Isolation and characterization of a previously undetected human cAMP phosphodiesterase by complementation of cAMP phosphodiesterase-deficient Saccharomyces cerevisiae.
We have established a highly sensitive functional screen for the isolation of cDNAs encoding cAMP phosphodiesterases (PDEs) by complementation of defects in a Saccharomyces cerevisiae strain lacking both endogenous cAMP PDE genes, PDE1 and PDE2. Three groups of cDNAs corresponding to three distinct human genes encoding cAMP-specific PDEs were isolated from a human glioblastoma cDNA library using this functional screen. Two of these genes are closely related to the Drosophila dunce cAMP-specific PDE. The third gene, which we named HCP1, encoded a novel cAMP-specific PDE. HCP1 has an amino acid sequence related to the sequences of the catalytic domains of all cyclic nucleotide PDEs. HCP1 is a high affinity cAMP-specific PDE (Km = 0.2 microM) that does not share other properties of the cAMP-specific PDE family, i.e. extensive sequence homology to the Drosophila dunce cAMP PDE and sensitivity to rolipram and R020-1724. The PDE activity of HCP1 is not sensitive to cGMP or other inhibitors of the cGMP-inhibitable PDEs, such as milrinone. The biochemical and pharmacological properties of HCP1 suggest that it is a member of a previously undiscovered cyclic nucleotide PDE family. Northern blot analysis indicates that high levels of HCP1 mRNA are present in human skeletal muscle.
Michaeli
T
T
Cold Spring Harbor Laboratory, New York 11724.
Bloom
T J
TJ
Martins
T
T
Loughney
K
K
Ferguson
K
K
Riggs
M
M
Rodgers
L
L
Beavo
J A
JA
Wigler
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D004330:Drosophila / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D009132:Muscles / Q000201:enzymology; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1