Intracellular localization of the PDE4A cAMP-specific phosphodiesterase splice variant RD1 (RNPDE4A1A) in stably transfected human thyroid carcinoma FTC cell lines.
Cells of two human follicular thyroid carcinoma cell lines (FTC133, FTC236) were stably transfected with a cDNA encoding the PDE4A cAMP-specific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to generate the cloned cell lines, FTC133A and FTC236A. This allowed the expression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin caused a profound activation (approx. 3-4-fold) of homogenate PDE activity, no such stimulation was evident in the RD1-expressing cell lines, indicating loss of PDE1 activity. Reverse transcriptase-PCR analysis indicated that this was due to the down-regulation of the PDE1C isoform. The novel PDE4 activity in transfected cells was located exclusively in the membrane fraction, as was immunoreactive RD1. Low concentrations of the detergent Triton X-100, but not high NaCl concentrations, allowed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear region of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex1, which specifically identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar redistribution of immunoreactive species detected using both the Tex1 and anti-RD1 antisera. It is suggested that the PDE4A splice variant RD1 contains a membrane-association signal which allows the targeted expression of RD1 within the Golgi complex of these human follicular thyroid carcinoma cell lines.
Pooley
L
L
Division of Biochemistry and Molecular Biology, University of Glasgow, Scotland, U.K.
Shakur
Y
Y
Rena
G
G
Houslay
M D
MD
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; D005455:Fluorescent Antibody Technique; D006801:Humans; D008969:Molecular Sequence Data; D017830:Octoxynol; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D012326:RNA Splicing; D013964:Thyroid Neoplasms / Q000201:enzymology*; D014162:Transfection; D014407:Tumor Cells, Cultured
D010726:Phosphodiesterase Inhibitors; D017830:Octoxynol; D010727:Phosphoric Diester Hydrolases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D054677:Cyclic Nucleotide Phosphodiesterases, Type 1; D054703:Cyclic Nucleotide Phosphodiesterases, Type 4; C517355:PDE4A protein, human