Elsevier

Gene

Volume 319, 13 November 2003, Pages 21-31
Gene

Molecular comparison of rat cyclic nucleotide phosphodiesterase 8 family: unique expression of PDE8B in rat brain

https://doi.org/10.1016/S0378-1119(03)00809-6Get rights and content

Abstract

Cyclic nucleotide phosphodiestease (PDE) type 8 is categorized into a family of 3-isobutyl-1-methylxanthine-insensitive PDE hydrolyzing cAMP with high affinity. We have isolated cDNAs encoding rat PDE8A and PDE8B from brain and testis, respectively. The sequence analysis demonstrated that rat PDE8A was a protein of 823 amino acid residues. Rat PDE8B protein was predicted as an N-terminal truncated form of 760 amino acid residues. Both of rat PDE8 proteins include REC, PAS and catalytic PDE domains. Tissue-specific expression patterns of rat PDE8A and PDE8B transcripts were demonstrated by Northern blot analysis. Rat PDE8A transcripts were rich in the liver and testis, and those of rat PDE8B were particularly abundant in the brain and were not expressed in the thyroid gland, while human thyroid gland contains PDE8B transcripts at a high level. Rat PDE8B transcripts were found in all brain regions other than cerebellum and shown to exist in the neuronal cells in in situ hybridization. Mouse PDE8B1 sequence was also identified by a database search and sequence alignment, revealing a protein of 885 amino acid residues, which is 99% and 96% identical to rat and human PDE8B1, respectively. As well as rat PDE8B, expression of mouse PDE8B transcripts was not confined to the thyroid gland. Species-dependent tissue expression pattern was quite unique features of PDE8B.

Introduction

Cyclic nucleotides, cAMP and cGMP, which are produced in response to extracellular stimulation through activation of adenylyl and guanylyl cyclases, work as second messengers regulating many cellular functions in various tissues. Cyclic nucleotide phosphodiesterases (PDEs) play a role in elimination of this signaling through hydrolysis of cyclic nucleotides. In mammalians, PDEs have been classified into 11 families (PDEs 1–11) based on their amino acid sequence homology, enzymatic characteristics and inhibitor sensitivity profiles Beavo, 1995, Conti and Jin, 1999, Fujishige et al., 1999, Hayashi et al., 1998, Houslay, 2001, Soderling et al., 1998a, Soderling et al., 1998b, Soderling and Beavo, 2000, Yuasa et al., 2000. The phylogenic tree analysis indicated that PDEs are classified into two groups; GAF-PDE families containing two GAF (for cGMP binding and stimulated phosphodiesterases, Anabaena adenyl cyclases and Escherichia coli FhlA) domains (Aravind and Ponting, 1997) in their N-terminal regions and non-GAF-PDE families lacking the domain (Yuasa et al., 2001). The former families include PDE2, PDE5, PDE6, PDE10 and PDE11. Half of PDE families such as PDE1, PDE3, PDE4, PDE6, PDE7 and PDE8 are composed of subfamily genes encoding highly similar but distinct products. In addition, mostly each PDE gene produces several splice variants. They show unique tissue expression, subcellular localization, alternative enzymatic regulation, or unique protein–protein interaction, and some of them are subject to transcriptional regulation (e.g., Bolger et al., 1997, Han et al., 1997, O'Connell et al., 1996, Sette et al., 1994, Swinnen et al., 1991, Yuasa et al., 2000). In some cases, difference in their expression levels among animal species has also been reported (e.g., Engels et al., 1994, Yuasa et al., 2001). Thus, the PDE superfamily is composed of more than 60 kinds of transcripts produced from 21 genes. PDEs 8–11 are identified using an approach with bioinformatics. Compared with classical PDEs such as PDEs 1–6, information of these recently discovered PDEs is poor.

In humans, PDE8 family is composed of two isogenes, PDE8A and PDE8B. Thus far, PDE8A cDNAs have been isolated from humans and mice Fisher et al., 1998, Soderling et al., 1998a, Wang et al., 2001. A full-length cDNA sequence and gene organization of human PDE8B have been reported by Hayashi et al. (2002) and Gamanuma et al. (2003). Very recently, we have documented comparison of enzymatic characteristics of human PDE8A1 and PDE8B1, which are a major full-length form (Gamanuma et al., 2003). Human PDE8A1 and PDE8B1 are 68% identical overall, and 80% identical in the PDE catalytic domain. PDE8A1 and PDE8B1 are cAMP-specific PDEs with Km values of 40 and 101 nM, respectively, and are insensitive to 3-isobutyl-1-methylxanthine and rolipram. Human PDE8A1 transcripts are observed in testis, spleen, colon, small intestine, ovary, placenta and kidney in decreasing order (Wang et al., 2001). The mouse PDE8A1, which is 79% identical to human PDE8A1 at the amino acid level, is expressed in several tissues as testis>eye>skeletal muscle>heart>embryo 7 days>kidney>ovary>brain (Soderling et al., 1998a). Expression levels of PDE8A transcripts were different in several tissues such as spleen, skeletal muscle, heart and liver between humans and mice. Human PDE8B exhibits quite unique tissue-specific expression pattern, which is confined to the thyroid gland. It is significant to explore tissue expression pattern of PDE8B transcripts in other mammalians from the view of understanding a physiological role of the enzyme. However, any other mammalian orthologues of PDE8B than humans have not been identified yet.

