEEE--EEEGARGGVFPGPRGGAMmPDE3A QLSSALCAGSLSVLLALLVRLVGGEVGGELEKSQEAAAEEE--EEEGARGGVFPGPRGGA

HsPDE3A PGGGARLSPWLQPSALLFSLLCAFFWMGLYLLRAGVRLPLAVALLAACCGGEALVQIGLGSsPDE3A --------------------------MGLYLLRAGVRLPLAVALLAACCGGEALVQIGLGRnPDE3A PGGGAQLSPWLQPAALLFSLLCAFFWMGLCLLRAGVRLPLAVALLAACCAGEALVQLSLGMmPDE3A PGGGAQLSPWLQPAALLFSLLCAFFWMGLCLLRAGVRLPLAVALLAACCAGEALVQLSLG *** *******************.******:.**

HsPDE3A VGEDHLLSLPAAGVVLSCLAAATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYSsPDE3A VGEDHLLSLPA----------ATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYRnPDE3A VGDGRLLSLPAAGVLLSCLGGATWLVLRLRLGVLMVALTSALRTVALVSLERFKVAWRPYMmPDE3A VGDGRLLSLPAAGVLLSCLGGATWLVLRLRLGVLMVAWTSVLRTVALVSLERFKVAWRPY **: :****** **************:* **.:***:*:************

HsPDE3A LAYLAGVLGILLARYVEQILPQSAE-AAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSASsPDE3A LAYLAGVLGILLARYVEQILPQSAG-AAPREHFGSQLLAGTKEDIPEFKRRRRSSSVVSARnPDE3A LAYLAAVLGLLLARYAEQLLPQCSGPAPPRERFGSQSSARTKEEIPGWKRRRRSSSVVAGMmPDE3A LAYLAAVLGLLLARYAEQILPQCSGPAPPRERFGSQLSARTKEEIPGWKRRRRSSSVVAG *****.***:*****.**:***.: * ***::*** * ***:** :**********:.

HsPDE3A EMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEATASLPSsPDE3A EMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIAVMGEAHGLITRnPDE3A EMSGCGGKSHRRTSLPCIPREQLMGHSEWDHKRGSRGSQ-SGTSVTVDIAVMGEAHGLITMmPDE3A EMSGCSGKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQ-SGTSITVDIAVMGEAHGLIT *****..*************************** **** ****:********** . :

HsPDE3A TSWQTLLFHQTCATSLRAVSNLLSTQLTFQAIHKPRVNPVTSLSENYTCSDSEESSEKDKSsPDE3A DLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPAVSFSENYTCSDSEESAEKDKRnPDE3A DLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPTVTFSENYTCSDSEEGLEKDKMmPDE3A DLLADPSLPPNVCTSLRAVSNLLSTQLTFQAIHKPRVNPTVTFSENYTCSDSEEGLEKDK : . .**************************..::***********. ****

HsPDE3A LAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSTSIKLQEAPSS---SSsPDE3A LAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPSPVRRDRSASIKLHEAPSSSAINRnPDE3A LAIPKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSASIKPHEAPSPSAVNMmPDE3A QAISKRLRRSLPPGLLRRVSSTWTTTTSATGLPTLEPAPVRRDRSASIKPHEAPSPSAVN ** *********************************:*******:*** :**** .

HsPDE3A PDSWNNPVMMTLTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPASsPDE3A PDSWKNPVMMTLTKSRSFTSSYAVSASNHVKAKKQSRPGSLVKISPLSSPCSSALQGTPARnPDE3A PDSWNAPVLMTLTKSRSFTSSYAVSAANHVKAKKQNRPAVADLTHKGILLMD---LQREVMmPDE3A PDSWNAPGLTTLTKSRSFTSSYAVSAANHVKAKKQNRPGGLAKISPVPSPSSSPPQGSPA ****: * : *************:**:********.**. . .

HsPDE3A SSLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVICSSCGRPYSQGSsPDE3A SSPVSKISTVQFPEPADATAKQGLSSHKALTYTQSAPDLSPHILTPPVICSSCGRPYSQGRnPDE3A AQPCRNQTEQMIL-------LKLPLIMRPTTTV-----------TAATSCRMMRKPSAREMmPDE3A SSPVSNSASQQFPESPEVTIKRGPGSHRALTYTQSAPDLSPQIPPPSVICSSCGRPYSQG :. : : : : : * . . * :* ::

HsPDE3A NPADEPLERSGVATRTPSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECLREPLRKASASsPDE3A NPADGPLERSGPAIQAQSRTDDTAQVTSDYETNNNSDSSDIVQNEDETECSREPLRKASARnPDE3A SH----------------------------------------------------------MmPDE3A NPADGPSERSGPAMLKPNRTDDTSQVTSDYETNNNSDSSDILQNEEEAECQREPQRKASA .

HsPDE3A CSTYAPETMMFLDKPILAPEPLVMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSSsPDE3A CSAYTPDTMMFLDKPILAPEPLVMDNLDSIMEHLNTWNFPIFDLVEKIGRKCGRILSQVSRnPDE3A ------------------------------------------------------------MmPDE3A CGTYTSQTMIFLDKPILAPEPLVMDNLDSIMDQLNTWNFPIFDLMENIGRKCGRILSQVS

HsPDE3A YRLFEDMGLFEAFKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGL

58

SsPDE3A YRLFEDMGLFEAFKIPIREFMNYFHALEIGYREIPYHNRIHATDVLHAVWYLTTQPIPGLRnPDE3A ------------------------------------------------------------MmPDE3A YRLFEDMGLFEAFKIPVREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGL

HsPDE3A STVINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMALYVAAAMSsPDE3A STVINDHGSTSDSDSDSGFTHGHMGYVFSKMYNVPDDKYGCLSGNIPALELMALYVAAAMRnPDE3A ------------------------------------------------------------MmPDE3A PSVIGDHGSASDSDSDSGFTHGHMGYVFSKMYHVPDDKYGCLSGNIPALELMALYVAAAM

HsPDE3A HDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLINLDHVEFKSsPDE3A HDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRTEYNFLVNLDHVEFKRnPDE3A ------------------------------------------------------------MmPDE3A HDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFLVNLDHVEFK

HsPDE3A HFRFLVIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNENDRLLVCQMCIKLADINGPASsPDE3A HFRFLVIEAILATDLKKHFDFVAKFNAKVNDEVGIDWTNENDRLLVCQMCIKLADINGPARnPDE3A ------------------------------------------------------------MmPDE3A HFRFLVIEAILATDLKKHFDFVAKFNAKVNDDVGIDWTNENDRLLVCQMCIKLADINGPA

HsPDE3A KCKELHLQWTDGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSSsPDE3A KCKELHLQWTEGIVNEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSRnPDE3A ------------------------------------------------------------MmPDE3A KCKELHLRWTEGIASEFYEQGDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCHS

HsPDE3A YDSAGLMPGKWVEDSDESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQISsPDE3A YDSAGLMPGKWVEDSDESGDTDDPEEEE----APKEEETCENNDSPRKKTFKRRKIYCQIRnPDE3A ------------------------------------------------------------MmPDE3A YDSAGLMPGKWVDDSDDSGDTDDPEEEEEEAETPHEDEACESSIAPRKKSFKRRRIYCQI

HsPDE3A TQHLLQNHKMWKKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPSsPDE3A TQHLLQNHKMWKKVIEEEQRLAGIESQSLDQAPQQHSSEQIQAIKEEDEDKGKPRGEETPRnPDE3A ------------------------------------------------------------MmPDE3A TQHLLQNHMMWKKVIEEEQCLSGTENQSLDQVPLQHPSEQIQAIKEEEEEKGKPRAEETL

HsPDE3A TQKPDQSsPDE3A TPKPNQRnPDE3A ------MmPDE3A APQPDL

PDE3B – Sequence Alignment [Homo sapiens, Rattus norvegicus, Mus musculus](44 amino-acid insert of catalytic domain is highlighted)

HsPDE3B MRRDERD--AKAMRS------LQPPDGAGSPPESLRNGYVKSCVSPLRQDPPRGFFFHLCRnPDE3A MRKDERERDTPAMRSPYAAAARPATATAASPPESLRNGYVKSCVSPLRQDPPRSFFFHLCMmPDE3A MRKDERERDAPAMRSPP-----PPPASAASPPESLRNGYVKSCVSPLRQDPPRSFFFHLC **:***: : **** *.************************.******

59

HsPDE3B RFCNVELRPPPASPQQPRRCSPFCRARLSLGDLAAFVLALLLGAEPESWAAGAAWLRTLLRnPDE3A RFCNVEPPA----------ASLRAGARLSLAALAAFVLAALLGAGPERWAAAATGLRTLLMmPDE3A RFCNVEPPA----------ASLRAGARLSLGVLAAFVLAALLGARPERWAAAAAGLRTLL ****** .* . *****. ******* **** ** ***.*: *****

HsPDE3B SVCSHSLSPLFSIACAFFFLTCFLTRTKRGPGPGRSCGSWWLLALPACCYLGDFLVWQWWRnPDE3A SACSLSLSPLFSIACAFFFLTCFLTRAQRGP--DRGAGSWWLLALPACCYLGDFAAWQWWMmPDE3A SACSLSLSPLFSIACAFFFLTCFLTRAQRGP--GRGAGSWWLLALPACCYLGDFAAWQWW *.** *********************::*** *..***************** .****

HsPDE3B SWPWGDGDAGSAAPHTPPEAAAGRLLLVLSCVGLLLTLAHPLRLRHCVLVLLLASFVWWVRnPDE3A SWLRGE----------PAAAAAGRLCLVLSCVGLL-TLAPRVRLRHGVLVLLFAGLVWWVMmPDE3A SWLRGE----------PA--AAGRLCLVLSCVGLL-TLAPRVRLRHGVLVLLFAGLVWWV ** *: * ***** ********* *** :**** *****:*.:****

HsPDE3B SFTSLGSLPSALRPLLSGLVGGAGCLLALGLDHFFQIREAPLHPRLSSAAEEKVPVIRPRRnPDE3A SFSGLGALPPALRPLLSCLVGGAGCLLALGLDHFFHVRGASPPPRSASTADEKVPVIRPRMmPDE3A SFSGLGALPPALRPLLSCLVGGAGCLLALGLDHFFHVRGASPPPRSASTAEEKVPVIRPR **:.**:** ******* *****************::* * ** :*:*:*********

HsPDE3B RRSSCVSLGETAASYYGSCKIFRRPSLPCISREQMILWDWDLKQWYKPHYQNSGGGNGVDRnPDE3A RRSSCVSLGESAAGYYGSGKMFRRPSLPCISREQMILWDWDLKQWCKPHYQNSGGGNGVDMmPDE3A RRSSCVSLGESAAGYYGSGKMFRRPSLPCISREQMILWDWDLKQWCKPHYQNSGGGNGVD **********:**.**** *:************************ **************

HsPDE3B LSVLNEARNMVSDLLTDPSLPPQVISSLRSISSLMGAFSGSCRPKINPLTPFPGFYPCSERnPDE3A LSVLNEARNMVSDLLIDPSLPPQVISSLRSISSLMGAFSGSCRPKINSFTPFPGFYPCSEMmPDE3A LSVLNEARNMVSDLLIDPSLPPQVISSLRSISSLMGAFSGSCRPKINSFTPFPGFYPCSE *************** ******************************* :***********

HsPDE3B IEDPAEKGDRKLNKGLN-RNSLPTPQLRRSSGTSGLLPVEQSSRWDRNNGKRPHQEFGISRnPDE3A VEDPVEKGDRKLHKGLSSKPSFPTAQLRRSSGASGLLTSEHHSRWDRSGGKRPYQELSVSMmPDE3A VEDPVEKGDRKLHKGLSGRTSFPTPQLRRSSGASSLLTNEHCSRWDRSSGKRSYQELSVS :***.*******.***. : *:** *******:*.** *: *****. *** :**:.:*

HsPDE3B SQGCYLNGPFNSNLLTIPKQRSSSVSLTHHVGLRRAGVLSSLSPVNSSNHGPVSTGSLTNRnPDE3A SHGCHLNGPFSSNLMTIPKQRSSSVSLTHHAGLRRAGALPSPSLLNSSSHVPVSAGCLTNMmPDE3A SHGCHLNGPFSSNLFTIPKQRSSSVSLTHHAGLRRAGALPSHSLLNSSSHVPVSAGSLTN *:**:*****.***:***************.******.* * * :***.* ***:*.***

HsPDE3B RSPIEFPDTADFLNKPSVILQRSLGNAPNTPDFYQQLRNSDSNLCNSCGHQMLKYVSTSERnPDE3A RSPVGFLDTSDFLTKPSVTLHRSLGSVSSAADFHQYLRNSDSSLCSSCGHQILKYVSTCEMmPDE3A RSPIGFPDTTDFLTKPNIILHRSLGSVSSAADFHQYLRNSDSNLCSSCGHQILKYVSTCE ***: * **:***.**.: *:****.. .: **:* ******.**.*****:******.*

