Way, Gemma Ann; (1998) The role of the phosphatidylinositol transfer protein and the small GTP-ASE ARF in regulated exocytosis from RBL-2H3 mast cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Two proteins have been identified previously, based on their ability to restore secretion from granulocytes (neutrophils and HL60 cells): the ADP-ribosylation factor (ARF), and the phosphatidyl-inositol transfer protein (PI-TP). If granulocytes are permeabilised before stimulation there is a decline in the secretory response. This is associated with the leakage of cytosolic proteins, and implies that some of the lost proteins are involved in the regulation of secretion. Previous work has relied upon the use of the artificial stimulus, GTPγS. RBL-2H3 mast cells may be stimulated experimentally via their tyrosine-kinase linked FcϵR1 receptors, using the physiological stimulus. Therefore the primary goal of this project was to demonstrate that the effects of myr.rARF-1 and rPI-TP on secretion were of physiological relevance. When conditions for depleting RBL-2H3 mast cell cytosol were established, ARF and PI-TP leakage were found to coincide temporally with the gradual decline in secretion. In reconstitution studies, cytosol-depleted cells were incubated with recombinant myr.ARF-1 and both α and β isoforms of rPI-TP; each of these proteins successfully restored lost responsiveness. Myr.rARF-1 was also found to restore lost phospholipase D activity following permeabilisation. The mechanisms through which these proteins contribute to exocytosis were investigated: it was found that while ARF appears to exert its effect via PLD activation, PI-TP operates via a different pathway. However, analysis of polyphosphoinositide levels in reconstituted cells indicates a possible common mechanism, namely the increased synthesis of phosphatidylinositol-4,5-bisphosphate (PIP2). To identify the site of PIP2 synthesis, the subcellular location of the PI-TP's was examined. It was discovered that antigen-stimulation mediates a translocation of PI-TPα to the cells' cortex.

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