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Human platelet cGI-PDE: expression in yeast and localization of the
catalytic domain by deletion mutagenesis
PP Cheung, H Xu, MM McLaughlin, FA Ghazaleh, GP Livi and RW Colman
Sol Sherry Thrombosis Research Center, Temple University School of
Medicine, Philadelphia, PA 19140, USA.
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.
Volume 88,
Issue 4,
pp. 1321-1329,
08/15/1996
Copyright © 1996 by The American Society of Hematology
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