HsPDE3B SDGTDCCSGKSGEEE-NIFSKESFKLMETQQEEETEKKDSRKLFQEGDKWLTEEAQSEQQRnPDE3A PDGTDHHNEKSGEEDSTVFSKERLNIVETQ-EEETVKEDCRELFLEGDDHLMEEA---QQMmPDE3A PDGTDHPSEKSGEEDSSVFSKEPLNIVETQ-EEETMKKACRELFLEGDSHLMEEA---QQ **** . *****: .:**** ::::*** **** *: .*:** ***. * *** **

HsPDE3B TNIEQEVSLDLILVEEYDSLIEKMSNWNFPIFELVEKMGEKSGRILSQVMYTLFQDTGLLRnPDE3A PNIDQEVLLDPMLVEDYDSLIEKMSNWNFQIFELVEKMGEKSGRILSQVMYTLFQDTGLLMmPDE3A PNIDQEVSLDPMLVEDYDSLIEKMNNWNFQIFELVEKMGEKSGRILSQVMYTLFQDTGLL **:*** ** :***:********.**** ******************************

HsPDE3B EIFKIPTQQFMNYFRALENGYRDIPYHNRIHATDVLHAVWYLTTRPVPGLQQIHNGCGTGRnPDE3A ETFKIPTQEFMNYFRALENGYRDIPYHNRVHATDVLHAVWYLTTRPIPGLQQLHNNHETEMmPDE3A ETFKIPTQEFMNYFRALENGYRDIPYHNRVHATDVLHAVWYLTTRPIPGLPQIHNNHETE * ******:********************:****************:*** *:** *

HsPDE3B NETDSDGRINHGRIAYISSKSCSNPDESYGCLSSNIPALELMALYVAAAMHDYDHPGRTNRnPDE3A TKADSDARLSSGQIAYLSSKSCCIPDKSYGCLSSNIPALELMALYVAAAMHDYDHPGRTNMmPDE3A TKADSDGRLGSGQIAYISSKSCCIPDMSYGCLSSNIPALELMALYVAAAMHDYDHPGRTN .::***.*: *:***:*****. ** *********************************

60

HsPDE3B AFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLHLDHVEFKRFRFLVIEAIRnPDE3A AFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLNLDHMEFKRFRFLVIEAIMmPDE3A AFLVATNAPQAVLYNDRSVLENHHAASAWNLYLSRPEYNFLLNLDHMEFKRFRFLVIEAI ******************************************.***:*************

HsPDE3B LATDLKKHFDFLAEFNAKANDVNSNGIEWSNENDRLLVCQVCIKLADINGPAKVRDLHLKRnPDE3A LATDLKKHFEFLAEFNAKANDVNSNGIEWSSENDRLLVCQVCIKLADINGPAKDRDLHLRMmPDE3A LATDLKKHFDFLAEFNAKANDVNSNGIEWSSENDRLLVCQVCIKLADINGPAKDRDLHLR *********:********************.********************** *****:

HsPDE3B WTEGIVNEFYEQGDEEANLGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPRnPDE3A WTEGIVNEFYEQGDEEATLGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPMmPDE3A WTEGIVNEFYEQGDEEAALGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLP ***************** ******************************************

HsPDE3B GQWLEAEEDNDTESGD------------DEDGEELDTEDEEMENNLNPKPPRRKSRRRIFRnPDE3A GQWIEAEEGDDTESDDDDDDDDDDDDDDDDDDEELDSDDEETEDNLNPKPQRRKGRRRIFMmPDE3A GQWIETEEGDDTESDDDDDD------DDGDGGEELDSDDEETEDNLNPKPQRRKGRRRIF ***:*:** :**** * : ****::*** *:****** ***.*****

HsPDE3B CQLMHHLTENHKIWKEIVEE-EEKCKADGNKLQVENSSLPQADEIQVIEEADEEE-----RnPDE3A CQLMHHLTENHKIWKEIIEE-EEKCKAEGNKLQVDNASLPQADEIQVIEEADEEEEQMFEMmPDE3A CQLMHHLTENHKIWKEIIEEEEEKCKAEGNKLQVDNASLPQADEIQVIEEADEEEEQMFE *****************:** ******:******:*:******************

GAF DOMAIN SEQUENCE ALIGNMENT

PDE2_GAF1 DASSLQLKVLQYLQQETRASRCCLLLVSEDNLQLSC---KVFG-------D---------PDE5_GAF1 DVTALCHKIFLHIHGLISADRYSLFLVCEDSSNDKFLISRLFDVAE---GSTL-------PDE6_GAF1 QTEKCIFNVMKKLCFLLQADRMSLFMYRT-RNGIAELATRLFNVHK---DAVLE------

61

PDE10_GAF1 DNQLLLYELSSIIKIATKADGFALYFLGECNNS-LC----IFT-------------PPGIPDE11_GAF1 DLTSLSYKILIFVCLMVDADRCSLFLVEGAAAGKKTLVSKFFDVHA---GTPL-------PDE2_GAF2 DVSVLLQEIITEARNLSNAEICSVFLLDQ---N--ELVAKVFDG----------------PDE5_GAF2 SLEVILKKIAATIISFMQVQKCTIFIVDEDCSD---SFSSVFHMECEELEKSS-------PDE6_GAF2 DIERQFHKALYTVRAFLNCDRYSVGLLDMTKQK------EFFDVWPVLMGEVPPYSGPRTPDE10_GAF2 AIDSLLEHIMIYAKNLVNADRCALFQVDH---KNKELYSDLFDIGE---EKEG-------PDE11_GAF2 DLEKIVKKIMHRAQTLLKCERCSVLLLEDIESPVVK-FTKSFELMS---PKCSA------ . . : :

PDE2_GAF1 ------------------------KVLGEEVSFPL-TGCLGQVVEDKKSIQLKDLTSEDVPDE5_GAF1 -------------------EEV----SNNCIRLEWNKGIVGHVAALGEPLNIKDAYEDPRPDE6_GAF1 -DC-L-------------------VMPDQEIVFPLDMGIVGHVAHSKKIANVPNTEEDEHPDE10_GAF1 KEG---------------KP-----RLIPAGPITQGTTVSAYVAKSRKTLLVEDILGDERPDE11_GAF1 -------------------LPCSSTENSNEVQVPWGKGIIGYVGEHGETVNIPDAYQDRRPDE2_GAF2 -------------------GVV--DDESYEIRIPADQGIAGHVATTGQILNIPDAYAHPLPDE5_GAF2 -----------------------DTLTREHDANKINYMYAQYVKNTMEPLNIPDVSKDKRPDE6_GAF2 PDGREINFYKVIDYILHGKEDIKVIPNPPPDHWALVSGLPAYVAQNGLICNIMNAPAEDFPDE10_GAF2 -------------------KPV--FKKTKEIRFSIEKGIAGQVARTGEVLNIPDAYADPRPDE11_GAF2 -DA-E-------------NSFKESMEKSSYSDWLINNSIAELVASTGLPVNISDAYQDPR * : : .

