Abstract
The atypical chemokine receptor 3, ACKR3, is a G protein-coupled receptor, which does not couple to G proteins but recruits βarrestins. At present, ACKR3 is considered a target for cancer and cardiovascular disorders, but less is known about the potential of ACKR3 as a target for brain disease. Further, mouse lines have been created to identify cells expressing the receptor, but there is no tool to visualize and study the receptor itself under physiological conditions. Here, we engineered a knock-in (KI) mouse expressing a functional ACKR3-Venus fusion protein to directly detect the receptor, particularly in the adult brain. In HEK-293 cells, native and fused receptors showed similar membrane expression, ligand induced trafficking and signaling profiles, indicating that the Venus fusion does not alter receptor signaling. We also found that ACKR3-Venus enables direct real-time monitoring of receptor trafficking using resonance energy transfer. In ACKR3-Venus knock-in mice, we found normal ACKR3 mRNA levels in the brain, suggesting intact gene transcription. We fully mapped receptor expression across 14 peripheral organs and 112 brain areas and found that ACKR3 is primarily localized to the vasculature in these tissues. In the periphery, receptor distribution aligns with previous reports. In the brain there is notable ACKR3 expression in endothelial vascular cells, hippocampal GABAergic interneurons and neuroblast neighboring cells. In conclusion, we have generated Ackr3-Venus knock-in mice with a traceable ACKR3 receptor, which will be a useful tool to the research community for interrogations about ACKR3 biology and related diseases.
Highlights
G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors
ACKR3‐Venus lacks G protein activity but recruits βarrestins To test the functionality of the ACKR3-Venus construct used to generate the KI mice, the ACKR3 receptor genetically fused to the fluorescent protein, Venus-GFP (Venus), at its C-tail was expressed in HEK-293 cells and it signaling properties compared with that of the wildtype (WT) ACKR3
Cells transfected with the delta opioid receptor (DOR) were used as a positive control for the assay and showed robust G protein activation for the Gi/Go family members upon treatment with MetEnkephalin (Fig. 1B and Additional file 1: Figure S1A)
Summary
G protein-coupled receptors (GPCRs) constitute the largest family of cell surface receptors. They are involved in a wide diversity of cellular and physiological processes and contribute to disease. GPCRs are the target of 35% of commercially marketed drugs [1]. Since this family of Ehrlich et al Mol Brain (2021) 14:151 to scavenge excess CXCL11 [8]. CXCR4 and ACKR3 binding to endogenous ligand CXCL12 has been shown to contribute to tumor growth, neovascularization, neurogenesis and brain inflammation [9,10,11]. ACKR3 ligands are considered promising drugs in the context of cancer and neuroinflammation yet mechanistic understanding of ACKR3 biology is incomplete, and the potential of ACKR3 as a therapeutic target has not been fully exploited
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