Immunological investigations into the properties and function of the phosphatidylinositol transfer protein

It is becoming increasingly apparent that the inositol lipids are involved in a wide range of cellular processes, ranging from transmembrane signal transduction, to modulation of the behaviour of the cytoskeleton. To date one of their best characterised functions is in the operation of the Phospholipase C signalling pathway, whereby inositol lipid components of the cell are degraded in response to ligand binding at membrane receptors to produce the second messenger molecules diacylglycerol, and inositol-1,4,5-trisphosphate. Studies conducted on this process have implicated the mammalian phosphatidylinositol transfer protein (PI-TP) as being essential to the functioning of this pathway. This thesis outlines studies on the nature and function of PI-TP in the leukaemic cell line HL60, and the neutrophil, for which the HL60 cell is often regarded as a model. In studies utilising HL60 cells permeabilised, and cytosol depleted using the bacterial toxin Streptolysin O (SLO), PI-TP was identified as being essential to the maintenance of the PLC signalling response. On the basis of these data, earlier work was extended by the development of immunochemicals against the two identified isoforms of mammalian PI-TP (α and β) and their use to study various characteristics of this protein. These reagents include polyclonal antibodies raised against a fusion protein of PI-TPα/GST, and a panel of monoclonal antibodies against native bovine brain PI-TPα, and bacterially expressed rat brain PI-TP β. Among the questions addressed are the kinetics with which the proteins involved in PLC signalling leak from SLO permeabilised cells, the quantities of PI- TPα and β present in the intact cell, and the phosphorylation state of the protein in response to ligand stimulation. In the course of this work it was discovered that the β isoform is by far the predominant of the two proteins in neutrophils, and HL60 cells, and that the two protein display distinct kinetics of leakage, with the a isoform leaking very rapidly from the cell while the P isoform is significantly retained. PI-TPα appears to undergo phosphorylation in response to treatment of the cell with both PMA, and the chemotactic peptide fMLP