Regulation of angiogenesis by the endothelial Rho GTP/GDP exchange factor, FGD5

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We seek to understand how pro-angiogenic G protein coupled receptor (GPCR) signals cooperate with vascular endothelial growth factor (VEGF)-receptor signals, to stimulate differentiation of the endothelial cells (EC) lining established blood vessels, and initiate new blood vessel growth. We focus on the phosphoinositide 3 kinase (PI3K) signal pathway. Endothelial PI3K activity is both necessary and rate limiting for new blood vessel growth in mice. We also know VEGF-stimulation of EC is insufficient, since dysfunctional vessel growth is seen in a variety of EC-selective GPCR-knockout mice, including CXCR4 and APLNR. Understanding the details of the cooperative signalling with the VEGF receptor will reveal new mechanisms of angiogenic EC differentiation and assembly of vessels in the embryo, and in cancer blood vessels in the adult.******Differentiation of the lead or “tip” EC during new-vessel sprouting includes protrusion of “fingers” (i.e. filopodia) to sample the tissue environment, and gene expression that can be recapitulated in 3D culture. Our earlier work has defined a critical role for the PI3K-b isoform, coupled to pro-angiogenic GPCRs in the EC for tip cell generation. We propose to examine the assembly of the PI3K signalling protein complex, and the regulation of PI3K-b activity by the Rho GTP-binding protein exchange factor, FGD5, in this application.******Aim 1: To characterize pro-angiogenic GPCR coupling to PI3K-b in EC. We will determine if CXCR4 assembles in a complex with PI3K-b, and if PI3K-b signal output differs from the endosome vs plasma membrane activity to elicit i) sprouting, and angiogenic outputs of ii) cytoskeletal remodeling, and iii) gene expression.***We will determine if APLNR, stimulated by the pro-angiogenic ligand, Apelin, behaves similarly to activated CXCR4 in EC. We will contrast the effect of the APLNR to the alternate, angiogenesis-inhibitory ligand, APELA.******Aim 2: To characterize how the Rac1GEF, FGD5, regulates endothelial PI3K activity. We will determine if FGD5 i) is recruited to the PI3K-b vs -a complex at the endosome, and ii) RhoGEF activity regulates PI3K-b vs a, exploiting expression of domain-deleted FDG5 mutants in ECs, iii) directly regulates PI3K-b via Rac1-GTP binding to PI3K-b.***We will study the effect of endothelial-selective FGD5 loss-of-function on angiogenesis in vivo, exploiting a novel FGD5 conditional knockout mouse we have recently generated.******Aim 3: To determine the function of putative lipid-binding domains of FGD5. The function of conserved PH and FYVE domains is not known. We will use domain-deletants and select point mutants of FGD5 expressed in EC to study FGD5 regulation of i) sprouting, ii) PI3K activity, and iii) subcellular localization.******Significance: Trainees will gain knowledge of vascular biology and reveal new insight into regulation of differential outputs of the GPCR PI3K pathway vs VEGF receptor signals in development, to inform new angiogenesis inhibitor drug discovery.*****