Identification, characterization and regional distribution in brain of RPDE-6 (RNPDE4A5), a novel splice variant of the PDE4A cyclic AMP phosphodiesterase family.
COS-7 cells were transfected with a plasmid encoding a putative splice variant of PDE4A cyclic AMP-specific phosphodiesterase, RPDE-6 (RNPDE4A5). This led to the expression of a novel, cyclic AMP-specific, rolipram-inhibited phosphodiesterase activity. In such transfected cells a novel approximately 109 kDa species was recognized by anti-peptide sera raised against a dodecapeptide whose sequence is found at the extreme C-terminus of both RPDE-6 and another PDE4A splice variant. RD1 (RNPDE4A1A). RPDE-6 activity and immunoreactivity was found distributed between both pellet (approximately 25%) and cytosol (approximately 75%) fractions of transfected COS-7 cells. Soluble and pellet RPDE-6 activities exhibited similar low Km values for cyclic AMP (approximately 2.4 microM) and were both inhibited by low concentrations of rolipram, with IC50 values for the soluble activity being lower (approximately 0.16 microM) than for the pellet activity (approximately 1.2 microM). Pellet RPDE-6 was resistant to release by either high NaCl concentrations or the detergent Triton X-100. Probing brain homogenates with the anti-(C-terminal peptide) sera identified two immunoreactive species, namely an approximately 79 kDa species reflecting RD1 and an approximately 109 kDa species that co-migrated with the immunoreactive species seen in COS cells transfected to express RPDE-6. The approximately 109 kDa species was found distributed between both the low-speed (P1) and high-speed (P2) pellet fractions as well as the cytosol fractions derived from both brain and RPDE-6-transfected COS cells. In contrast, RD1 was found exclusively in the P2 fraction. Phosphodiesterase (PDE) activity immuno-precipitated by these antisera from brain cytosol had the characteristics of COS cell-expressed RPDE-6 with KmcyclicAMP approximately 3.7 microM and IC50rolipram approximately 0.12 microM. The distribution of PDE activity immunoprecipitated from the cytosol of various brain regions paralleled that seen for the distribution of the approximately 109 kDa immunoreactive species. It is suggested that the 109 kDa species identified in brain cytosol and pellet fractions is the native form of RPDE-6. The PDE4A splice variants, RD1 and RPDE-6, were shown to have distinct patterns of expression among various brain regions. PDE4A and PDE4B activities appear to provide the major source of PDE4 activity in brain membranes, whereas the cytosolic PDE4 activity is suggested to reflect predominantly the activity of the PDE4D family. Alternative splicing of the PDE4A gene confers distinct N-terminal domains on RPDE-6 and RD1, which attenuates the Vmax. of these enzymes and defines their distinct subcellular distribution pattern.
McPhee
I
I
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Pooley
L
L
Lobban
M
M
Bolger
G
G
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002522:Chlorocebus aethiops; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003600:Cytosol / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D014644:Genetic Variation; D007668:Kidney; D007700:Kinetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D010902:Pituitary Gland / Q000201:enzymology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism; D020889:Rolipram; D014162:Transfection
D004791:Enzyme Inhibitors; D011760:Pyrrolidinones; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C095813:Pde4a protein, mouse; C521385:Pde4a protein, rat; D020889:Rolipram