PDE2_GAF1 Q-----QLQSMLGCELQAMLCVPVISRATDQVVALACAFNKLE-----GDLFTDEDEHVIPDE5_GAF1 F--NAEVD-QITGYKTQSILCMPIKNHR-EEVVGVAQAINKKS---GNGGTFTEKDEKDFPDE6_GAF1 F--CDFVD-ILTEYKTKNILASPIMNG--KDVVAIIMAVNKVD-----GSHFTKRDEEILPDE10_GAF1 FPRG-QMVN-T-GLESGTRIQSVLCLPIVTAI-GDLIGINELYRHWG-KEAFCLSHQEVAPDE11_GAF1 F--NDEID-KLTGYKTKSLLCMPIRSSD-GEIIGVAQAINKI----PEGAPFTEDDEKVMPDE2_GAF2 F--YRGVD-DSTGFRTRNILCFPIKNEN-QEVIGVAELVNKIN-----GPWFSKFDEDLAPDE5_GAF2 FPWTTENTGNVNQQCIRSLLCTPIKNGKKNKVIGVCQLVNKMEENTGKVKPFNRNDEQFLPDE6_GAF2 FAFQKEPL-DESGWMIKNVLSMPIVNKK-EEIVGVATFYNRKD-----GKPFDEMDETLMPDE10_GAF2 F--NREVD-LYTGYTTRNILCMPIVSR--GSVIGVVQMVNKIS-----GSAFSKTDENNF PDE11_GAF2 F--DAEAD-QISGFHIRSVLCVPIWNSN-HQIIGVAQVLNRLD-----GKPFDDADQRLF

: :*. *: . .::.: .: * .:

PDE2_GAF1 QHCFHYTSTVLPDE5_GAF1 AAYLAFCGIVLPDE6_GAF1 LKYLNFANLIMPDE10_GAF1 TANLAWASVAIPDE11_GAF1 QMYLPFCGIAIPDE2_GAF2 TAFSIYCGISIPDE5_GAF2 EAFVIFCGLGIPDE6_GAF2 ESLTQFLGWSVPDE10_GAF2 KMFAVFCALALPDE11_GAF2 EAFVIFCGLGI : :

Raw Data - Amino Acid Frequency – PDE3A & PDE3B(Using ExPASy ProtParam tool)

PDE3A

62

Number of amino acids: 1141

Molecular weight: 125109.5

Theoretical pI: 5.73

Amino acid composition: 

Ala (A) 102 8.9%

Arg (R) 65 5.7%

Asn (N) 39 3.4%

Asp (D) 59 5.2%

Cys (C) 27 2.4%

Gln (Q) 44 3.9%

Glu (E) 78 6.8%

Gly (G) 70 6.1%

His (H) 31 2.7%

Ile (I) 49 4.3%

Leu (L) 119 10.4%

Lys (K) 47 4.1%

Met (M) 22 1.9%

Phe (F) 34 3.0%

Pro (P) 75 6.6%

Ser (S) 114 10.0%

Thr (T) 63 5.5%

Trp (W) 16 1.4%

Tyr (Y) 25 2.2%

Val (V) 62 5.4%

Pyl (O) 0 0.0%

Sec (U) 0 0.0%

(B) 0 0.0%

(Z) 0 0.0%

(X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 137Total number of positively charged residues (Arg + Lys): 112

63

A 102 8.9395267309377

R 65 5.6967572304995

N 39 3.4180543382997

D 59 5.1709027169149

C 27 2.3663453111305

Q 44 3.8562664329535

E 78 6.8361086765994

G 70 6.1349693251533

H 31 2.7169149868536

I 49 4.2944785276073

L 119 10.429447852760

K 47 4.1191936897458

M 22 1.9281332164767

F 34 2.9798422436459

P 75 6.5731814198071

S 114 9.9912357581069

T 63 5.5214723926380

W 16 1.4022787028922

Y 25 2.1910604732690

V 62 5.4338299737072

O 0 0

U 0 0

B 0 0

Z 0 0

X 0 0

PDE3B

Number of amino acids: 1112

Molecular weight: 124377.4

Theoretical pI: 5.57

Amino acid composition:

Ala (A) 71 6.4%

Arg (R) 67 6.0%

Asn (N) 59 5.3%

Asp (D) 56 5.0%

Cys (C) 32 2.9%

Gln (Q) 42 3.8%

Glu (E) 82 7.4%

Gly (G) 73 6.6%

His (H) 30 2.7%

Ile (I) 43 3.9%

Leu (L) 133 12.0%

Lys (K) 42 3.8%

Met (M) 16 1.4%

Phe (F) 46 4.1%

Pro (P) 74 6.7%

Ser (S) 108 9.7%

Thr (T) 40 3.6%

Trp (W) 23 2.1%

Tyr (Y) 25 2.2%

Val (V) 50 4.5%

Pyl (O) 0 0.0%

64

A 71 6.3848920863309

R 67 6.0251798561151

N 59 5.3057553956834

D 56 5.0359712230215

C 32 2.8776978417266

Q 42 3.7769784172661

E 82 7.3741007194244

G 73 6.5647482014388

H 30 2.6978417266187

I 43 3.8669064748201

L 133 11.960431654676

K 42 3.7769784172661

M 16 1.4388489208633

F 46 4.1366906474820

P 74 6.6546762589928

S 108 9.7122302158273

T 40 3.5971223021582

W 23 2.0683453237410

Y 25 2.2482014388489

V 50 4.4964028776978

Sec (U) 0 0.0%

(B) 0 0.0%

(Z) 0 0.0%

(X) 0 0.0%

Total number of negatively charged residues (Asp + Glu): 138Total number of positively charged residues (Arg + Lys): 109

APPENDIX – CPairwise Sequence Alignment of PDE3 using Smith-Waterman Algorithm (Calculated using: http://www.ebi.ac.uk/Tools/psa/emboss_water/) – Key data highlighted

######################################### Program: water# Rundate: Sat 18 Apr 2015 19:19:10# Commandline: water# -auto# -stdout# -asequence emboss_water-I20150425-191909-0597-76098860-pg.asequence# -bsequence emboss_water-I20150425-191909-0597-76098860-pg.bsequence# -datafile EBLOSUM62# -gapopen 10.0# -gapextend 0.5# -aformat3 pair# -sprotein1# -sprotein2# Align_format: pair# Report_file: stdout########################################

