title: Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2.
abstract: BACKGROUND
  Phosphatidylinositol transfer protein (PI-TP), which has the ability to transfer phosphatidylinositol (PI) from one membrane compartment to another, is required in the inositol lipid signalling pathway through phospholipase C-beta (PLC-beta) that is regulated by GTP-binding 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 substrate for PLC-beta.
  
  
  RESULTS
  We have designed an experimental protocol that takes advantage of the different rates of release of endogenous PI-TP and PLC-beta from HL60 cells permeabilized with streptolysin O. We have examined the kinetics of stimulated inositol lipid hydrolysis 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 observed in the absence of any added protein, and the rate was accelerated by PI-TP using either guanosine 5'[gamma-thio]-triphosphate (GTP gamma S) or the receptor-directed agonist fMetLeuPhe as activators. In addition, depletion from the cells of both PI-TP and PLC-beta isoforms by extended permeabilization (40 minutes) allowed us to control the levels of PLC-beta present in the cells. Once again, PI-TP increased the rates of reactions. To identify whether the role of PI-TP was to make available the substrate phosphatidylinositol bisphosphate (PIP2) for the PLC, we examined the synthesis of PIP2 in cells depleted of PI-TP. We found that PI-TP was essential for the synthesis of PIP2.
  
  
  CONCLUSIONS
  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 inositol lipid signalling and acts by interacting with the inositol lipid kinases. We hypothesize that the preferred substrate for PLC-beta is not the lipid that is resident in the membrane but that provided through PI-TP.
authors.affiliation: Department of Physiology, University College London, UK.
authors.forename: E
authors.initials: E
authors.lastname: Cunningham
authors.forename: G M
authors.initials: GM
authors.lastname: Thomas
authors.forename: A
authors.initials: A
authors.lastname: Ball
authors.forename: I
authors.initials: I
authors.lastname: Hiles
authors.forename: S
authors.initials: S
authors.lastname: Cockcroft
mesh_terms: D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D003067:Coenzymes / Q000378:metabolism; D017931:DNA Primers; D019204:GTP-Binding Proteins / Q000378:metabolism; D018922:HL-60 Cells; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis*; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis*; D010716:Phosphatidylinositols / Q000378:metabolism*; D054799:Phospholipase C beta; D049294:Phospholipid Transfer Proteins; D051381:Rats; D015398:Signal Transduction; D013379:Substrate Specificity; D010738:Type C Phospholipases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D003067:Coenzymes; D017931:DNA Primers; D007295:Inositol Phosphates; D007527:Isoenzymes; D008565:Membrane Proteins; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D010716:Phosphatidylinositols; D049294:Phospholipid Transfer Proteins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D019204:GTP-Binding Proteins