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Fig. 4. PDE4D4 localizes with spectrin at cell-cell borders in PMVECs. (A) Confocal microscopy using antibodies to PDE4 and βII spectrin indicates both proteins localize to cell-cell borders in PMVECs (top panel), but not in PAECs (bottom panel). (B) Schematic depicts comparison of full-length PDE4D4 to the catalytically inactive construct comprised of the N-terminal (1-166 residues) of PDE4D4 fused to GFP, which was expressed in PMVECs. (C) Confocal microscopy of PMVECs expressing PDE4D4-GFP fusion protein (top panel) indicates the peptide localized to cell-cell borders with βII spectrin, similarly to endogenous PDE4D4. GFP vector control, bottom panel. (D) Control and PDE4D41-166-expressing cells were grown to confluence and separated into pellet, cytosolic and membrane fractions. Western analysis revealed that most PDE4D4 enzyme was present in caveolin-containing membrane fractions. The PDE4D41-166 fragment did not shift the location of the endogenous PDE4D4 enzyme. (E) In PMVEC membranes expressing the catalytically inactive PDE4D4-GFP fusion protein, cAMP PDE activity was reduced by 
30% compared with that in control cell membranes. Rolipram (10 µM for 10 minutes) inhibited 85% of the PDE4 activity in control PMVECs and 65% of the PDE4 activity in PDE4D41-166 expressing cells (*P<0.05, n=3). (F) Radioimmunoassay revealed that PDE4D41-166-expressing cells possessed higher basal cAMP concentrations, and greater forskolin-stimulated (1 µM, 3 minutes) cAMP responses (P<0.05, n=3). (G) Expression of PDE4D4 in PAECs resulted in co-immunoprecipitation of 
II spectrin with PDE4 (right panel), which was not observed in control cells (left panel). (H) The PDE4D41-166 construct was expressed in PAECs, which express little PDE4D4. Cells treated with rolipram (10 µM for 10 minutes) show similarly reduced phosphodiesterase activity in control and PDE4D41-166-expressing cells (P<0.05, n=3).
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