Balliol College, University of Oxford, Oxford, OX1 3BJ, UK.
Department of Oncology and Weatherall Institute of Molecular Medicine, University of Oxford, Oxford, OX3 7DQ, UK.
Sci Rep. 2021 Apr 23;11(1):8870. doi: 10.1038/s41598-021-85408-x.
ADGRL4/ELTD1 is an orphan adhesion GPCR (aGPCR) expressed in endothelial cells that regulates tumour angiogenesis. The majority of aGPCRs are orphan receptors. The Stachel Hypothesis proposes a mechanism for aGPCR activation, in which aGPCRs contain a tethered agonist (termed Stachel) C-terminal to the GPCR-proteolytic site (GPS) cleavage point which, when exposed, initiates canonical GPCR signalling. This has been shown in a growing number of aGPCRs. We tested this hypothesis on ADGRL4/ELTD1 by designing full length (FL) and C-terminal fragment (CTF) ADGRL4/ELTD1 constructs, and a range of potential Stachel peptides. Constructs were transfected into HEK293T cells and HTRF FRET, luciferase-reporter and Alphascreen GPCR signalling assays were performed. A stable ADGRL4/ELTD1 overexpressing HUVEC line was additionally generated and angiogenesis assays, signalling assays and transcriptional profiling were performed. ADGRL4/ELTD1 has the lowest GC content in the aGPCR family and codon optimisation significantly increased its expression. FL and CTF ADGRL4/ELTD1 constructs, as well as Stachel peptides, did not activate canonical GPCR signalling. Furthermore, stable overexpression of ADGRL4/ELTD1 in HUVECs induced sprouting angiogenesis, lowered in vitro anastomoses, and decreased proliferation, without activating canonical GPCR signalling or MAPK/ERK, PI3K/AKT, JNK, JAK/HIF-1α, beta catenin or STAT3 pathways. Overexpression upregulated ANTXR1, SLC39A6, HBB, CHRNA, ELMOD1, JAG1 and downregulated DLL4, KIT, CCL15, CYP26B1. ADGRL4/ELTD1 specifically regulates the endothelial tip-cell phenotype through yet undefined signalling pathways.
ADGRL4/ELTD1 是一种在血管内皮细胞中表达的孤儿黏附 GPCR（aGPCR），它可以调节肿瘤血管生成。大多数 aGPCR 是孤儿受体。Stachel 假说提出了一种 aGPCR 激活的机制，其中 aGPCR 包含一个连接的激动剂（称为 Stachel），位于 GPCR 蛋白水解位点（GPS）切割点的 C 端，当暴露时，它会启动典型的 GPCR 信号传导。越来越多的 aGPCR 已经证明了这一点。我们通过设计全长（FL）和 C 末端片段（CTF）ADGRL4/ELTD1 构建体以及一系列潜在的 Stachel 肽来测试 ADGRL4/ELTD1 上的这个假设。将构建体转染到 HEK293T 细胞中，并进行 HTRF FRET、荧光素酶报告基因和 AlphaScreen GPCR 信号转导测定。此外，还生成了稳定过表达 ADGRL4/ELTD1 的 HUVEC 系，并进行了血管生成测定、信号转导测定和转录谱分析。ADGRL4/ELTD1 在 aGPCR 家族中的 GC 含量最低，密码子优化显著提高了其表达。FL 和 CTF ADGRL4/ELTD1 构建体以及 Stachel 肽均不能激活典型的 GPCR 信号。此外，ADGRL4/ELTD1 在 HUVEC 中的稳定过表达诱导了发芽血管生成，降低了体外吻合度，并降低了增殖，而没有激活典型的 GPCR 信号或 MAPK/ERK、PI3K/AKT、JNK、JAK/HIF-1α、β连环蛋白或 STAT3 途径。过表达上调了 ANTXR1、SLC39A6、HBB、CHRNA、ELMOD1、JAG1，并下调了 DLL4、KIT、CCL15、CYP26B1。ADGRL4/ELTD1 通过尚未定义的信号通路特异性调节内皮尖端细胞表型。
