Skippen, Alison Jane; (2004) Regulation of phosphoinositide dynamics in cell signalling by ADP-ribosylation factor and phosphatidylinositol transfer protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important source of second messengers and can act as a signalling molecule in its own right. This study has investigated the mechanism of ADP-ribosylation factor (ARF)-stimulated PI(4,5)P2 synthesis. ARF proteins can directly activate type I phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase) and phospholipase D (PLD) in vitro. The role of PLD in the regulation of PI(4,5)P2 synthesis was examined in cytosol-depleted HL60 cells using butan- l-ol to diminish the PLD-derived phosphatidic acid (PA), and an ARFl point mutant, N52R-ARF1, that can selectively activate type I PIP 5-kinase but not PLD activity. This work concluded that both PA derived from the PLD pathway, and ARF proteins by directly stimulating type I PIP 5-kinase activity, contribute to the regulation of PI(4,5)P2 synthesis at the plasma membrane in HL60 cells. The PI(4,5)P2 synthesized upon ARF addition was available for hydrolysis by phospholipase C (PLC) only in the presence of a phosphatidylinositol transfer protein a (PITPa). PITP[alpha] was previously identified as a cytosolic factor which can reconstitute agonist-stimulated PLC signalling in cytosol-depleted cells. This study has examined the importance of PITP[alpha] to bind and transfer PI or PC, as well as interact with a membrane surface, for PI delivery required for PLC signalling. Using structural information obtained from the recently elucidated crystal structure of human PITP[alpha] bound to PI, amino acid residues were identified that were predicted to be important for PITPa to bind to PI, and to interact with a membrane surface. Residues T59, K61, E86, and N90 were responsible for interacting with the inositol ring of PI and single point mutants at these residues were sufficient to cause a selective loss in PI but not PC binding and transfer. These mutants were also unable to restore PLC signalling. These amino acid residues were conserved in all proteins with a PITP domain identified in many organisms including mammals, flies, worms, amphibian, fish, and several unicellular organisms. Two tryptophan residues (W203 and W204) were required to dock at the membrane, allowing a model to be described for how PITP[alpha] may undergo lipid exchange at a membrane surface.

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