Characterization of two recombinant PDE3 (cGMP-inhibited cyclic nucleotide phosphodiesterase) isoforms, RcGIP1 and HcGIP2, expressed in NIH 3006 murine fibroblasts and Sf9 insect cells.
cDNAs encoding PDE3 [cGMP-inhibited cyclic nucleotide phosphodiesterase (cGI PDE)] isoforms, cGIP1 and cGIP2, have been cloned from rat (R) and human (H) cDNA libraries. The deduced amino acid sequences of RcGIP1 and HcGIP2 are very similar in their conserved catalytic domains but differ in their N-terminal regulatory domains [Meacci, E., et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 3721-3725; Taira, M., et al. (1993) J. Biol. Chem. 268, 18573-18579]. cDNAs encoding both rat adipocyte RcGIP1 and human myocardial HcGIP2 (full-length forms and truncated forms lacking much of the putative N-terminal domain) were expressed in NIH 3006 fibroblasts and in Sf9 insect cells. The recombinant proteins exhibited the expected subunit molecular mass, immunologic reactivities, and characteristics of native membrane-associated forms of the enzymes, e.g., high affinity for cAMP (Km), sensitivity to the selective cGI PDE inhibitors OPC 3689 and OPC 3911 and to cGMP. The full-length recombinants were predominantly particulate, whereas the truncated HcGIP2 forms were cytosolic suggesting that N-terminal domains contain structural determinants important for membrane association. Both fibroblast RcGIP1 and authentic adipocyte cGI PDE were phosphorylated in vitro by cAMP-dependent protein kinase; tryptic [32P]peptides released from rat adipocyte 32P-cGI PDE and 32P-RcGIP1 exhibited identical electrophoretic profiles suggesting that the same peptides are phosphorylated in both.
Leroy
M J
MJ
Department of Cell and Molecular Biology, Lund University, Sweden.
Degerman
E
E
Taira
M
M
Murata
T
T
Wang
L H
LH
Movsesian
M A
MA
Meacci
E
E
Manganiello
V C
VC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D017667:Adipocytes / Q000201:enzymology; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006152:Cyclic GMP / Q000494:pharmacology*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D017931:DNA Primers; D018076:DNA, Complementary; D005347:Fibroblasts; D015723:Gene Library; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D015363:Quinolones / Q000494:pharmacology; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera; D014162:Transfection
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D010726:Phosphodiesterase Inhibitors; D015363:Quinolones; D011994:Recombinant Proteins; C021294:cilostamide; C060853:OPC 3911; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; C517287:PDE3A protein, human; C517291:PDE3B protein, human; D006152:Cyclic GMP