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

我们试图了解促血管生成的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，以刺激内皮细胞（EC）内衬已建立的血管的分化，并启动新的血管生长。我们专注于磷脂酰肌醇3激酶（PI 3 K）信号通路。内皮PI 3 K活性对于小鼠中的新血管生长是必需的并且是速率限制性的。我们也知道VEGF刺激EC是不够的，因为在各种EC选择性GPCR敲除小鼠中观察到功能障碍的血管生长，包括CXCR 4和APLNR。了解与VEGF受体的合作信号的细节将揭示血管生成EC分化和组装的血管在胚胎中的新机制，并在成人的癌血管。 在新血管发芽期间，引线或“尖端”EC的分化包括“指状物”（即丝状伪足）的突出以取样组织环境，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PI 3 K-b亚型的关键作用，其与EC中用于尖端细胞生成的促血管生成GPCR偶联。我们建议检查的PI 3 K信号蛋白复合物的组装，和调节PI 3 K-b活性的Rho GTP结合蛋白交换因子，FGD 5，在本申请中。 目的1：表征EC中促血管生成GPCR与PI 3 K-b的偶联。我们将确定CXCR 4是否与PI 3 K-b组装成复合物，以及PI 3 K-b信号输出是否不同于内体与质膜活性，以引发i）发芽和ii）细胞骨架重塑的血管生成输出，以及iii）基因表达。 我们将确定是否APLNR，刺激促血管生成配体，爱帕琳，行为类似于激活CXCR 4在EC。我们将对比APLNR与替代的血管生成抑制配体APELA的作用。 目的2：研究Rac 1GEF，FGD 5如何调节内皮细胞PI 3 K活性。我们将确定FGD 5 i）是否被募集到内体处的PI 3 K-b vs -a复合物，和ii）RhoGEF活性是否调节PI 3 K-b vs a，利用EC中结构域缺失的FDG 5突变体的表达，iii）是否通过Rac 1-GTP结合PI 3 K-b直接调节PI 3 K-b。 我们将研究内皮选择性FGD 5功能丧失对体内血管生成的影响，利用我们最近产生的一种新的FGD 5条件性敲除小鼠。 目的3：确定FGD 5的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EC中表达的FGD 5的结构域缺失体和选择点突变体来研究FGD 5对i）发芽、ii）PI 3 K活性和iii）亚细胞定位的调节。 重要性：学员将获得血管生物学知识，并揭示对GPCR PI 3 K通路与VEGF受体信号差异输出调节的新见解，以告知新的血管生成抑制剂药物发现。

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.