#=======================================## Aligned_sequences: 2# 1: HsPDE3A# 2: HsPDE3B# Matrix: EBLOSUM62

65

O 0 0

U 0 0

B 0 0

Z 0 0

X 0 0

# Gap_penalty: 10.0# Extend_penalty: 0.5## Length: 1212# Identity: 526/1212 (43.4%)# Similarity: 687/1212 (56.7%)# Gaps: 204/1212 (16.8%)# Score: 2116.0# ##=======================================

HsPDE3A 4 PGDAA-----RVRDKPVHSGVS---QAPTAG------RDCH--HRADPAS 37 |.|.| .:|:..|.|.|| |.|..| |.|: .|..|||HsPDE3B 16 PPDGAGSPPESLRNGYVKSCVSPLRQDPPRGFFFHLCRFCNVELRPPPAS 65

HsPDE3A 38 PRDSGCRGCWGDLVLQPLRSSRKLSSALCAGSL-SFLLALLVRLVRGEVG 86 |: ||.|.|....:.|..|.| :|:||||: HsPDE3B 66 PQ-------------QPRRCSPFCRARLSLGDLAAFVLALLL-------- 94

HsPDE3A 87 CDLEQCKEAAAAEEEEAAPGAEGGVFPGPRGGAPGGGARLSPWLQ----- 131 .||.|..|.|| .||: HsPDE3B 95 ----------GAEPESWAAGA--------------------AWLRTLLSV 114

HsPDE3A 132 ---PSALLFSLLCAFFWMGLYLLRAGVRLP---------LAVALLAACCG 169 ..:.|||:.||||::..:|.|. .|.| ..:||.|.|..HsPDE3B 115 CSHSLSPLFSIACAFFFLTCFLTRT-KRGPGPGRSCGSWWLLALPACCYL 163

HsPDE3A 170 GEALV---------QIGLGVGEDHLLSLPAAG---VVLSCLAAATWLV-- 205 |:.|| ....|....|.....||| :||||:.....|. HsPDE3B 164 GDFLVWQWWSWPWGDGDAGSAAPHTPPEAAAGRLLLVLSCVGLLLTLAHP 213

HsPDE3A 206 LRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGILLARYV 255 ||||..||::.|.|.|..||..||.....|.||.|:.|.|..|.|||..:HsPDE3B 214 LRLRHCVLVLLLASFVWWVSFTSLGSLPSALRPLLSGLVGGAGCLLALGL 263

HsPDE3A 256 EQILPQSAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSAEMSGC-- 303 :... |..||. |..:|.:..:|.:||.:.|||||.|...|.:.. HsPDE3B 264 DHFF-QIREAP----LHPRLSSAAEEKVPVIRPRRRSSCVSLGETAASYY 308

HsPDE3A 304 -SSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGS-QSSGTSITVDIAVMG 351 |.|..||.|||||.|||:: ..:||.|:..:.. |:||....||::|:.HsPDE3B 309 GSCKIFRRPSLPCISREQMI-LWDWDLKQWYKPHYQNSGGGNGVDLSVLN 357

HsPDE3A 352 EA---------TASLPTSWQTLLFHQTCATSLRAVSNLLSTQLTFQAIHK 392 || ..|||.. ..:|||::|:|:. .|....:HsPDE3B 358 EARNMVSDLLTDPSLPPQ---------VISSLRSISSLMG---AFSGSCR 395

HsPDE3A 393 PRVNPVTSLSENYTCSDSEESSEKDKLAIPKRL-RRSLPPGLLRRVSSTW 441 |::||:|.....|.||:.|:.:||....:.|.| |.|||...|||.|.| HsPDE3B 396 PKINPLTPFPGFYPCSEIEDPAEKGDRKLNKGLNRNSLPTPQLRRSSGT- 444

HsPDE3A 442 TTTTSATGLPTLEPAPVRRDRSTSIK-LQEAPSSSPDSW-NNPV---MMT 486 :||..:|.:. |.||:...: .||...||...: |.|. ::|HsPDE3B 445 ------SGLLPVEQSS-RWDRNNGKRPHQEFGISSQGCYLNGPFNSNLLT 487

HsPDE3A 487 LTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPAS 536 :.|.| |.::|..:||..: |.|.|:.:||::|....|:. ..HsPDE3B 488 IPKQR----SSSVSLTHHVGLR---RAGVLSSLSPVNSSNHGPVS---TG 527

HsPDE3A 537 SLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQIL-TPPVIC 585 ||.:: |.::||::||...|.|:...|:|....:.||...|:. :...:|HsPDE3B 528 SLTNR-SPIEFPDTADFLNKPSVILQRSLGNAPNTPDFYQQLRNSDSNLC 576

HsPDE3A 586 SSCGRPY------SQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETNN 629 :|||... |:.:..|....:||......|:.......|...|...HsPDE3B 577 NSCGHQMLKYVSTSESDGTDCCSGKSGEEENIFSKESFKLMETQQEEETE 626

66

HsPDE3A 630 NSDSSDIVQNEDE--TECLREPLRKASACSTYAPETMMFLDKPILAPEPL 677 ..||..:.|..|: || :|.:......|..:.||. :HsPDE3B 627 KKDSRKLFQEGDKWLTE-------EAQSEQQTNIEQEVSLDL-------I 662

HsPDE3A 678 VMDNLDSIMEQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEA 727 :::..||::|:::.||||||:|||.:|.|.|||||||.|.||:|.||.|.HsPDE3B 663 LVEEYDSLIEKMSNWNFPIFELVEKMGEKSGRILSQVMYTLFQDTGLLEI 712

HsPDE3A 728 FKIPIREFMNYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLST 777 ||||.::|||||.|||.|||||||||||||||||||||||||:|:|||..HsPDE3B 713 FKIPTQQFMNYFRALENGYRDIPYHNRIHATDVLHAVWYLTTRPVPGLQQ 762

HsPDE3A 778 VINDHGSTSDSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELM 827 :.|..|:.:::|||....||.:.|:.||:.:..|:.|||||.||||||||HsPDE3B 763 IHNGCGTGNETDSDGRINHGRIAYISSKSCSNPDESYGCLSSNIPALELM 812

HsPDE3A 828 ALYVAAAMHDYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLF 877 ||||||||||||||||||||||||:|||||||||||||||||||:||||:HsPDE3B 813 ALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYNDRSVLENHHAASAWNLY 862

