The characterization of phospholipase D in FRTL-5 thyroid cells

We previously demonstrated that TSH activates phospholipase D (PLD) in Fisc her rat thyroid line (FRTL)-5 cells. To date, two types of mammalian phosph atidylcholine-specific PLD cDNAs, designated as PLD-I 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) an d RhoA family members, while PLD-2 is relatively independent of such stimul i. We established the presence of PLD-I and PLD-2 by Western blot analysis and compared PLD activity in cytosol, membranes and combined fractions in t he presence and absence of GTP gamma S. The membrane fraction showed very l ittle activity in the absence of GTP gamma S, but this activity increased a pproximate to 5-fold (P < 0.05, ANOVA) in the presence of GTP gamma S. Maxi mal PLD activity was seen with the combination of membrane plus cytosolic f ractions (which contained ARF and RhoA) where the addition of GTP gamma S i ncreased PLD activity approximate to 8-fold (P < 0.05, ANOVA). To determine the relative activities of PLD-I and PLD-2 in FRTL-5 thyroid cells, cell-f ree PLD assays were performed in the presence of GTP gamma S or GDP beta S with varying concentrations of phosphatidylinositol 4,5-bisphosphate (PIP2) . PLD-2 contributed only approximate to 19% of the total amount of PLD acti vity in the membranes and PLD-I was the predominant PLD isoform. TSH stimul ated PLD-I activity by up to 2.3-fold over control values (P < 0.01, ANOVA) . To establish the dependence of PLD-I on small molecular weight G-proteins , the translocations of ARF and RhoA to the membrane fractions was determin ed after stimulation by TSH. Both ARF and RhoA were maximally translocated to the membrane fraction after 10 min incubation with 100 mu U/ml TSH by ap proximate to 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 a nd RhoA to the membrane fraction. (C) 2000 Elsevier Science Ireland Ltd. Al l rights reserved.