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

我们试图了解促血管生成G蛋白偶联受体（GPCR）信号如何与血管内皮生长因子（VEGF）受体信号合作，刺激血管内皮细胞（EC）的分化，并启动新血管的生长。我们的重点是磷酸肌肽3激酶（PI3K）信号通路。内皮PI3K活性是小鼠新生血管生长的必要条件和速率限制。我们也知道vegf对EC的刺激是不够的，因为在多种EC选择性gpcr敲除小鼠（包括CXCR4和APLNR）中可以看到血管生长功能障碍。了解与VEGF受体合作信号传导的细节将揭示胚胎血管生成EC分化和血管组装的新机制，以及成人肿瘤血管。在新血管发芽期间，铅状或“尖端”EC的分化包括“手指”（即丝状足）向组织环境的突出，以及可以在3D培养中重现的基因表达。我们早期的工作已经确定了PI3K-b异构体的关键作用，它与EC中促血管生成的gpcr偶联，用于尖端细胞的生成。在这个应用中，我们建议研究PI3K信号蛋白复合物的组装，以及Rho gtp结合蛋白交换因子FGD5对PI3K-b活性的调节。目的1：表征EC中促血管生成GPCR与PI3K-b的偶联。我们将确定CXCR4是否与PI3K-b在复合体中组装，以及PI3K-b信号输出是否与内核体和质膜活性不同，从而引发i)发芽，ii)细胞骨架重塑的血管生成输出，以及iii)基因表达。我们将确定applnr在促血管生成配体Apelin的刺激下，在EC中的行为是否与活化的CXCR4相似。我们将对比applnr与另一种血管生成抑制配体APELA的效果。目的2：表征Rac1GEF、FGD5如何调节内皮细胞PI3K活性。我们将确定FGD5 i)是否被招募到内体的PI3K-b vs -a复合体，ii) RhoGEF活性调节PI3K-b vs . a，利用ECs中结构域缺失的FDG5突变体的表达，iii)通过与PI3K-b结合的Rac1-GTP直接调节PI3K-b。我们将利用我们最近培育的新型FGD5条件敲除小鼠，研究内皮选择性FGD5功能丧失对体内血管生成的影响。目的3：确定FGD5脂质结合结构域的功能。保守的PH和FYVE结构域的功能尚不清楚。我们将使用结构域删除剂和选择FGD5在EC中表达的点突变体来研究FGD5对i)发芽，ii) PI3K活性和iii)亚细胞定位的调节。意义：学员将获得血管生物学知识，揭示GPCR PI3K通路与VEGF受体信号的差异输出调控的新见解，为新的血管生成抑制剂药物的发现提供信息。

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.