Garner, KL; (2012) An investigation into protein and lipid binding by the phosphatidylinositol transfer protein RdgBβ. Doctoral thesis , UCL (University College London).

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Abstract

The phosphatidylinositol transfer proteins (PITPs) are a family of lipid carrier proteins that bind and transfer phosphatidylinositol (PI) and phosphatidylcholine (PC) between membranes. PITPs are commonly involved in phosphoinositide-requiring processes, including phospholipase C and PI 3-kinase signalling, and membrane trafficking. In this study I focus on the uncharacterised soluble PITP, RdgBβ (PITPNC1). The lipid binding and transfer properties of RdgBβ have scarcely been characterised, and the function of RdgBβ is completely unknown. I uncover that RdgBβ interacts with 14-3-3 through its long, disordered C-terminus. RdgBβ is ubiquitinated and subject to rapid degradation in cells, and binding of 14-3-3 via two phosphorylated residues may serve to protect the protein from protease digestion. Whereas RdgBβ binds 14-3-3 under basal conditions, I deduce that, upon stimulation of cells with phorbol ester, RdgBβ binds the Angiotensin II receptor (AT1R)-associated protein, ATRAP, via its N-terminal PITP domain. Others have shown that ATRAP suppresses Angiotensin II signalling by uncoupling AT1R from G proteins and promoting AT1R internalisation. I find that the RdgBβ-ATRAP interaction is blocked by inhibition of protein kinase C or protein synthesis, and may function to re-localise RdgBβ to the membrane in stimulated cells. Unexpectedly, I find that RdgBβ binds PI and phosphatic acid (PA), rather than PI and PC, and that binding of PA is increased by stimulation of cells with GTPγS. Mass spectrometry is used to analyse the molecular species of PI and PA bound by RdgBβ, and reveals that whereas RdgBβ is non-selective in its binding of PI, it selects short-chain monounsaturated or saturated PA species, likely derived from the hydrolysis of PC by phospholipase D.

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