Fig 1.
Characterization of ORF74-Rluc8.
HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-Rluc8 (Rluc8) or empty vector DNA (mock-transfected). (A) Cell surface expression was determined by ELISA. (B-D) Whole cell binding experiments with 100 pM 125I-CXCL8 (B, C) or 125I-CXCL10 (D) were performed in the presence of increasing concentrations unlabeled CXCL1 (B), CXCL8 (C) or CXCL10 (D). HEK293T cells transfected with ORF74-Rluc8 (E) or WT-ORF74 (F) were stimulated with increasing concentrations of CXCL1 (open squares), CXCL8 (filled squares) or CXCL10 (open circles) and InsP accumulation was quantified. Data are shown as the mean ± SEM of at least three independent experiments each performed in triplicate and are presented as fold over mock-transfected cells (dotted line) (A), percentage of specific 125I-CXCL8 (B, C) or 125I-CXCL10 binding (D) or fold over basal (E, F). Statistical differences in cell surface expression (A) were determined by a Student t test. NS = not significant.
Table 1.
Pharmacological characterization of ORF74-Rluc8.
Fig 2.
ORF74 recruits β-arrestin1 and β-arrestin2 in response to human chemokines.
HEK293T cells co-expressing ORF74-Rluc8 and β-arrestin1-eYFP (A, C) or β-arrestin2-eYFP (B, D) were stimulated with increasing concentrations of CXCL1 (open squares), CXCL8 (filled squares) or CXCL10 (open circles) (A, B) or co-stimulated with CXCL1 and CXCL10 (C, D). β-arrestin recruitment to the receptor was measured as an increase in BRET ratio (BRETr). Data are shown as fold over basal and represent the mean of pooled data from at least three independent experiments each performed in triplicate. Error bars indicate SEM values. Significant differences between vehicle and chemokine-stimulation were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001). NS = not significant.
Fig 3.
Characterization and β-arrestin recruitment to ORF74-R3.50A.
(A, B) HEK293T cells were transiently transfected with WT-ORF74 (WT), ORF74-R3.50A (R3.50A) or empty vector DNA (mock-transfected). Relative receptor expression at the cell surface was determined by ELISA (A) and constitutive activation of PLC was determined by measuring InsP accumulation (B). Data are presented as fold over mock-transfected cells (dotted line). (C, D) HEK293T cells expressing ORF74-Rluc8 (WT) (filled circles) or ORF74-R3.50A-Rluc8 (R3.50A) (open squares) in combination with β-arrestin1-eYFP (C) or β-arrestin2-eYFP (D) were stimulated with increasing concentrations of CXCL1. Data are shown as fold over basal. All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (A) or constitutive PLC activation (B) between WT-ORF74 and ORF74-R3.50A were determined by a Student t test (**** p ≤ 0.0001, *** p ≤ 0.001).
Fig 4.
Characterization and β-arrestin recruitment to ORF74-ST/A.
(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine in ORF74-ST/A are shown in bold brown. The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B-F) HEK293T cells were transiently transfected with WT-ORF74 (WT) (B-E) or ORF74-ST/A (ST/A) (B-F) or empty vector DNA (mock-transfected) (B, E). (B) Relative receptor expression at the cell surface was determined by ELISA. Binding of 125I-CXCL10 (C) or 125I-CXCL8 (D) to intact HEK293T cells was measured in the presence of increasing concentrations unlabeled homologous chemokines. Constitutive (E) or chemokine-induced (F) activation of PLC was determined by measuring InsP accumulation. (G) HEK293T cells expressing ORF74-Rluc8 (WT) or ORF74-ST/A-Rluc8 (ST/A) in combination with β-arrestin1-eYFP (βarr1) or β-arrestin2-eYFP (βarr2) were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) before measurement of BRET. Data are presented as fold over mock-transfected cells (dotted line) (B, E), percentage of specific binding (C, D) or fold over basal (F, G). All data are represented as the mean of pooled data from at least three independent experiments each performed in triplicate and error bars indicate SEM values. Statistical differences of cell surface expression (B) or constitutive PLC activation (E) between WT-ORF74 and ORF74-ST/A or between vehicle- and corresponding CXCL1-treated cells (G) were determined by a Student t test (**** p ≤ 0.0001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.
Fig 5.
Serines and threonines at the distal end of the C-tail are essential for β-arrestin recruitment.
