Identification of cyclic AMP‐phosphodiesterase variants from the PDE4D gene expressed in human peripheral mononuclear cells

Abstract

To determine whether the expression of different PDE4D variants is unique to the rat or conserved through evolution, we have characterized the different PDE4D mRNAs expressed in human peripheral blood mononuclear cells. RT-PCR was performed using primers based on rat sequences and mRNAs from mononuclear cells. The specifically amplified fragments had a size identical to that predicted for rat PDEAD1, PDE4D2 and PDE4D3. Sequencing confirmed that these fragments are derived from the human PDE4D gene. Their sequence was highly homologous to that reported for the rat variants, cDNAs corresponding to the entire ORF of human PDE4D2 and PDFAD3 were expressed in mammalian cells, causing a large increase in PDE activity. Western blot analysis of human peripheral blood mononuclear cell extracts demonstrated the presence of proteins corresponding to the recombinant PDE4D1 and PDE4D2. The pattern of splicing and different promoter usage of the PDE4D gene is therefore conserved during evolution, which indicates an important physiological role.

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene

“…The structure of these cDNAs demonstrate that transcripts from the PDE4D gene undergo a complex pattern of alternative splicing, which resembles that of transcripts from the D. melanogaster dunce gene. Our data demonstrate that the PDE4D gene has a more complex structure than that reported previously [13,16]. We have also studied the biochemical properties of the various PDE4D isoforms, and for this purpose utilized a single system, expression in mammalian COS-7 cells, in which the properties of the five individual isoforms could be compared.…”

“…For example, the deduced amino acid sequences of the proteins encoded by pDun411 and pPDE82 are identical with those of the human PDE4D1 and PDE4D2 isoforms respectively cloned by PCR by Nemoz et al [13]. The truncated human PDE4D cDNA isolated by Baecker et al [36] could represent any of our pPDE43, pPDE39 or pPDE79 cDNAs.…”

“…Accession numbers with an asterisk (*) correspond to the clones of Nemoz et al [13]. All other accession numbers are from Nemoz et al [13], or the present study. The letters HS, for homo sapiens, are usually prefixed to the isoform name (e.g.…”

“…We have reported previously [12] the cloning of two human PDE4D mRNA transcripts : PDE4D3 and PDE4D4. The cDNAs representing the human PDE4D1 and PDE4D2 transcripts have also been reported [13]. A comparison of the sequences of the rat and human PDE4D1, PDE4D2 and PDE4D3 proteins reveals them to be highly conserved : each of the human proteins is nearly identical in length with its rat counterpart and has greater than 90 % amino acid identity.…”

Expression of mRNA encoding cAMP‐specific phosphodiesterase isoforms in rat parotid glands

“…It has been reported that one subtype of the PDE4 subfamily, PDE4D, is activated by phosphorylation with A-kinase, and the PDE4D gene of the rat codes for at least three alternative splicing variants; ratPDE3.1(PDE4D 1), ratPDE3.2(PDFAD2) and ratPDE3.3(PDE4D3) (25,26). Only ratPDE3.3(PDE4D3) has been phosphorylated by A-kinase to be activated in a eeU free system (25).…”