Phosphatidylinositol transfer protein dictates the rate of inositol trisphosphate production by promoting the synthesis of PIP2

Curr Biol. 1995 Jul 1;5(7):775-83. doi: 10.1016/s0960-9822(95)00154-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Carrier Proteins / metabolism*
  • Coenzymes / metabolism
  • DNA Primers
  • GTP-Binding Proteins / metabolism
  • HL-60 Cells
  • Humans
  • Inositol Phosphates / biosynthesis*
  • Isoenzymes / metabolism
  • Membrane Proteins*
  • Molecular Sequence Data
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphatidylinositol Phosphates / biosynthesis*
  • Phosphatidylinositols / metabolism*
  • Phospholipase C beta
  • Phospholipid Transfer Proteins
  • Rats
  • Signal Transduction
  • Substrate Specificity
  • Type C Phospholipases / metabolism

Substances

  • Carrier Proteins
  • Coenzymes
  • DNA Primers
  • Inositol Phosphates
  • Isoenzymes
  • Membrane Proteins
  • Phosphatidylinositol 4,5-Diphosphate
  • Phosphatidylinositol Phosphates
  • Phosphatidylinositols
  • Phospholipid Transfer Proteins
  • Type C Phospholipases
  • Phospholipase C beta
  • GTP-Binding Proteins