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)受体信号协同作用，刺激已建立的血管内皮细胞(EC)的分化，并启动新的血管生长。我们关注的是磷脂酰肌醇3激酶(PI3K)信号通路。内皮细胞PI3K活性对小鼠新生血管的生长既是必需的，也是限速的。我们也知道，血管内皮生长因子对血管内皮细胞的刺激是不够的，因为在多种血管内皮细胞选择性GPCR基因敲除的小鼠中都可以看到血管生长障碍，包括CXCR4和APLNR。了解与血管内皮生长因子受体协同信号的细节，将揭示胚胎和成人肿瘤血管中血管生成EC分化和组装的新机制。*在新血管萌发过程中，引线或“尖端”EC的分化包括伸出“手指”(即丝状足)以采样组织环境，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亚型在EC中与促血管生成GPCRs结合在TIP细胞生成中的关键作用。我们建议在这一应用中检测PI3K信号蛋白复合体的组装，以及Rho GTP结合蛋白交换因子FGD5对PI3K-b活性的调节。*目标1：表征EC中PI3K-b的促血管生成GPCR偶联。我们将确定CXCR4是否与PI3K-b组装在一个复合体中，以及PI3K-b信号输出是否与内体和质膜活性不同，从而引发i)发芽和血管生成输出，ii)细胞骨架重塑，iii)基因表达。*我们将确定APLNR是否在促血管生成配体Apelin的刺激下，在EC中表现为类似于激活的CXCR4。我们将比较APLNR和另一种血管生成抑制配体apela的作用。*目标2：表征rac1gef，FGD5，如何调节内皮PI3K活性。我们将确定FGD5 i)是否被内体上的PI3K-b vs-a复合体招募，ii)Rhogef的活性调节PI3K-b vs a，利用结构域缺失的FDG5突变体在内皮细胞中的表达，iii)通过rac1-GTP与PI3K-b结合直接调节PI3K-b。*我们将利用我们最近建立的一种新的FGD5条件基因敲除小鼠，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可能的脂结合域的功能。保守的PH和FYVE结构域的功能尚不清楚。我们将使用在EC中表达的FGD5的结构域缺失和选择点突变来研究FGD5对I)萌芽、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，并揭示GPCRPI3K途径的差异输出与开发中的血管内皮生长因子受体信号之间的差异输出的调节，从而为新的血管生成抑制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.*****