Isolation and characterization of PDE8A, a novel human cAMP-specific phosphodiesterase.
As part of our efforts to understand the regulation of intracellular cAMP and to generate new targets for pharmacological intervention, we have cloned and characterized the first isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE8A. PDE8A is most similar to PDE4 (38.5% amino acid identity in the catalytic domain), but is clearly not a member of any of the seven known PDE families. We report the cloning of human cDNA encoding the C-terminal 713 amino acids of the protein, including a 283 amino acid region located near the C-terminus that is homologous to the approximately 270 amino acid catalytic domain of other PDEs. In addition, we found cDNA sequences consistent with alternative 5' mRNA splicing analogous to that seen in other PDE genes. PDE8A is expressed in a wide variety of tissues as a approximately 4.5 kb mRNA, with highest levels in testis, ovary, small intestine, and colon. The C-terminal 545 amino acids of PDE8A (the region shared among all splice variants) were expressed in baculovirus. Kinetic analysis of the baculovirus expressed enzyme shows it to be a very high affinity cAMP specific PDE with a Km of 55 nM for cAMP and 124 microM for cGMP. The Vmax (150 pmol/min/microgram recombinant enzyme) is about 10 times slower than that of PDE4. The cAMP hydrolytic activity of PDE8A is not modulated by cGMP at concentrations up to 100 microM. The enzyme requires the presence of at least 1 mM Mn2+ or Mg2+ for maximal activity in vitro, while 100 mM Ca2+ restores only about 20% activity. PDE8A is insensitive (up to 100 microM) to a variety of PDE inhibitors including rolipram, zaprinast, vinpocetine, SKF-94120, and IBMX, but is inhibited (IC50 = 9 microM) by the PDE inhibitor dipyridimole. To give PDE8A a descriptive name that distinguishes it from the other two known high affinity cAMP-specific phosphodiesterases (PDE4 and PDE7), we denote PDE8A as the high affinity cAMP-specific and IBMX-insensitive PDE.
Fisher
D A
DA
Department of Molecular Sciences, Pfizer Central Research, Groton, Connecticut 06340, USA. douglas_a_fisher@groton.pfizer.com
Smith
J F
JF
Pillar
J S
JS
St Denis
S H
SH
Cheng
J B
JB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001483:Base Sequence; D002412:Cations / Q000494:pharmacology; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D005810:Multigene Family; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014018:Tissue Distribution
D002412:Cations; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C112770:PDE8A protein, human