ARAP3 is a phosphoinositide 3’-kinase- (PI3K) and Rap-regulated dual GTPase activating protein (GAP) for Arf6 and RhoA, that was identified from porcine leukocyte cytosol in a screen for phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P<sub>3</sub>) binding proteins. Firstly, we show that ARAP3 binds its regulator, PtdIns(3,4,5)P<sub>3</sub>, in an unusual PH domain-dependent manner. A point mutation in the most N-terminal of its five PH domains abrogates PtdIns(3,4,5)P<sub>3</sub> binding and interferes with ARAP3’s catalytic activities. However, we demonstrate that none of the five PH domains is sufficient to bind PtdIns(3,4,5)P<sub>3</sub> in isolation. We proposed a mechanism by which basic residues in two PH domains, the SAM domain and elsewhere in the protein synergise to mediate affinity PtdIns(3,4,5)P<sub>3</sub> binding. Secondly, we have investigated the role of ARAP3 <i>in vivo </i>using chemotaxing neutrophils as a model system. Previous work in the laboratory showed that neutrophils lacking ARAP3 were unable to undergo net movement towards the source of a local gradient of chemoattractant. We have used novel methodologies to analyse the local distribution of ARAP3, and the local distributions and global levels of phosphoinositides in primary neutrophils from ARAP3 mouse models. We show that ARAP3 localises to the leading edge, and directly or indirectly regulates the local distribution of PtdIns(3,4,5)P<sub>3</sub> during chemotaxis. We propose that PtdIns(3,4,5)P<sub>3</sub> recruits ARAP3 to the leading edge of a chemotaxing neutrophil, where it is part of a feedback loop that is responsible for the maintenance of polarisation in the direction of a chemoattractant gradient.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:598120 |
| Date | January 2010 |
| Creators | Craig, H. E. |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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