A low-Km, rolipram-sensitive, cAMP-specific phosphodiesterase from human brain. Cloning and expression of cDNA, biochemical characterization of recombinant protein, and tissue distribution of mRNA.
We have isolated cDNA clones from human frontal cortex cDNA libraries that encode a unique subtype of the low-Km, cAMP-specific phosphodiesterases (PDEs IV). The 564-amino acid sequence of the protein (human brain PDE IV (hPDE IVB)) shows significant homology to a PDE IV subtype expressed in human monocytes (hPDE IVA), particularly within the approximately 300-amino acid PDE IV catalytic domain. The degree of protein sequence identity is much greater between hPDE IVB and a homolog derived from rat brain (92% over 562 amino acids) than between hPDE IVB and hPDE IVA (76% over 538 amino acids), suggesting a greater subtype-specific versus species-specific conservation of protein sequence. Analysis of the distribution of hPDE IVB mRNA expression revealed a restricted pattern, with an approximately 4-kilobase mRNA detected in brain, heart, lung, and skeletal muscle and not in placenta, liver, kidney, or pancreas. An additional approximately 5-kilobase hPDE IVB-related mRNA species was detected in brain tissue. Recombinant hPDE IVB displayed all of the expected kinetic characteristics for a PDE IV, including sensitivity to the isozyme-selective inhibitor rolipram (Ki = 0.085 microM). Scatchard analysis of (R)-[3H]rolipram binding data suggested the presence of two noninteracting high affinity rolipram-binding sites (Kd = 0.4 and 6 nM) or a negatively cooperative interaction among multiple binding sites.
McLaughlin
M M
MM
Department of Gene Expression Sciences, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406.
Cieslinski
L B
LB
Burman
M
M
Torphy
T J
TJ
Livi
G P
GP
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D003001:Cloning, Molecular; D004247:DNA; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009928:Organ Specificity / Q000235:genetics; D011760:Pyrrolidinones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D020889:Rolipram; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid
D011760:Pyrrolidinones; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D020889:Rolipram