ELTD1 is a highly conserved, non-canonically signalling adhesion GPCR regulating NOTCH, KIT and endothelial sprouting


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David Favara1,Madhulika Nambiar2,Helen Sheldon3,Massimo Masiero4,Demin Li4,Ines Liebscher5,Ali Jazayeri2,Alison Banham4,Adrian Harris3
1,2Heptares Therapeutics, UK,3Department of Oncology, University of Oxford,4NDCLS, Radcliffe Department of Medicine, University of Oxford,5Institute of Biochemistry, Medical Faculty, University of Leipzig

Abstract

Background

ELTD1/ADGRL4, an orphan family 1 adhesion GPCR (aGPCR) expressed in endothelial cells, was identified as a novel regulator of angiogenesis and a potential anti-cancer therapeutic target by our laboratory. How ELTD1 functions remains unknown. Our aim was to analyse ELTD1’s function in endothelial cells.

Method

62 sequenced vertebrate genomes were interrogated for ELTD1. Conservation and domain alterations were analysed. A phylogenetic timetree was assembled to establish time estimates for ELTD1’s evolution. An ELTD1 silencing mRNA array was performed on primary human umbilical-vein endothelial cells (HUVEC), validated with qPCR and FACS. Stable codon-optimised ELTD1 overexpressing HUVEC cell lines were generated, validated with qPCR, WB and FACS. Functional angiogenesis assays and a mRNA microarray were performed. For signalling, stable ELTD1 overexpressing HUVECS and transiently transfected HEK293TS were assayed with FRET and luciferase-reporter systems.

Results

ELTD1 arose 435 million years ago (mya) in bony fish and is present in all subsequent vertebrates. Due to a deletion approximately 177mya, ELTD1 has 2 evolutionary variants: variant with 3 EGFS and a variant with 2 EGFS. Additionally, ELTD1 may be ancestral to members of aGPCR family 2. HUVEC ELTD1 silencing revealed effects on NOTCH (DDL4 upregulation, JAG1 and HES2 downregulation). Silencing also upregulated KIT. HUVEC ELTD1 overexpression revealed that ELTD1 promotes vessel sprouting, inhibits lumen anastomoses in mature vessels, and lowers proliferation rate. There was no effect on cell migration or adhesion. Gene expression changes included upregulation of angiogenesis associated ANTRX1 & ANGPT2 and downregulation of migration associated CCL15 and CYP26B. KIT & DLL4 were downregulated. Signalling experiments revealed that ELTD1 does not couple to canonical GPCR pathways.

Conclusion

ELTD1 has two evolutionary variants (both highly conserved) which regulate NOTCH genes, ANTRX1, ANGPT2, CCL15, CYP26B, regulating endothelial function. Repression of KIT suggests a role maintaining endothelial differentiation. Unlike other aGPCRS, ELTD1’s signalling is non-canonical, requiring further investigation.