Human platelet cGI-PDE: expression in yeast and localization of the catalytic domain by deletion mutagenesis.
Cyclic adenosine monophosphate (cAMP) is an important modulator of platelet responses to agonists. Cyclic nucleotide phosphodiesterase (PDE) controls intracellular cAMP concentrations by hydrolyzing it to AMP. The major PDE activity in platelets is PDE3A (cyclic guanosine monophosphate [cGMP]-inhibited PDE). To obtain structural information on platelet PDE3A, we cloned the enzyme cDNA from a human erythroleukemia cell (HEL) library since the cell line expresses many platelet proteins. This clone consists of 87% of the full-length human myocardial PDE3A cDNA, spanning from nucleotides 456 to 4606, and is identical in sequence. The nucleotide coding for the N terminal 179 amino acid sequence (nt 1-536) as well as four other cDNAs (nt 1459-1632, nt 1765-1986, nt 2152-2538, and nt 2978-3375) obtained by RT-PCR of platelet RNA are also identical to the myocardial sequences, indicating that the HEL, myocardial, and platelet PDE3As are the same. Northern blot analysis of HEL cell RNA detected two mRNAs of 7.5 and 4.4 kb. Four new deletion mutants are reported. PDE 3A delta 1 and PDE 3A delta 2, encoding amino acids 665 to 1141 and amino acids 679 to 1141, respectively, were expressed in a PDE-deficient yeast. They displayed PDE activities of 172 and 79 pmol/mg/min, respectively. PDE 3A delta 3 and PDE 3A delta 4, encoding amino acids 686 to 1141 and 700 to 1141, had no detectable PDE activity. All mutant proteins were expressed as determined by Western blot analysis. These findings localize the PDE3A catalytic domain to within amino acid residues 679 to 1141.
Cheung
P P
PP
Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Xu
H
H
McLaughlin
M M
MM
Ghazaleh
F A
FA
Livi
G P
GP
Colman
R W
RW
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001792:Blood Platelets / Q000201:enzymology*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D006152:Cyclic GMP / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D017931:DNA Primers; D018076:DNA, Complementary; D010726:Phosphodiesterase Inhibitors; D012333:RNA, Messenger; D011994:Recombinant Proteins; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP