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Title: Identification of ADP-ribosylation factor as a requirement for G-protein-mediated phospholipase D activation and secretion in HL60 cells
Author: Fensome, Amanda Caroline
ISNI:       0000 0001 3459 786X
Awarding Body: University of London
Current Institution: University College London (University of London)
Date of Award: 1996
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Ligand binding to both G-protein coupled and tyrosine kinase receptors has been shown to activate phospholipase D (PLD). PLD catalyses the hydrolysis of phosphatidylcholine resulting in the production of choline and the potential second messenger phosphatidic acid (PA). In vitro studies have demonstrated that factors from both the particulate and cytosolic fractions are required for G-protein-mediated PLD activation. Permeabilisation of cells with the cytolytic toxin Streptolysin O (SLO) allows cytosolic proteins to leak out. G- protein-dependent PLD activity is almost completely permeabilised HL60 cells which are depleted of their cytosolic proteins Subsequent addition of cytosol to these cells restores G-protein-mediated PLD activity, indicating that a cytosolic factor is required. In this thesis, cytosol-depleted HL60 cells were used to identify the cytosolic factors required for G-protein dependent PLD activation. The factors were purified from bovine brain cytosol and identified as two monomeric G-protein -ADP- ribosylation factor 1 and 3 (ARF 1 and 3). ARF is involved in vesicular trafficking and is an essential component of the constitutive secretory pathway in yeast. PLD activation has been linked to regulated secretion in both neutrophils and HL60 cells. In view of the established role of ARF, and the link between PLD and secretion, the ability of ARF to restore secretion in cytosol-depleted cells was investigated. ARF restored GTPγS-stimulated secretion; and throughout the characterisation a correlation between PLD activity and secretion was observed indicating that these responses are linked. A second protein -PITP- also restored GTPγS-stimulated secretion. Simultaneous addition of ARF and PITP produced an additive response indicating that they act via a common mechanism. PIP2 is demonstrated to be required for both ARF and PITP restored secretion and it is proposed that both these proteins restore secretion by promoting the synthesis of PIP2.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Biochemistry