HUMAN PHOSPHODIESTERASE 4A - CHARACTERIZATION OF FULL-LENGTH AND TRUNCATED ENZYMES EXPRESSED IN COS CELLS

The type 4 phosphodiesterase (PDE) family comprises four enzymes (4A, 4B, 4C and 4D) that are characterized by their specificity for cAMP an d selective inhibition by the antidepressant drug rolipram [3-(cyclope ntoxyl)-4-methoxyphenyl]2-pyrrolidone}. In common with other PDEs, the y consist of a central conserved domain associated with catalytic acti vity in addition to two N-terminal upstream conserved regions (UCR1 an d UCR2) that are unique to the type 4 enzymes. We have isolated a 2 kb cDNA encoding a full-length type 4A PDE {HS PDE4A4B [Bolger, Michaeli , Martins, St.John, Steiner, Rodgers, Riggs, Wigler and Ferguson (1993 ) Mol. Cell Biol. 13, 6558-6571]} from a human frontal cortex cDNA lib rary. Northern blot analysis showed that the major PDE4A mRNA of 4.5 k b was widely distributed in different human tissues. The recombinant P DE4A expressed in COS cells had a molecular mass of approx. 117 kDa as revealed by SDS/PAGE/Western blotting with a PDE4A-specific antibody and was specific for cAMP with a K-m of 4.8 mu M. The enzyme activity was potently inhibited by R-rolipram (IC50 204 nM) and showed a 2.7-fo ld stereoselectivity over the S enantiomer. Analysis of the kinetics o f inhibition indicated that R-rolipram did not behave as a simple comp etitive inhibitor. Dixon replots suggested that there was more than on e mode of interaction consistent with the detection in the enzyme of a high-affinity binding site for R-rolipram with a K-d of 2.3 nM. Trunc ation of the PDE4A enzyme by deletion mutagenesis showed that neither of the UCRs was required for catalytic activity and identified an appr ox. 71 kDa core enzyme with a K-m for cAMP of 3.3 mu M. In contrast wi th the full-length PDE4A, R-rolipram behaved as a simple competitive i nhibitor of this form of the enzyme with decreased potency (IC50 1022 nM) and no stereoselectivity. In addition, no high-affinity rolipram-b inding site was detected in the truncated enzyme, indicating that this interaction involves sequences upstream of the catalytic domain of th e enzyme.