HsPDE3A 878 MSRPEYNFLINLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNGKVND- 926 :||||||||::|||||||.||||||||||||||||||||:|:||.|.|| HsPDE3B 863 LSRPEYNFLLHLDHVEFKRFRFLVIEAILATDLKKHFDFLAEFNAKANDV 912

HsPDE3A 927 -DVGIDWTNENDRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQ 975 ..||:|:||||||||||:||||||||||||.::|||:||:|||||||||HsPDE3B 913 NSNGIEWSNENDRLLVCQVCIKLADINGPAKVRDLHLKWTEGIVNEFYEQ 962

HsPDE3A 976 GDEEASLGLPISPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGK 1025 |||||:||||||||||||:||||.||||||:|||||||||||:|||:||:HsPDE3B 963 GDEEANLGLPISPFMDRSSPQLAKLQESFITHIVGPLCNSYDAAGLLPGQ 1012

HsPDE3A 1026 WV---EDSD-ESGDTDDPEEEEEEAPAPNEEETCENNESPKKKTFK-RRK 1070 |: ||:| ||||.:|.||.: .|:|..|||.:||....| ||:HsPDE3B 1013 WLEAEEDNDTESGDDEDGEELD------TEDEEMENNLNPKPPRRKSRRR 1056

HsPDE3A 1071 IYCQITQHLLQNHKMWKKVIEEEQRLAG------IENQSLDQTPQSHSSE 1114 |:||:..||.:|||:||:::|||::... :||.||.| ::HsPDE3B 1057 IFCQLMHHLTENHKIWKEIVEEEEKCKADGNKLQVENSSLPQ------AD 1100

HsPDE3A 1115 QIQAIKEEEEEK 1126 :||.|:|.:||:HsPDE3B 1101 EIQVIEEADEEE 1112

67

######################################### Program: water# Rundate: Sat 18 Apr 2015 19:16:03# Commandline: water# -auto# -stdout# -asequence emboss_water-I20150425-191602-0681-15585880-pg.asequence# -bsequence emboss_water-I20150425-191602-0681-15585880-pg.bsequence# -datafile EBLOSUM62# -gapopen 10.0# -gapextend 0.5# -aformat3 pair# -sprotein1# -sprotein2# Align_format: pair# Report_file: stdout########################################

#=======================================## Aligned_sequences: 2# 1: HsPDE3A# 2: MmPDE3A# Matrix: EBLOSUM62# Gap_penalty: 10.0# Extend_penalty: 0.5## Length: 1154# Identity: 945/1154 (81.9%)# Similarity: 1006/1154 (87.2%)# Gaps: 28/1154 ( 2.4%)# Score: 4830.5#

68

##=======================================

HsPDEA 1 MAVPGDAARVRDKPVHSGVSQAPTAGRDCHHRADPA-SPRDSGCRGCWGD 49 |||.|:||:...||...|.|.|..|..:..||.:.: |||.||| ||..MmPDEA 1 MAVRGEAAQDLAKPGLGGASPARVARGNHRHRGESSPSPRGSGC--CWRA 48

HsPDEA 50 LVLQPLRSSRKLSSALCAGSLSFLLALLVRLVRGEVGCDLEQCKEAAAAE 99 |.|||||.|.:|||||||||||.|||||||||.||||.:||:.:||||.|MmPDEA 49 LALQPLRRSPQLSSALCAGSLSVLLALLVRLVGGEVGGELEKSQEAAAEE 98

HsPDEA 100 EEEAAPGAEGGVFPGPRGGAPGGGARLSPWLQPSALLFSLLCAFFWMGLY 149 ||| .||.||||||||||||||||:|||||||:|||||||||||||||.MmPDEA 99 EEE--EGARGGVFPGPRGGAPGGGAQLSPWLQPAALLFSLLCAFFWMGLC 146

HsPDEA 150 LLRAGVRLPLAVALLAACCGGEALVQIGLGVGEDHLLSLPAAGVVLSCLA 199 |||||||||||||||||||.||||||:.||||:..|||||||||:||||.MmPDEA 147 LLRAGVRLPLAVALLAACCAGEALVQLSLGVGDGRLLSLPAAGVLLSCLG 196

HsPDEA 200 AATWLVLRLRLGVLMIALTSAVRTVSLISLERFKVAWRPYLAYLAGVLGI 249 .||||||||||||||:|.||.:|||:|:|||||||||||||||||.|||:MmPDEA 197 GATWLVLRLRLGVLMVAWTSVLRTVALVSLERFKVAWRPYLAYLAAVLGL 246

HsPDEA 250 LLARYVEQILPQ-SAEAAPREHLGSQLIAGTKEDIPVFKRRRRSSSVVSA 298 |||||.|||||| |..|.|||..||||.|.|||:||.:||||||||||:.MmPDEA 247 LLARYAEQILPQCSGPAPPRERFGSQLSARTKEEIPGWKRRRRSSSVVAG 296

HsPDEA 299 EMSGCSSKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQSSGTSITVDIA 348 ||||||.|||||||||||||||||||||||||||||||| ||||||||||MmPDEA 297 EMSGCSGKSHRRTSLPCIPREQLMGHSEWDHKRGPRGSQ-SGTSITVDIA 345

HsPDEA 349 VMGE---------ATASLPTSWQTLLFHQTCATSLRAVSNLLSTQLTFQA 389 |||| |..|||.: .| ||||||||||||||||||MmPDEA 346 VMGEAHGLITDLLADPSLPPN--------VC-TSLRAVSNLLSTQLTFQA 386

HsPDEA 390 IHKPRVNPVTSLSENYTCSDSEESSEKDKLAIPKRLRRSLPPGLLRRVSS 439 ||||||||..:.|||||||||||..||||.||.|||||||||||||||||MmPDEA 387 IHKPRVNPTVTFSENYTCSDSEEGLEKDKQAISKRLRRSLPPGLLRRVSS 436

HsPDEA 440 TWTTTTSATGLPTLEPAPVRRDRSTSIKLQEAPSSS---PDSWNNPVMMT 486 ||||||||||||||||||||||||.|||..||||.| |||||.|.:.|MmPDEA 437 TWTTTTSATGLPTLEPAPVRRDRSASIKPHEAPSPSAVNPDSWNAPGLTT 486

HsPDEA 487 LTKSRSFTSSYAISAANHVKAKKQSRPGALAKISPLSSPCSSPLQGTPAS 536 ||||||||||||:|||||||||||:|||.||||||:.||.|||.||:|||MmPDEA 487 LTKSRSFTSSYAVSAANHVKAKKQNRPGGLAKISPVPSPSSSPPQGSPAS 536

