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, FT C236) were stably transfected with a cDNA encoding the PDE4A cAMP-spec ific phosphodiesterase (PDE) splice variant RD1 (RNPDE4A1A) so as to g enerate the cloned cell lines, FTC133A and FTC236A. This allowed the e xpression of a novel rolipram-inhibited cAMP-specific PDE activity in these cells. Unlike the parent cell lines in which Ca2+/calmodulin cau sed a profound activation (approx. 3-4-fold) of homogenate PDE activit y, no such stimulation was evident in the RD1-expressing cell lines, i ndicating loss of PDE1 activity. Reverse transcriptase-PCR analysis in dicated 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 concentration s of the detergent Triton X-100, but not high NaCl concentrations, all owed RD1 to be solubilized. Laser scanning confocal immunofluorescence analyses identified RD1 immunoreactivity in a discrete perinuclear re gion of these RD1-expressing transfected cell lines. A similar pattern of labelling was observed using the antiserum Tex 1, which specifical ly identified the Golgi apparatus. Treatment of FTC133A cells with the Golgi-perturbing agents monensin and brefeldin A led to a similar red istribution 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 expre ssion of RD1 within the Golgi complex of these human follicular thyroi d carcinoma cell lines.