EXPRESSION AND MUTAGENESIS OF THE CATALYTIC DOMAIN OF CGMP-INHIBITED PHOSPHODIESTERASE (PDE3) CLONED FROM HUMAN PLATELETS

We have used reverse transcriptase PCR, platelet mRNA and degenerate p rimers based on platelet peptide sequences, to amplify a fragment of p latelet cGMP-inhibited phosphodiesterase (cGI-PDE; PDE3). Sequence ana lysis of this clone established that both the platelet and the cardiac forms of PDE3 were derived from the same gene (PDE3A). A RT-PCR produ ct representing the C-terminal half of platelet PDE3 cDNA and correspo nding to amino acid residues 560-1141 of the cardiac enzyme, was clone d and expressed in Escherichia coli cGI-PDE Delta 1. Further deletion mutants were constructed by removing either an additional 100 amino ac ids from the N-terminus (cGI-PDE Delta 2) or the 44-amino-acid insert characteristic of the PDE3 family, from the catalytic domain (cGI-PDE Delta 1 Delta i). In addition, site-directed mutagenesis was performed to explore the function of the 44-amino-acid insert. All mutants were evaluated for their ability to hydrolyse cAMP and cGMP, their ability to be photolabelled by [P-32]cGMP and for the effects of PDE3 inhibit ors. The K-m values for hydrolysis of cAMP and cGMP by immunoprecipita tes of cGI-PDE Delta 1 (182 +/- 12 nM and 153 +/- 12 nM respectively) and cGI-PDE Delta 2 (131 +/- 17 nM and 99 +/- 1 nM respectively) were significantly lower than those for immunoprecipitates of intact platel et PDE3 (398 +/- 50 nM and 252 +/- 16 nM respectively). Moreover, N-te rminal truncations of platelet enzyme increased the ratio of V-max for cGMP/V-max for cAMP from 0.16 +/- 0.01 in intact platelet enzyme, to 0.37 +/- 0.05 in cGI-PDE Delta 1 and to 0.49 +/- 0.04 in cGI-PDE Delta 2. Thus deletion of the N-terminus enhanced hydrolysis of cGMP relati ve to cAMP, suggesting that N-terminal sequences may exert selective e ffects on enzyme activity. Removal of the 44-amino-acid insert generat ed a mutant with a catalytic domain closely resembling those of other PDE gene families but despite a limited ability to be photolabelled by [P-32]cGMP, no cyclic nucleotide hydrolytic activities of the mutant were detectable. Mutation of amino acid residues in putative beta-turn s at the beginning and end of the 44-amino-acid insert to alanine resi dues markedly reduced the ability of the enzyme to hydrolyse cyclic nu cleotides. The PDE3 inhibitor, lixazinone, retained the ability to inh ibit cAMP hydrolysis and [P-32]cGMP binding by the N-terminal deletion mutants and the site-directed mutants, suggesting that PDE3 inhibitor s may interact exclusively with the catalytic domain of the enzyme.