The characterization of phospholipase D in FRTL-5 thyroid cells

Abstract

We previously demonstrated that TSH activates phospholipase D (PLD) in Fischer rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosphatidylcholine-specific PLD cDNAs, designated as PLD-1 and PLD-2, have been cloned. The present study determined the PLD isoform composition in FRTL-5 thyroid cells and which isoform is regulated by TSH. PLD-1 is activated by small molecular weight G-proteins, such as ADP-ribosylation factor (ARF) and RhoA family members, while PLD-2 is relatively independent of such stimuli. We established the presence of PLD-1 and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in the presence and absence of GTPγS. The membrane fraction showed very little activity in the absence of GTPγS, but this activity increased ≈5-fold ( P<0.05, ANOVA) in the presence of GTPγS. Maximal PLD activity was seen with the combination of membrane plus cytosolic fractions (which contained ARF and RhoA) where the addition of GTPγS increased PLD activity ≈8-fold ( P<0.05, ANOVA). To determine the relative activities of PLD-1 and PLD-2 in FRTL-5 thyroid cells, cell-free PLD assays were performed in the presence of GTPγS or GDPβS with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP 2). PLD-2 contributed only ≈19% of the total amount of PLD activity in the membranes and PLD-1 was the predominant PLD isoform. TSH stimulated PLD-1 activity by up to 2.3-fold over control values ( P<0.01, ANOVA). To establish the dependence of PLD-1 on small molecular weight G-proteins, the translocations of ARF and RhoA to the membrane fractions was determined after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 μU/ml TSH by ≈1.7- and 2.3-fold over control values, respectively ( P<0.02 and P<0.03, ANOVA). It is concluded that TSH stimulates PLD-1 activity in FRTL-5 thyroid cells and this is accompanied by the translocation of ARF and RhoA to the membrane fraction.