Molecular dissection of the role of ARAP3 in angiogenesis

During embryonic development, specialised tissues form. For example, a circulatory system is required to nourish the developing organism. This process is called angiogenesis. Angiogenesis is also important in cancer, where new tumours need to be provided with blood so they can be nourished. We found out that ARAP3, a new signalling protein, is needed for angiogenesis when we studied a mouse that lacked ARAP3 and saw that the embryos died because they couldn’t form blood vessels. We want to establish why ARAP3 is required for angiogenesis, and whether it could be a good target for cancer treatment. For this, we will look at the way in which ARAP3 is controlled and in term controls other cellular proteins. We will analyse vessels in mouse embryos, which either lack ARAP3 or where it contains a small change (mutation). Secondly, we will study endothelial cells (which form blood vessels) lacking ARAP3, or containing the mutant ARAP3 protein, to see how they differ from normal cells. Finally, we will put ARAP3, or its mutants back into endothelial cells, to ‘rescue’ them. In this way we will learn why ARAP3 is required for angiogenesis, and whether it is a good cancer drug target.

Phosphoinositide 3-OH kinases (PI3Ks) control a multitude of fundamental cellular responses after appropriate stimulation. One such response is angiogenesis, both during embryogenesis and in tumorigenesis. ARAP3 was identified in a screen for novel PI3K effectors as a PI3K- and Rap- regulated dual GTPase activating protein (GAP) for Rho and Arf. To understand its physiological function, we recently created an ARAP3 knock-out mouse. Loss of ARAP3 leads to embryonic lethality in mid-gestation: ARAP3-/- embryos are retarded, anaemic and devoid of blood vessels; their yolk sacs contain no blood vessels and their placentas do not develop the labyrinth of maternal and foetal capillaries. We conclude that embryonic death is due to an angiogenesis defect, hence ARAP3 is required for angiogenesis in the developing embryo. To elucidate the signalling upstream of ARAP3 in the control of angiogenesis, we created a second mouse model (ARAP3R302/3A), in which ARAP3 can no longer be activated by PI3K. We propose to analyse embryogenesis in ARAP3R302/3A embryos and compare them to ARAP3-/- embryos. We propose further to analyse endothelial cells derived from ARAP3-/- and ARAP3R302/3A embryos in terms of their shape, cellular junctions, focal adhesions, ability to chemotax and to form tubular structures in vitro, to define the precise cellular defects arising from lack (or mutation) of ARAP3. Together, these studies will establish the importance of ARAP3 as an effector of PI3K for control of angiogenesis. To clarify the signalling pathways downstream of ARAP3, we will dissect out whether particular defects are due to ARAP3?s Rho or Arf GAP activity. This will be done by rescuing ARAP3-/- endothelial cells by retroviral transduction with individual ARAP3 GAP domain point mutants. Thereby, we will increase our understanding of the cross-talk between Rho and Arf small GTPases, which is mediated by ARAP3 and controlled by PI3K during angiogenesis. Furthermore, we will address modulation of ARAP3?s Rho GAP activity by Rap by rescue with a Ras binding domain point mutant; to see whether ARAP3?s scaffold function is important for its angiogenic potential, we will use a ?SAM domain truncation construct. In summary, this project will elucidate PI3K-controlled signalling to Rho and Arf GTPases mediated by ARAP3 in angiogenesis in developing embryos and in endothelial cells. Whilst our work concentrates on embryogenesis, the results obtained are likely to be applicable to the angiogenic processes fundamental to the growth of a range of human cancers.