Cloning and characterization of novel PDE4D isoforms PDE4D6 and PDE4D7☆
Introduction
Many types of extracellular cell signals elicit a significant part of their effects by regulating levels of the intracellular signaling molecule cyclic AMP (cAMP). Conventional descriptions of the cAMP-signaling pathway depict G protein-coupled receptors that either stimulate or inhibit the activity of the enzyme adenylyl cyclase. Cyclic AMP regulates the activity of a kinase, protein kinase A (PKA). Substrates of PKA include transcription factors, ion channels, enzymes and other proteins essential for signal transduction.
Recently, it has become apparent that cAMP signaling is more complex than previously appreciated. At least nine types of adenylyl cyclase have been cloned [1], each with different modes of regulation. For example, AC 1 may be stimulated directly by Ca2+ and calmodulin without the involvement of G protein-coupled receptors [2]. PKA is coupled to specific signaling pathways through a family of proteins called A-kinase anchoring proteins (AKAPS) [3], [4]. Cyclic AMP itself is now known to bind directly to some proteins, including EPAC [5] and CNG channels [6], without the intervention of PKA. Just as important as adenylyl cyclases for maintaining cAMP levels are the enzymes that degrade cAMP, the phosphodiesterases.
To date, 11 gene families of cyclic nucleotide-specific phosphodiesterases (PDEs) have been discovered [7]. These families differ with respect to their regulation and their specificity for either cGMP or cAMP. The PDE4 gene family is characterized by high specificity for cAMP and for selective inhibition by rolipram. Because rolipram has demonstrated efficacy in various in vivo models (e.g., anti-inflammation [8], cognition [9] and depression [10]), the PDE4 family of genes has attracted a large amount of interest from pharmacologists. The PDE4 family is composed of four genes, PDE4 A, B, C and D. Additional specificity is rendered by generation of splice variants from the genes [3], [7]. To date, five variants of the PDE4D gene have been identified and characterized each with its own specific pattern of distribution and potential to couple to specific signaling pathways [3], [11], [12]. PDE4 gene products are distinguished by two structural motifs, namely the upstream conserved region (UCR) 1 and 2 [13], which may play a role in regulation of PDE activity [3], [14].
Two important areas of current research on the role of PDEs in signal transduction are the mechanisms that regulate activity of PDE4 enzymes and the targeting of PDE4 enzymes to specific signaling complexes. For the regulation of activity, it is now well established that cAMP, through a PKA-dependent mechanism, increases the activity of PDEs containing upstream conserved region 1 (UCR1) [14], [15]. The mechanism is thought to involve relief from a constitutive inhibitory activity. Short variants of PDEs that lack the complete UCR1 are not regulated by an increase in PKA activity.
Targeting of PDEs to specific signaling pathways has been shown using both two hybrid approaches and immunoprecipitation. Each variant may play a specific role in cells by coupling to these signaling pathways [16], [17], [18]. Coupling usually results from interactions of the variants with the N-terminals of the proteins. This is the one variable region for the splice variants. In order to determine the specific role of a given PDE4 variant, it is important to examine the structure of the N-terminal as well as its regulation by PKA.
In this paper, we have examined in more detail the splice variants that are transcribed off the PDE4D gene. By identifying the N-terminals of these splice variants, we may be able to identify the signaling complexes to which each of them belong. This information should aid in identifying specific physiological roles for each of the variants. In addition, by making the recombinant enzymes, we show differential regulation of the PDE4Ds depending on the presence of UCR1 and UCR2.
Section snippets
Reagents, chemicals and software
All cell culture media were from GIBCO-BRL (Invitrogen). Rolipram was purchased from AG Scientific (San Diego, CA). All other chemicals are from Sigma (St. Louis, MO). [3H]-cAMP, [3H]-Rolipram and α-[32P]-dCTP are from Amersham (Piscataway, NJ). Antibody to human PDE4D was from FabGennix (Shreveport, LA). Protein sequence alignments were generated using DNAStar (ClustalV). PDE4D6 and PDE4D7 enzymatic assay data were analyzed using Prism software.
Cloning of PDE4D isoforms
Novel 5′ sequences of rat/human PDE4D6 and PDE4D7
Sequences of PDE4D isoforms
Fig. 1A shows a protein sequence alignment of all the PDE4D isoforms reported here. The isoforms in the PDE4 gene family are classified as long, short and supershort forms, dependent on existence of UCR1 and UCR2 domain in the gene [3], [19], [23]. For example, PDE4D1 containing only UCR2 domain is a short-form enzyme. PDE4D3, PDE4D4 and PDE4D5 containing both UCR1 and UCR2 domains are long-form enzymes. Supershort-form PDE4, represented by PDE4A1, lacks the UCR1 and has a truncated UCR2 [3],
Acknowledgements
We would like to thank Dr. James Barrett for comments on the manuscript and Dr. Joerg Heyer for helpful discussions.
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The sequences in the report have been deposited into GenBank with the following accession numbers: rat PDE4D6 (AF536974), human PDE4D6 (AF536975), rat PDE4D7 (AF536979), mouse PDE4D7 (AF536978), human PDE4D7 (AF536976), human PDE4D8 (AF536977) and human PDE4D 3′ UTR (AF536980).
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Current address: 351 Middle Road, Farmington, CT 06032, USA.
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Current address: Genomics Institute of the Novartis Research Foundation, 3115 Merryfield Row, San Diego, CA 92121, USA.