(A) Schematic representation of the C-tail of ORF74, starting at the conserved VPxxY-motif in TM7. Serine and threonine residues mutated to alanine are shown in bold brown and clustered to indicate the different ORF74-ST/A mutants (ST/A1, ST/A2 and ST/A3). The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) [35]. (B) HEK293T cells were transiently transfected with ORF74-Rluc8 (WT), ORF74-ST/A1-Rluc8 (ST/A1), ORF74-ST/A2-Rluc8 (ST/A2) or ORF74-ST/A3-Rluc8 (ST/A3) or empty vector DNA (mock-transfected) and receptor cell surface expression was determined by ELISA. (C-F) HEK293T cells expressing ORF74-Rluc8 (WT) or one of the Rluc8-tagged ORF74-ST/A mutants in combination with β-arrestin1-eYFP (C, E) or β-arrestin2-eYFP (D, F) were treated with increasing concentrations CXCL1 (C, D) or were vehicle-stimulated (white bars) or stimulated with 300 nM CXCL1 (black bars) (E, F) before measurement of BRET. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over mock-transfected cells (dotted line) (B) or fold over basal (C-F) and error bars indicate SEM values. Statistical differences between ORF74 WT and mutant cell surface expression (B) or difference between vehicle- and corresponding CXCL1-treated cells (E, F) were determined by one-way ANOVA followed by a Bonferroni test (B) or a Student t test (E, F), respectively (**** p ≤ 0.0001, ** p ≤ 0.01). NS = not significant.
Fig 6.
Homology model of the phosphorylated ORF74 C-tail bound to β-arrestin1.
(A) Sequence alignment of V2R, CCR5, CXCR2, and ORF74 with a focus on serine (S) and threonine (T) residues (bold, brown) and aspartate (D) and glutamate (E) residues (bold). Underlined S/T residues are experimentally determined to be phosphorylated [37–45] or are phosphorylated in the active β-arrestin1 crystal structure [20]. The location of TM7 (delineated) and helix 8 (marked red) are based on the CCR5 crystal structure (PDB-code 4MBS) (35). Residues of the V2R-peptide solved in the active β-arrestin1 crystal structure are marked light green (the grey residues were not solved in the crystal structure), and the final 19 C-terminal residues of ORF74 that were used to build the model are marked light blue. (B) 3D model of the C-tail of ORF74 (blue) based on the crystal structure of the C-tail of V2R (green) in β-arrestin1 (gold) (PDB-code 4JQI). Spheres indicate the Cα-atoms of S/T residues of V2R and ORF74. A detailed view of S335/S338 (C) and T341/T342 (D) interacting with β-arrestin1 highlighting interactions between phosphorylated S/T residues in ORF74 with several key residues [36] in β-arrestin1 (i.e. K10, K11, K107, R165, K294). Dashed lines indicate H-bonds or ionic interactions. β-arrestin1-carbon, ORF74-carbon, nitrogen, oxygen, and phosphate atoms are colored gold, slate, blue, red, and orange respectively. Water molecules are depicted as red spheres.
Fig 7.
ORF74 internalizes and traffics via early, recycling and late endosomes.
HEK293T cells were transiently transfected with ORF74-Rluc8 (WT) (A-D) or ORF74-ST/A2-Rluc8 (ST/A2) (E-H) in combination with Venus-K-Ras (plasma membrane marker) (A, E), Venus-Rab5a (early endosome marker) (B, F), Venus-Rab7a (late endosome/lysosome marker) (C, G) or Venus-Rab11 (recycling endosome marker) (D, H) and stimulated with CXCL1, CXCL8 or CXCL10 for indicated time and BRET was measured. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over vehicle-stimulated cells (dotted line) and error bars indicate SEM values. Statistical differences between the area under the curve of vehicle- and corresponding CXCL1-, CXCL8- or CXCL10-treated cells (baseline = 1) were determined by one-way ANOVA followed by a Bonferroni test (**** p ≤ 0.0001, *** p≤ 0.001, ** p ≤ 0.01, * p ≤ 0.05). NS = not significant.
Fig 8.
ORF74 trafficking is β-arrestin-dependent.
HEK293T cells were transiently transfected with ORF74-Rluc8 and Venus-K-Ras (plasma membrane marker) (A, C) or Venus-Rab5a (early endosome marker) (B, D) in combination with control (Contr) or β-arrestin1/2 (βarr1/2) siRNA. (A, B) Downregulation of β-arrestin1/2 levels was determined by immunoblotting. STAT3 levels were determined as loading control. (C, D) Cells were stimulated with CXCL1 for indicated time and BRET was measured. Data are shown as the mean of pooled data from three independent experiments each performed in triplicate. Data is presented as fold over vehicle-stimulated cells (dotted line) and error bars indicate SEM values. Statistical differences between the area under the curve of cells treated with control or β-arrestin1/2 siRNA (baseline = 1) were determined by a Student t test (** p ≤ 0.01).