The human cyclic AMP-specific phosphodiesterase PDE-46 (HSPDE4A4B) expressed in transfected COS7 cells occurs as both particulate and cytosolic species that exhibit distinct kinetics of inhibition by the antidepressant rolipram

Transfection of COS7 cells with a plasmid encoding the human cyclic A-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed similar to-96% of the total COS cell PDE activity, A fusion protein was generated which encompassed residues (788-886) at the extreme C terminus of PDE-46 and was used to generate an antiserum that detected PDE-46 in transfected COS7 cells, Immunoblotting studies identified PDE-46 as a similar to 125-kDa species that was associated with both the soluble and particulate fractions. The relative V-max of particulate PDE-46 was similar to 56% that of cytosolic PDE-46. Particulate PDE-46 was not solubilized using Triton X-100 or high NaCl concentrations, Immunofluorescence analysis by laser scanning confocal micros copy showed that PDE-46 was located at discrete margins of the cen, indicative of association with membrane cortical regions. The human PDE4A species, h6.1 (HSPDE4A4C), which lacks the N-terminal extension of PDE-46, was found as an entirely soluble species when expressed in COS7 cells, h6.1 was shown to have an similar to 11-fold higher V-max relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0.195 mu M) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 mu M). The basis of this difference lag in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (K-i = 1.6 mu M) but as a partial competitive inhibitor of the particulate enzyme (K-i = 0.037 mu M; K-i' = 2.3 mu M) Particulate PDE-46 thus showed a similar to 60-fold higher affinity for rolipram than cytosolic PDE-46.