Molecular cloning and subcellular distribution of the novel PDE4B4 cAMP-specific phosphodiesterase isoform.
We have isolated cDNAs encoding PDE4B4, a new cAMP-specific phosphodiesterase (PDE4) isoform with novel properties. The amino acid sequence of PDE4B4 demonstrates that it is encoded by the PDE4B gene, but that it differs from the previously isolated PDE4B1, PDE4B2 and PDE4B3 isoforms by the presence of a novel N-terminal region of 17 amino acids. PDE4B4 contains both of the upstream conserved region 1 (UCR1) and UCR2 regulatory units that are characteristic of 'long' PDE4 isoforms. RNase protection demonstrated that PDE4B4 mRNA is expressed preferentially in liver, skeletal muscle and various regions of the brain, which differs from the pattern of tissue distribution of the other known PDE4B long forms, PDE4B1 and PDE4B3. Expression of PDE4B4 cDNA in COS7 cells produced a protein of 85 kDa under denaturing conditions. Subcellular fractionation of recombinant, COS7-cell expressed PDE4B4 showed that the protein was localized within the cytosol, which was confirmed by confocal microscopic analysis of living COS7 cells transfected with a green fluorescent protein-PDE4B4 chimaera. PDE4B4 exhibited a K(m) for cAMP of 5.4 microM and a V(max), relative to that of the long PDE4B1 isoform, of 2.1. PDE4B4 was inhibited by the prototypical PDE4 inhibitor rolipram [4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidinone] with an IC(50) of 83 nM. Treatment of COS7 cells with forskolin, to elevate cAMP levels, produced activation of PDE4B4, which was associated with the phosphorylation of PDE4B4 on Ser-56 within UCR1. The unique tissue distribution and intracellular targeting of PDE4B4 suggests that this isoform may have a distinct functional role in regulating cAMP levels in specific cell types.
Shepherd
Malcolm
M
Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biology and Life Sciences, University of Glasgow, Davidson Building, Glasgow G12 8QQ, Scotland, Uk.
McSorley
Theresa
T
Olsen
Aileen E
AE
Johnston
Lee Ann
LA
Thomson
Neil C
NC
Baillie
George S
GS
Houslay
Miles D
MD
Bolger
Graeme B
GB
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D019556:COS Cells; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D018076:DNA, Complementary / Q000302:isolation & purification; D004789:Enzyme Activation / Q000502:physiology; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D051381:Rats
D018076:DNA, Complementary; D007527:Isoenzymes; D017868:Cyclic AMP-Dependent Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517360:PDE4B protein, human