PHOSPHATIDYLINOSITOL TRANSFER PROTEIN DICTATES THE RATE OF INOSITOL TRISPHOSPHATE PRODUCTION BY PROMOTING THE SYNTHESIS OF PIP2

Background: Phosphatidylinositol transfer protein (PI-TP), which has t he ability to transfer phosphatidylinositol (PI) from one membrane com partment to another, is required in the inositol lipid signalling path way through phospholipase C-beta (PLC-beta) that is regulated by GTP-b inding protein(s) in response to extracellular signals. Here, we test the hypothesis that the principal role of PI-TP is to couple sites of lipid hydrolysis to sites of synthesis, and so to replenish depleted s ubstrate for PLC-beta. Results: We have designed an experimental proto col that takes advantage of the different rates of release of endogeno us PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolys is in cells depleted of PI-TP, but not of endogenous PLC-beta, in the presence and absence of exogenous PI-TP. Linear time-courses were obse rved in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'[gamma-thio]-triphosphate (GTP gamm a S) or the receptor-directed agonist fMetLeuPhe as activators. In add ition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the leve ls of PLC-beta present in the cells. Once again, PI-TP increased the r ates of reactions. To identify whether the role of PI-TP was to make a vailable the substrate phosphatidylinositol bisphosphate (PIP2) for th e PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. W e found that PI-TP was essential for the synthesis of PIP2. Conclusion s: The predicted function of PI-TP in inositol lipid signalling is the provision of substrate for PLC-beta from intracellular sites where PI is synthesized. We propose that PI-TP is in fact a co-factor in inosi tol lipid signalling and acts by interacting with the inositol lipid k inases. We hypothesize that the preferred substrate for PLC-beta is no t the lipid that is resident in the membrane but that provided through PI-TP.