HsPDEA 537 SLVSKISAVQFPESADTTAKQSLGSHRALTYTQSAPDLSPQILTPPVICS 586 |.||..::.|||||.:.|.|:..|||||||||||||||||||..|.||||MmPDEA 537 SPVSNSASQQFPESPEVTIKRGPGSHRALTYTQSAPDLSPQIPPPSVICS 586

HsPDEA 587 SCGRPYSQGNPADEPLERSGVATRTPSRTDDTAQVTSDYETNNNSDSSDI 636 |||||||||||||.|.||||.|...|:|||||:|||||||||||||||||MmPDEA 587 SCGRPYSQGNPADGPSERSGPAMLKPNRTDDTSQVTSDYETNNNSDSSDI 636

HsPDEA 637 VQNEDETECLREPLRKASACSTYAPETMMFLDKPILAPEPLVMDNLDSIM 686 :|||:|.||.|||.||||||.||..:||:|||||||||||||||||||||MmPDEA 637 LQNEEEAECQREPQRKASACGTYTSQTMIFLDKPILAPEPLVMDNLDSIM 686

HsPDEA 687 EQLNTWNFPIFDLVENIGRKCGRILSQVSYRLFEDMGLFEAFKIPIREFM 736 :||||||||||||:|||||||||||||||||||||||||||||||:||||MmPDEA 687 DQLNTWNFPIFDLMENIGRKCGRILSQVSYRLFEDMGLFEAFKIPVREFM 736

HsPDEA 737 NYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLSTVINDHGSTS 786 |||||||||||||||||||||||||||||||||||||||.:||.||||.|MmPDEA 737 NYFHALEIGYRDIPYHNRIHATDVLHAVWYLTTQPIPGLPSVIGDHGSAS 786

HsPDEA 787 DSDSDSGFTHGHMGYVFSKTYNVTDDKYGCLSGNIPALELMALYVAAAMH 836

69

|||||||||||||||||||.|:|.||||||||||||||||||||||||||MmPDEA 787 DSDSDSGFTHGHMGYVFSKMYHVPDDKYGCLSGNIPALELMALYVAAAMH 836

HsPDEA 837 DYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFL 886 ||||||||||||||||||||||||||||||||||||||||||||||||||MmPDEA 837 DYDHPGRTNAFLVATSAPQAVLYNDRSVLENHHAAAAWNLFMSRPEYNFL 886

HsPDEA 887 INLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNGKVNDDVGIDWTNEN 936 :||||||||||||||||||||||||||||||||||.||||||||||||||MmPDEA 887 VNLDHVEFKHFRFLVIEAILATDLKKHFDFVAKFNAKVNDDVGIDWTNEN 936

HsPDEA 937 DRLLVCQMCIKLADINGPAKCKELHLQWTDGIVNEFYEQGDEEASLGLPI 986 ||||||||||||||||||||||||||:||:||.:||||||||||||||||MmPDEA 937 DRLLVCQMCIKLADINGPAKCKELHLRWTEGIASEFYEQGDEEASLGLPI 986

HsPDEA 987 SPFMDRSAPQLANLQESFISHIVGPLCNSYDSAGLMPGKWVEDSDESGDT 1036 |||||||||||||||||||||||||||:|||||||||||||:|||:||||MmPDEA 987 SPFMDRSAPQLANLQESFISHIVGPLCHSYDSAGLMPGKWVDDSDDSGDT 1036

HsPDEA 1037 DDPEEEEEEAPAPNEEETCENNESPKKKTFKRRKIYCQITQHLLQNHKMW 1086 ||||||||||..|:|:|.||::.:|:||:||||:|||||||||||||.||MmPDEA 1037 DDPEEEEEEAETPHEDEACESSIAPRKKSFKRRRIYCQITQHLLQNHMMW 1086

HsPDEA 1087 KKVIEEEQRLAGIENQSLDQTPQSHSSEQIQAIKEEEEEKGKPRGEEIPT 1136 ||||||||.|:|.|||||||.|..|.||||||||||||||||||.||...MmPDEA 1087 KKVIEEEQCLSGTENQSLDQVPLQHPSEQIQAIKEEEEEKGKPRAEETLA 1136

######################################### Program: water# Rundate: Sat 18 Apr 2015 19:17:35# Commandline: water# -auto# -stdout# -asequence emboss_water-I20150425-191734-0220-70784086-es.asequence# -bsequence emboss_water-I20150425-191734-0220-70784086-es.bsequence# -datafile EBLOSUM62# -gapopen 10.0# -gapextend 0.5# -aformat3 pair# -sprotein1# -sprotein2# Align_format: pair# Report_file: stdout########################################

#=======================================## Aligned_sequences: 2# 1: HsPDE3B# 2: MmPDE3B# Matrix: EBLOSUM62# Gap_penalty: 10.0# Extend_penalty: 0.5## Length: 1124# Identity: 905/1124 (80.5%)# Similarity: 964/1124 (85.8%)# Gaps: 41/1124 ( 3.6%)# Score: 4631.0#

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HsPDE3B 1 MRRD--ERDAKAMRSLQPPD-GAGSPPESLRNGYVKSCVSPLRQDPPRGF 47 ||:| ||||.||||..||. .|.||||||||||||||||||||||||.|MmPDE3B 1 MRKDERERDAPAMRSPPPPPASAASPPESLRNGYVKSCVSPLRQDPPRSF 50

HsPDE3B 48 FFHLCRFCNVELRPPPASPQQPRRCSPFCRARLSLGDLAAFVLALLLGAE 97 ||||||||||| ||.||.: ..||||||.|||||||.||||.MmPDE3B 51 FFHLCRFCNVE--PPAASLR--------AGARLSLGVLAAFVLAALLGAR 90

HsPDE3B 98 PESWAAGAAWLRTLLSVCSHSLSPLFSIACAFFFLTCFLTRTKRGPGPGR 147 ||.|||.||.||||||.||.|||||||||||||||||||||.:| ||||MmPDE3B 91 PERWAAAAAGLRTLLSACSLSLSPLFSIACAFFFLTCFLTRAQR--GPGR 138

HsPDE3B 148 SCGSWWLLALPACCYLGDFLVWQWWSWPWGDGDAGSAAPHTPPEAAAGRL 197 ..|||||||||||||||||..||||||..|: .||||||MmPDE3B 139 GAGSWWLLALPACCYLGDFAAWQWWSWLRGE------------PAAAGRL 176

HsPDE3B 198 LLVLSCVGLLLTLAHPLRLRHCVLVLLLASFVWWVSFTSLGSLPSALRPL 247 .||||||| |||||..:||||.|||||.|..||||||:.||:||.|||||MmPDE3B 177 CLVLSCVG-LLTLAPRVRLRHGVLVLLFAGLVWWVSFSGLGALPPALRPL 225

