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 phosphodiesterase variants encoded by the PDE4D gene are targeted to discrete subcellular structures. In quiescent FRTL-5 thyroid cells, the rolipram-sensitive 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 compartments, a 105-kDa variant with the properties of PDE4D4 was recovered mostly in the particulate fraction. The PDE4D3 form was readily solubilized with nonionic detergents. Conversely, the PDE4D4 form required 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 activation of the cAMP-PDE in both compartments and was correlated with a shift in electrophoretic mobility of the PDE4D3 polypeptide. Long term incubation with TSH caused an increase of the PDE activity in the soluble fraction and the appearance of a 68-kDa immunoreactive polypeptide with the properties of PDE4D2. Immunofluorescence analysis showed, in addition to diffuse staining, a signal localized on regions adjacent to the plasma membrane on cytoskeletal structures and in a perinuclear region of quiescent cells. Long term incubation with TSH caused an increase in the immunofluorescence signal in the soluble compartment. These data demonstrate that three PDE4D splicing variants are targeted to discrete subcellular compartments and that hormones cause the activation of these isoforms in a temporally and spatially dependent manner.
Jin
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SL
Division of Reproductive Biology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, California 94305-5317, USA.
Bushnik
T
T
Lan
L
L
Conti
M
M
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D002458:Cell Fractionation; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D003902:Detergents / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D015151:Immunoblotting; D007150:Immunohistochemistry; D011760:Pyrrolidinones / Q000494:pharmacology*; D051381:Rats; D020889:Rolipram; D013961:Thyroid Gland / Q000201:enzymology*; D013972:Thyrotropin / Q000494:pharmacology*
D003902:Detergents; D011760:Pyrrolidinones; D013972:Thyrotropin; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D020889:Rolipram