SUBCELLULAR-LOCALIZATION OF ROLIPRAM-SENSITIVE, CAMP-SPECIFIC PHOSPHODIESTERASES - DIFFERENTIAL TARGETING AND ACTIVATION OF THE SPLICING VARIANTS DERIVED FROM THE PDE4D GENE

Biochemical and immunofluorescence analyses revealed that phosphodiest erase variants encoded by the PDE4D gene are targeted to discrete subc ellular structures. In quiescent FRTL-5 thyroid cells, the rolipram-se nsitive phosphodiesterase (PDE) activity (cAMP-PDE) was recovered both in the soluble and particulate fractions of the homogenate, Although an immunoreactive 93-kDa PDE (PDE4D3) variant was recovered in both co mpartments, a 105-kDa variant with the properties of PDE4D4 was recove red mostly in the particulate fraction. The PDE4D3 form was readily so lubilized with nonionic detergents. Conversely, the PDE4D4 form requir ed buffers containing ionic detergents for extraction, suggesting that different mechanisms target these variants to insoluble structures. A 15-min stimulation with thyroid-stimulating hormone (TSH) led to an a ctivation of the cAMP-PDE in both compartments and was correlated with a shift in electrophoretic mobility of the PDE4D3 polypeptide, Long t erm incubation with TSH caused an increase of the PDE activity in the soluble fraction and the appearance of a 68-kDa immunoreactive polypep tide with the properties of PDE4D2. Immunofluorescence analysis showed , in addition to diffuse staining, a signal localized on regions adjac ent to the plasma membrane on cytoskeletal structures and in a perinuc lear region of quiescent cells. Long term incubation with TSH caused a n increase in the immunofluorescence signal in the soluble compartment . These data demonstrate that three PDE4D splicing variants are target ed to discrete subcellular compartments and that hormones cause the ac tivation of these isoforms in a temporally and spatially dependent man ner.