HsPDE3B 248 LSGLVGGAGCLLALGLDHFFQIREAPLHPRLSSAAEEKVPVIRPRRRSSC 297 ||.|||||||||||||||||.:|.|...||.:|.||||||||||||||||MmPDE3B 226 LSCLVGGAGCLLALGLDHFFHVRGASPPPRSASTAEEKVPVIRPRRRSSC 275

HsPDE3B 298 VSLGETAASYYGSCKIFRRPSLPCISREQMILWDWDLKQWYKPHYQNSGG 347 |||||:||.||||.|:||||||||||||||||||||||||.|||||||||MmPDE3B 276 VSLGESAAGYYGSGKMFRRPSLPCISREQMILWDWDLKQWCKPHYQNSGG 325

HsPDE3B 348 GNGVDLSVLNEARNMVSDLLTDPSLPPQVISSLRSISSLMGAFSGSCRPK 397 ||||||||||||||||||||.|||||||||||||||||||||||||||||MmPDE3B 326 GNGVDLSVLNEARNMVSDLLIDPSLPPQVISSLRSISSLMGAFSGSCRPK 375

HsPDE3B 398 INPLTPFPGFYPCSEIEDPAEKGDRKLNKGLN-RNSLPTPQLRRSSGTSG 446 ||..|||||||||||:|||.|||||||:|||: |.|.||||||||||.|.MmPDE3B 376 INSFTPFPGFYPCSEVEDPVEKGDRKLHKGLSGRTSFPTPQLRRSSGASS 425

HsPDE3B 447 LLPVEQSSRWDRNNGKRPHQEFGISSQGCYLNGPFNSNLLTIPKQRSSSV 496 ||..|..|||||::|||.:||..:||.||:|||||:|||.||||||||||MmPDE3B 426 LLTNEHCSRWDRSSGKRSYQELSVSSHGCHLNGPFSSNLFTIPKQRSSSV 475

HsPDE3B 497 SLTHHVGLRRAGVLSSLSPVNSSNHGPVSTGSLTNRSPIEFPDTADFLNK 546 |||||.||||||.|.|.|.:|||:|.|||.|||||||||.||||.|||.|MmPDE3B 476 SLTHHAGLRRAGALPSHSLLNSSSHVPVSAGSLTNRSPIGFPDTTDFLTK 525

HsPDE3B 547 PSVILQRSLGNAPNTPDFYQQLRNSDSNLCNSCGHQMLKYVSTSESDGTD 596 |::||.||||:..:..||:|.|||||||||:|||||:||||||.|.||||MmPDE3B 526 PNIILHRSLGSVSSAADFHQYLRNSDSNLCSSCGHQILKYVSTCEPDGTD 575

HsPDE3B 597 CCSGKSGEEE-NIFSKESFKLMETQQEEETEKKDSRKLFQEGDKWLTEEA 645 ..|.|||||: ::||||...::|| |||||.||..|:||.|||..|.|||MmPDE3B 576 HPSEKSGEEDSSVFSKEPLNIVET-QEEETMKKACRELFLEGDSHLMEEA 624

HsPDE3B 646 QSEQQTNIEQEVSLDLILVEEYDSLIEKMSNWNFPIFELVEKMGEKSGRI 695 ||.||:||||||.:|||:||||||||:||||.|||||||||||||||MmPDE3B 625 ---QQPNIDQEVSLDPMLVEDYDSLIEKMNNWNFQIFELVEKMGEKSGRI 671

HsPDE3B 696 LSQVMYTLFQDTGLLEIFKIPTQQFMNYFRALENGYRDIPYHNRIHATDV 745 ||||||||||||||||.||||||:||||||||||||||||||||:|||||MmPDE3B 672 LSQVMYTLFQDTGLLETFKIPTQEFMNYFRALENGYRDIPYHNRVHATDV 721

HsPDE3B 746 LHAVWYLTTRPVPGLQQIHNGCGTGNETDSDGRINHGRIAYISSKSCSNP 795 |||||||||||:|||.||||...|..:.|||||:..|:|||||||||..|MmPDE3B 722 LHAVWYLTTRPIPGLPQIHNNHETETKADSDGRLGSGQIAYISSKSCCIP 771

HsPDE3B 796 DESYGCLSSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYN 845

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|.||||||||||||||||||||||||||||||||||||||||||||||||MmPDE3B 772 DMSYGCLSSNIPALELMALYVAAAMHDYDHPGRTNAFLVATNAPQAVLYN 821

HsPDE3B 846 DRSVLENHHAASAWNLYLSRPEYNFLLHLDHVEFKRFRFLVIEAILATDL 895 |||||||||||||||||||||||||||:|||:||||||||||||||||||MmPDE3B 822 DRSVLENHHAASAWNLYLSRPEYNFLLNLDHMEFKRFRFLVIEAILATDL 871

HsPDE3B 896 KKHFDFLAEFNAKANDVNSNGIEWSNENDRLLVCQVCIKLADINGPAKVR 945 |||||||||||||||||||||||||:||||||||||||||||||||||.|MmPDE3B 872 KKHFDFLAEFNAKANDVNSNGIEWSSENDRLLVCQVCIKLADINGPAKDR 921

HsPDE3B 946 DLHLKWTEGIVNEFYEQGDEEANLGLPISPFMDRSSPQLAKLQESFITHI 995 ||||:|||||||||||||||||.|||||||||||||||||||||||||||MmPDE3B 922 DLHLRWTEGIVNEFYEQGDEEAALGLPISPFMDRSSPQLAKLQESFITHI 971

HsPDE3B 996 VGPLCNSYDAAGLLPGQWLEAEEDNDTESGDDED------GEELDTEDEE 1039 ||||||||||||||||||:|.||.:||||.||:| |||||::|||MmPDE3B 972 VGPLCNSYDAAGLLPGQWIETEEGDDTESDDDDDDDDGDGGEELDSDDEE 1021

HsPDE3B 1040 MENNLNPKPPRRKSRRRIFCQLMHHLTENHKIWKEIV-EEEEKCKADGNK 1088 .|:||||||.|||.||||||||||||||||||||||: ||||||||:|||MmPDE3B 1022 TEDNLNPKPQRRKGRRRIFCQLMHHLTENHKIWKEIIEEEEEKCKAEGNK 1071

HsPDE3B 1089 LQVENSSLPQADEIQVIEEADEEE 1112 |||:|:||||||||||||||||||MmPDE3B 1072 LQVDNASLPQADEIQVIEEADEEE 1095

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