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Annexin A3 is necessary for artery-vein alignment in the mouse retinas (RNA-seq analysis of isolated retinal endothelial cells from Anxa3;Tie2-cre mutant mice)

ABSTRACT: Annexin A3 (Anxa3) is a member of the calcium-regulated, cell membrane binding family of Annexin proteins. We previously confirmed that Anxa3 is expressed in the endothelial lineage in vertebrates and that loss of Anxa3 in Xenopus laevis leads to embryonic blood vessel defects. However, the biological function of Anxa3 in mammals is completely unknown. In order to investigate Anxa3 vascular function in mammals, we generated an endothelial cell specific Anxa3 conditional knockout mouse model (Anxa3f/f;Tie2-Cre).

ORGANISM(S): Mus musculus

PROVIDER: GSE135528 | GEO | 2019/08/08

REPOSITORIES: GEO

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Project description:Vascular endothelial protein tyrosine phosphatase (VE-PTP, PTPRB) is a receptor type phosphatase that is crucial for the regulation of endothelial junctions and blood vessel development. VE-PTP regulates vascular integrity by dephosphorylating substrates which are key players in endothelial junction stability, such as the angiopoietin receptor TIE2, the endothelial adherens junction protein VE-cadherin and the vascular endothelial growth factor receptor VEGFR2. Here, we have systematically searched for novel substrates of VE-PTP in endothelial cells by utilizing two approaches. First, we studied changes in the endothelial phosphoproteome upon exposing cells to a highly VE-PTP-specific phosphatase inhibitor followed by affinity isolation and mass-spectrometric analysis of phosphorylated proteins by phosphotyrosine-specific antibodies. Second, we used a substrate trapping mutant of VE-PTP to pull down phosphorylated substrates in combination with SILAC-based quantitative mass spectrometry measurements. We identified a set of substrate candidates of VE-PTP, of which a remarkably large fraction is related to cell junctions (48/165; 29.1%). Several of those were found in both screens and displayed very high connectivity in predicted functional interaction networks. The receptor protein tyrosine kinase EPHB4 was the most prominently phosphorylated protein upon VE-PTP inhibition among those VE-PTP targets that were identified by both proteomic approaches. Further analysis revealed that EPHB4 forms a ternary complex with VE-PTP and TIE2 in endothelial cells. VE-PTP controls the phosphorylation of each of these two tyrosine kinase receptors. Despite of their simultaneous presence in a ternary complex, stimulating each of the receptors with their own specific ligand did not cross-activate the respective partner receptor. Our systematic approach has led to the identification of novel substrates of VE-PTP, of which many are relevant for the control of cellular junctions further promoting the importance of VE-PTP as a key player of junctional signalling.

Dataset Information

Annexin A3 is necessary for artery-vein alignment in the mouse retinas (RNA-seq analysis of isolated retinal endothelial cells from Anxa3;Tie2-cre mutant mice)

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