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

Abstract

Transfection of COS7 cells with a plasmid encoding the human cyclic AMP-specific PDE4A phosphodiesterase PDE-46 (HSPDE4A4B) led to the expression of a rolipram-inhibited PDE4 activity, which contributed ϳ96% 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 ϳ125-kDa species that was associated with both the soluble and particulate fractions. The relative V max of particulate PDE-46 was ϳ56% 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 microscopy showed that PDE-46 was located at discrete margins of the cell, 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 ϳ11-fold higher V max relative to that of PDE-46. In doseresponse studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC 50 ‫؍‬ 0.195 M) than those needed to inhibit the cytosolic enzyme (IC 50 ‫؍‬ 1.6 M). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (K i ‫؍‬ 1.6 M) but as a partial competitive inhibitor of the particulate enzyme (K i ‫؍‬ 0.037 M; K i ‫؍‬ 2.3 M). Particulate PDE-46 thus showed a ϳ60-fold higher affinity for rolipram than cytosolic PDE-46.

Characterization of five different proteins produced by alternatively spliced mRNAs from the human cAMP-specific phosphodiesterase PDE4D gene

“…It is possible that this may reflect particular conformational properties, or the anomalous binding to SDS, of these proteins in SDS\PAGE. Similar anomalous migration in SDS\PAGE has been observed for recombinant rat and human PDE4A proteins [21,26,35]. It is intriguing that PDE4D2 migrates anomalously, whereas PDE4D1 migrates as predicted, as PDE4D2 differs from PDE4D1 only by the deletion of amino acids 1-78 of PDE4D1.…”

“…Previous analysis of the inhibition of various human PDE4 isoforms by rolipram has demonstrated that the dose-response curves deviate from those expected for simple Michaelian kinetics [12,26,[44][45][46]. Therefore we analysed the dose-response curves for the inhibition of our human PDE4D isoforms by rolipram ( Figure 6) and derived Hill coefficients from these curves ( Table 4).…”

“…In immunocompetition studies (carried out as described previously for PDE4A antibodies [26]) recognition of PDE4D forms by the polyclonal antibody was blocked by treatment with either the immunogen GST-CT(1-40)4D or by GST-CT(1-66)4D but not by GST itself. For the monoclonal antibody, recognition of PDE4D forms was blocked by GST-CT(1-66)4D (see the Results section) but not by treatment with either GST or GST-CT(1-40)4D (results not shown).…”

“…This sequence corresponds to a portion of the C-terminal region common to all of the known human PDE4D proteins (see Figure 2). This fusion protein was used to raise and purify polyclonal antibodies as described before by us [26] for PDE4A.…”

ERK2 Mitogen-activated Protein Kinase Binding, Phosphorylation, and Regulation of the PDE4D cAMP-specific Phosphodiesterases

“…fied by three different procedures as described before by us (29). For two of these, SDS-polyacrylamide gels were run with a range of different protein amounts/lane and then subjected to immunoblotting.…”