Elevated expression of the adhesion GPCR ADGRL4/ELTD1 promotes endothelial sprouting angiogenesis without activating canonical GPCR signalling

David M Favara,Madhulika Nambiar,Adrian L Harris,Ines Liebscher,Ali Jazayeri,Helen Sheldon,Alison H Banham

doi:10.1038/s41598-021-85408-x

Abstract

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.

Highlights

ADGRL4/ELTD1 is an orphan adhesion GPCR which is expressed in endothelial c ells[1,2] and smooth muscle cells[3] where it regulates both physiological and tumour angiogenesis[4]
Wild type (WT) full length (FL) ADGRL4/ELTD1 was codon optimised retaining its amino acid coding sequence and increasing its GC content to 58%, a level higher than the majority of compared receptors (Figure S1A) with the resulting co FL ADGRL4/ELTD1 producing a much higher level of protein expression compared to the original sequence when transfected into HEK293T cells. (Fig. 1A)
We investigated whether a peptide analogous to ADGRL4/ELTD1’s putative tethered agonist region could elicit canonical GPCR signalling. 21 peptides of varying length corresponding to the amino acid sequence beginning at ADGRL4/ELTD1’s consensus GPCR-proteolysis site (GPS) cleavage site and extending C-terminally to the predicted start of the first 7TM loop (Fig. 2A) were tested

Summary

Introduction

ADGRL4/ELTD1 is an orphan adhesion GPCR which is expressed in endothelial c ells[1,2] and smooth muscle cells[3] where it regulates both physiological and tumour angiogenesis[4]. Cells in several common tumour types (head and neck, renal, colorectal, ovarian and brain cancers)[4,5] as well as in glioblastoma tumour cells[9]. It is not expressed in the majority of tumour cell lines[10]. A potential explanation is that ADGRL4/ELTD1 is associated with a vascular stabilising effect with better tumoural drug delivery. This makes ADGRL4/ELTD1 a complex target to consider for therapy. We tested whether the Stachel hypothesis applied to ADGRL4/ELTD1, and investigated the effect of ADGRL4/ELTD1 upregulation on angiogenesis

Methods

Results

Conclusion