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Volume 271, Number 49, Issue of December 6, 1996 pp. 31334-31344
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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

(Received for publication, March 14, 1996, and in revised form, August 26, 1996)

Elaine Huston , Linda Pooley , Pascale Julien , Grant Scotland , Ian McPhee , Michael Sullivan , Graeme Bolger and Miles D. Houslay

From the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, IBLS, Wolfson Link Building, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom and  VA Medical Center, Huntsman Cancer Institute, Departments of Medicine and Oncologic Science, University of Utah, Salt Lake City, Utah 84148

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 ~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 ~125-kDa species that was associated with both the soluble and particulate fractions. The relative Vmax of particulate PDE-46 was ~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 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 ~11-fold higher Vmax relative to that of PDE-46. In dose-response studies rolipram inhibited particulate PDE-46 at much lower concentrations (IC50 = 0.195 µM) than those needed to inhibit the cytosolic enzyme (IC50 = 1.6 µM). The basis of this difference lay in the fact that rolipram served as a simple competitive inhibitor of the cytosol enzyme (Ki = 1.6 µM) but as a partial competitive inhibitor of the particulate enzyme (Ki = 0.037 µM; Ki' = 2.3 µM). Particulate PDE-46 thus showed a ~60-fold higher affinity for rolipram than cytosolic PDE-46.


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