Fig. 6. Uptake of opioid peptides and atypical localization and trafficking of ACKR3.

a Uptake of dynorphin A (1–13) by ACKR3-expressing cells visualized by imaging flow cytometry. Left panels: U87, U87-ACKR3 or U87-ACKR3 cells pretreated with LIH383 (3 µM) stimulated with (FAM)-labeled dynorphin A (1–13) (250 nM, dA13-FAM, green channel). Five representative cells per condition are shown. Scale bar: 10 µm. Right panel: Percentage of cells with a given number of distinguishable vesicle-like structures (spots) and the geometrical mean fluorescence intensity (MFI, green channel) (inset). Data are representative of three independent experiments. b Uptake of opioid peptides (dynorphin A (1–13)-FAM (250 nM), big dynorphin-Cy5 (400 nM), BAM22-Cy5 (400 nM) or nociceptin-FAM (1 µM)) by U87 cells (NT) or U87 cells transfected with ACKR3 or classical opioid receptors analyzed by imaging flow cytometry as described in a. c ACKR3-mediated depletion of extracellular dynorphin A. U87 or U87-ACKR3 cells pretreated with LIH383 (400 nM) or LIH383ctrl were incubated with dynorphin A. Cell supernatant was added on U87 cells expressing KOR-SmBiT and LgBiT-beta-arrestin-1. (inset): EC₅₀ values. d Cellular localization of ACKR3 and classical opioid receptors fused to Neongreen fluorescent protein (green) stimulated or not by opioid peptides (1 µM) monitored by fluorescent confocal microscopy. Nuclear DNA was Hoechst-stained (blue). Pictures are representative of 10 acquired images from two independent experiments. Scale bar: 5 µm. e Ligand-induced receptor-arrestin delivery to endosomes monitored by β-galactosidase complementation assay in U2OS cells stably expressing ACKR3. Results are expressed as mean ± SD of three technical replicates. f–h Kinetics of ligand-induced internalization of ACKR3 and classical opioid receptors (f and h, HiBiT technology) and (g, flow cytometry). f U87 cells expressing N-terminally HiBiT-tagged receptors were stimulated with opioid peptides (1 µM) or CXCL12 (300 nM) for indicated times. Remaining membrane receptors were quantified with soluble LgBiT protein. g ACKR3 and KOR internalization and recycling in U87 cells after ligand stimulation (1 µM for opioid peptides, 300 nM for CXCL12) followed by acid wash, monitored by flow cytometry. h Effect of bafilomycin A1 (1.5 µM) on endosomal trafficking/cycling of ACKR3 and KOR following ligand stimulation (1 µM) monitored by HiBiT technology. If not otherwise indicated, results are presented as mean ± S.E.M of three to five independent experiments (n = 3 to 5). *p < 0.05, **p < 0.01, ***p < 0.001 by one-way ANOVA with Bonferroni correction (b), by two-way ANOVA: interaction between cell line and LIH383 treatment with Tukey’s post hoc test (c), and by two-tailed unpaired t-test (h). Source data and statistical analysis parameters are provided as Source Data file.