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Phosphatidylinositol transfer protein is a requirement for G-protein-mediated inositol lipid signalling in HL60 cells

Cunningham, Emer Mary; (1995) Phosphatidylinositol transfer protein is a requirement for G-protein-mediated inositol lipid signalling in HL60 cells. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

Ligand binding to G-protein-linked receptors triggers the activation of the phosphoinositide specific phospholipase C (PLC). Activated PLC catalyses the hydrolysis of phosphatidylinositol(4,5)bisphosphate to generate two intracellular second messengers, inositol 1,4,5- trisphosphate and diacylglycerol. Purification and molecular cloning have revealed that PLCs are made up of at least three families β, γ and δ. Members of the PLC β family are regulated by heterotrimeric G- proteins, either a-subunits of the Gq family or by β γ subunits. It has previously been established that when HL60 cells are permeabilized with the cytolytic toxin, streptolysin O, cytosolic proteins leak out of the cells and this is associated with a loss of GTPγS and fMetLeuPhe-mediated activation of PLC. In this thesis cytosol-depleted cells were used as a reconstitution system to identify the cytosolic component(s) necessary for inositol lipid signalling. Two reconstituting factors were isolated from rat brain cytosol. The major reconstituting factor was identified as a 35 KDa protein phosphatidylinositol-transfer protein (PI-TP). The minor reconstituting factor proved to be PLC β1. PI-TP is a ubiquitous protein found in all mammalian tissues examined to date. It was originally identified by its ability to bind and transfer PI and PC from one membrane compartment to another in vitro. Here it is shown that PI-TP can restore responsiveness to GTPγS and fMetLeuPhe to activate inositol lipid signalling and this reconstitution is Ca2+ and Mg.ATP dependent. PI-TP and PLC β1 synergise to restore GTPγS-stimulated inositol phosphate production. Examination of the kinetics of reconstitution with PI-TP shows that PI-TP increases the rate of inositol phosphate production by GTPγS or fMetLeuPhe-regulated PLC β. The data suggest that the function of PI-TP in inositol lipid signalling cannot be entirely explained by its established biochemical in vitro transfer activity but plays a more direct but as yet undetermined role. Substrate presentation to the inositol lipid kinases) as a mechanism to allow PI-TP to enhance G-protein-regulated PLC β activity is discussed.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Phosphatidylinositol transfer protein is a requirement for G-protein-mediated inositol lipid signalling in HL60 cells
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; G-protein-mediated; HL60; Inositol lipid signalling; Phosphatidylinositol; Protein
URI: https://discovery.ucl.ac.uk/id/eprint/10105995
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