Expression and characterization of deletion recombinants of two cGMP-inhibited cyclic nucleotide phosphodiesterases (PDE-3).
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1) and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal truncation mutants were expressed in Escherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited a high affinity (< 1 microM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of sensitivity of PDE-3s to cGMP.
He
R
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Department of Cellular and Molecular Biology, Lund University, Sweden.
Komas
N
N
Ekholm
D
D
Murata
T
T
Taira
M
M
Hockman
S
S
Degerman
E
E
Manganiello
V C
VC
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002384:Catalysis; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000502:physiology*; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D017434:Protein Structure, Tertiary; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000737:chemistry*; D017384:Sequence Deletion
D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054684:Cyclic Nucleotide Phosphodiesterases, Type 3; D006152:Cyclic GMP