Here we demonstrate cDNA sequences encoding rat PDE8A and PDE8B. Analysis of tissue expression patterns of these transcripts in rats reveals unique tissue expression patterns of PDE8 orthologues. Detailed expression of rat PDE8B transcripts in the brain is investigated by Northern blot and in situ hybridization analyses. These findings contribute to our better understanding of physiological functions of PDE8 proteins.

Section snippets

Materials

E. coli DH5α (Sambrook et al., 1989) was used as a host for plasmid construction. Restriction endonucleases, DNA-modifying enzymes, LA Taq and Ex Taq were obtained from Takara Bio (Kyoto, Japan). Marathon-Ready cDNA (rat testis and brain), Advantage 2 Polymerase Mix, Rat Multiple Tissue Northern blot, Mouse RNA Master Blot and ExpressHyb were purchased from Clontech (Palo Alto, CA). The cloning vectors pGEM-T Easy and pBluescriptII KS(+) were from Promega (Madison, WI) and Stratagene (La Jolla,

cDNA cloning of rodent PDE8

Fig. 1A shows cDNA fragments coding for the rat PDE8A1 and PDE8B1, which were obtained by a combination of PCR, 3′-RACE and database search as described in Materials and methods. An N-terminal sequence of rat PDE8A1, which was similar to the corresponding region of human PDE8A1, was found in rat EST databases using the 5′-sequence carried by pPDER8A2 (accession no. BE110619). A full-length cDNA of rat PDE8A1 of 2.6 kb was generated by PCR using primer sets based on the 5′- and 3′-sequences of

Discussion

Characteristic structural features of PDE8 proteins are the presence of the PAS and REC domains in their N-terminal parts. The PAS domain is observed in the N-terminal part of many proteins involved in the signal transduction. The REC domain is implicated in receiving the signal from sensor proteins in bacterial two-component systems, and sensor kinase-catalyzed aspartyl phosphorylation of the domain leads to regulation of the effector domain, which is covalently linked with the REC domain Pao

Acknowledgements

The authors thank Dr. Fujimura and Ms. Kurabe for their technical assistance and kind advice for in situ hybridization analysis.

References (36)

Cited by (48)

  • cAMP-PKA cascade: An outdated topic for depression?

    2022, Biomedicine and Pharmacotherapy
  • Role of cyclic nucleotides and their downstream signaling cascades in memory function: Being at the right time at the right spot

    2020, Neuroscience and Biobehavioral Reviews
    Citation Excerpt :

    PDE8A mRNA is expressed in a variety of peripheral tissues and with regard to its brain expression, it can be found in the substantia nigra, thalamus and to a lesser extend in the hippocampus and cortex (Kruse et al., 2011). PDE8B mRNA is mainly expressed in the brain and more precisely in the hippocampus, cortex, striatum and midbrain (Kobayashi et al., 2003). The N-terminal region contains REC (receiver) and PAS (Per, Arnst and Sim) regulatory domains that participate in a communication system and in protein interaction in prokaryotes.

  • Select 3',5'-cyclic nucleotide phosphodiesterases exhibit altered expression in the aged rodent brain

    2014, Cellular Signalling
    Citation Excerpt :

    PDE8B expression was nearly identical across species, with the highest expression in striatum followed by frontal cortex and then hippocampus and hypothalamus (lowest in cerebellum) (Figs. 1–3; [33]). Together, our rodent PDE8 findings were relatively consistent with what has been previously described in rodent [56]. PDE9A expression is highest in cerebellum of all species (Figs. 1–3; [33]), with particular enrichment in the Purkinje and granule cell layers (Figs. 2, 3; [57]).

  • The role of phosphodiesterases in hippocampal synaptic plasticity

    2013, Neuropharmacology
    Citation Excerpt :

    The PDE8 family selectively hydrolyses cAMP (Fisher et al., 1998a) and as the PDE4 family, which is also selective for cAMP, has been found to have such powerful effects on synaptic plasticity, it is important to determine if PDE8 shares a similar role. PDE8 transcripts are expressed in the hippocampus, and where a direct comparison has been made between different PDE families it is found at similar levels to PDE4 (Kobayashi et al., 2003; Lakics et al., 2010; Tsai et al., 2012). Indeed the expression of PDE8B has been found to be highly specific in the brain and to be highly enriched in the dentate gyrus and area CA1 of the hippocampus in particular (Tsai et al., 2012).

View all citing articles on Scopus

The nucleotide and deduced amino acid sequences reported in this paper have been deposited with the DDBJ database under accession nos. AB092696 and AB092697.

1

Both authors contributed equally to this work.

View full text