CXCR7 protein, human

Agonist-mediated internalization of ACKR3 was measured by BRET2 between ACKR3_RlucII and rGFP_CAAX (a gift from M. Bouvier, Université de Montréal) as previously described^{24 (link)}. Samples were prepared as described for β-arrestin2 recruitment time courses with the exception of the transfected DNA amounts. HEK293 cells were transfected with 42 ng ACKR3_RlucII and 170 ng rGFP_CAAX DNA, with empty pcDNA3.1 to bring the total DNA amount to 2.5 µg/well. Data presented in Figs. 1, 3, and 4C were collected with a PerkinElmer Victor Luminometer, while the time courses in Fig. 4D and 7 were measured on a Tecan Spark luminometer. All settings were identical to those used for arrestin association (described above). Data is presented as percent change compared to mock treated wells and are a composite of three independent experiments. The percent changes after 30 min were compared for statistical analysis.

Expression and purification of ACKR3 from Sf9 cells was performed as previously described^{27 (link), 56 (link)}. Briefly, Sf9 cells were transfected with ACKR3 or CXCL12_LRHQ, a high-affinity variant of CXCL12 with residues 1–3 replaced by the motif LRHQ, in pFasbBac vectors using X-tremeGene transfection reagent (Roche) to produce baculovirus. The receptors and CXCL12_LRHQ were co-expressed by infecting Sf9 cells at a density 2×10⁶ cells/ml with a multiplicity of infection (MOI) of 6 for each virus. After 48 hours, cell pellets were collected and stored at −80 °C. Membranes were prepared by dounce-homogenization in hypotonic buffer (10 mM HEPES pH 7.5, 10 mM MgCl₂, 20 mM KCl) and then three times with hypotonic buffer with 1 M NaCl. Between each cycle of douncing, the membranes were pelleted by centrifugation at 50k x g for 30 min and resuspended. The prepared membranes were then solubilized in 50 mM HEPES pH 7.5, 400 mM NaCl, 0.75%/0.15% dodecyl maltoside/cholesteryl hemisuccinate (DDM/CHS) with 2 mg/ml iodoacetamide and a Protease Inhibitor tablet (Roche). After 4 hours, the insoluble material was removed by 50k x g centrifugation for 30 min and the supernatant was added to Talon resin (Clontech) with 20 mM imidazole to bind overnight at 4°C. The resin was transferred to a column and washed with WB1 (50 mM HEPES pH 7.5, 400 mM NaCl, 0.1/0.02% lauryl maltose neopentyl glycol (LMNG)/CHS, 10% glycerol, 20 mM imidizole), followed by WB2 (WB1 with 0.025/0.005% LMNG/CHS), and finally eluted with EB (WB2 with 250 mM imidizole). The elutions were pooled and concentrated to 500 µl before passing over a PD MiniTrap G-25 desalting column (GE Healthcare) equilibrated with 50 mM HEPES pH 7.5, 100 mM NaCl, 0.025/0.005% LMNG/CHS, 10% glycerol. The final protein concentration was calculated using an A₂₈₀ extinction coefficient of 85000, snap frozen in liquid nitrogen, and stored at −80°C until use.

Phosphosite mapping was performed at the Purdue University Proteomics Facility. Briefly, ACKR3 in LMNG or nanodisc was first phosphorylated by GRK2 and GRK5 and then digested with trypsin. The fragments were analyzed via high-resolution MS without TiO₂ enrichment, and phosphorylation sites identified through peptide ionization patterns compared to the non-phosphorylated primary amino acid sequence.