Proposed model for regulation of CXCR7 trafficking.

<p>CXCR7 requires ubiquitination of the Lys residues of its C-tail in order to reach the cell surface. Receptor activation by CXCL12 and subsequent phosphorylation of the C-terminal Ser/Thr residues results in β-arrestin recruitment by CXCR7 and receptor internalization in CCPs. In addition, β-arrestin scaffolds the interaction of CXCR7 with an unknown de-ubiquitinating enzyme (DUB) responsible for receptor deubiquitination. After chemokine degradation in early endosomes and due to the transient interaction of CXCR7 with β-arrestin, release of β-arrestin (and DUB) from the endocytosed receptor results in a CXCR7 able to undergo ubiquitination by a specific E3 ligase (E3) and subsequent delivery of the recycled receptor to the cell surface.</p

Real-time monitoring of receptor ubiquitination using BRET².

<p>HEK293T cells were transfected with Ub-GFP² (white bars) or (G75A,G76A)-Ub-GFP² (filled bars) and (<b>A</b>) CXCR7-RLuc, CXCR7 ΔC-RLuc, or CXCR7 ST/A-Rluc, (<b>B</b>) CXCR4-RLuc, or (<b>C</b>) CXCR3-RLuc. BRET² was measured 30 min after stimulation with 10⁻⁸ M of CXCL12 (CXCL11 for CXCR3) by addition of coelenterazine 400a and immediate read out. Results are expressed in Net BRET normalized to basal as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>. Data represent the mean ± SEM of 3 experiments each performed in triplicate. **, p<0.01, and ***, p<0.001, by Student t test.</p

The C-terminus of CXCR7 is constitutively ubiquitinated.

<p><b>(A) CXCR7 gets deubiquitinated by CXCL12-stimulation.</b> HEK293T cells were transfected as indicated and processed for immunoprecipitation of the HA-Ub (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>). (<b>A</b>) CXCR7 was stimulated with 10⁻⁸ M CXCL12 for 30 min, and removal of CXCL12 was performed by two washes of the cells and additional 30 min incubation with fresh chemokine-free media. Detection of the immunoprecipitated CXCR7 was done with the 11G8 antibody. HA-Ub expression was confirmed by blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the right of the blot. (<b>B</b>) Detection of total CXCR7 protein expression by ELISA in the same cells.</p

(A) CXCR7 internalization depends on CCPs and is G protein-independent.

<p>HEK293T cells were transfected with wt CXCR7 (and β-arrestin (319–418) were indicated) and cell surface levels of the receptor after CXCL12 stimulation was detected by ELISA using the CXCR7-specific antibody 11G8. Incubation with 0.4 M Sucrose was done 30 min prior and during stimulation. PTX was incubated overnight at 25 ng/ml final concentration. <b>(B) β-arrestin1/2 knock-down prevents CXCR7 internalization.</b> HEK293/CXCR7 cells transfected with control siRNAs (white bars) or pools targeting β-arrestin1/2 (filled bars), were stimulated with CXCL12 (10⁻⁸ M) or vehicle for 45 min and receptor surface expression was determined. Knockdown of β-arrestin1 and -2, 48 hrs after transfection, was assessed in western blot using an anti-β–arrestin1/2 antibody (inset). Anti-STAT3 (mAb 79D7, Cell Signaling Technologies) was used as loading control. <b>(C) CXCR7 C-terminus is essential for receptor internalization.</b> HEK293T cells were transfected with wt CXCR7 (filled bars), CXCR7 ΔC (grey bars) or CXCR7 ST/A (white bars) and cell surface receptor levels were assessed as above. Data represent the mean ± SEM of at least 3 experiments each performed in triplicate. ***, p<0.001 by one-way ANOVA and Bonferroni post test.</p

CXCR7/CXCR3 tail switch alters ubiquitination properties of the receptors.

<p>(<b>A</b>) Immunoprecipitation experiments were performed in cells expressing chimeric receptors consisting on CXCR7 with CXCR3 C-terminus (CXCR7-X3) or the reciprocal CXCR3 with CXCR7 C-terminus (CXCR3-X7). Detection of the immunoprecipitated CXCR7 and CXCR3 was done with the 11G8 and mAB160 antibodies, respectively. HA-Ub expression was confirmed blotting lysates using an anti-HA antibody and equal loading was controlled by detection of actin on the same blot. Molecular weight markers (kDa) are indicated on the sides of the blots. (<b>B</b>) Detection of total CXCR7 protein expression by ELISA in the same cells.</p

β-arrestin2 recruitment to CXCR7 is dependent on C-terminal Ser/Thr residues.

<p>(<b>A</b>) CXCL11 or CXCL12-mediated β-arrestin2 recruitment to CXCR7. HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL11 (open circles) or CXCL12 (filled circles) (<b>B</b>) HEK293T co-expressing RLuc-tagged CXCR7 and YFP-tagged β-arrestin2 were incubated overnight with 25 ng/ml of PTX or for 30 min with the CXCR7-specific antibody 8F11 prior to the BRET measurement. (<b>C</b>) CXCL12-induced β-arrestin2 recruitment to CXCR7 wt (filled circles), a truncated CXCR7 lacking the C-terminus (CXCR7 ΔC, filled triangles) or a mutant CXCR7 for which all the Ser and Thr residues were mutated to Ala (CXCR7 ST/A, open squares). HEK293T cells coexpressing <i>RLuc</i>-tagged CXCR7 mutants and YFP-tagged β-arrestin2 were stimulated with increasing concentrations of CXCL12 prior to BRET measurements. Data represent the mean ± SEM of 4 experiments each performed in triplicate. Results are expressed in Net BRET as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034192#s4" target="_blank">Materials and Methods</a>. ***, p<0.001 by one-way ANOVA and Bonferroni post test.</p

CXCR7 recycles to the cell surface after internalization.

<p>(<b>A</b>) HEK293T stably expressing CXCR7 were stimulated with 10⁻⁸ M CXCL11, CXCL12 or vehicle for 45 min or 3 h and fixed immediately. CXCR7 was detected using the specific 11G8 antibody and an Alexa-488-conjugated secondary antibody. Scale bar represents 10 µm. (<b>B</b>) HEK293T cells expressing CXCR7 (filled symbols) or CXCR3 (open symbols) were incubated with CXCL11 (10⁻⁸ M, squares), CXCL12 (10⁻⁸ M, triangles) or vehicle (circles) for the indicated times. Cell surface receptor levels were detected by ELISA using CXCR7- or CXCR3-specific antibodies (11G8 and mAB160, respectively). Results were normalized to basal surface protein levels, and data represent the mean ± SEM of 4 experiments each performed in triplicate. (<b>C</b>) ELISA was performed as in B in cells pre-incubated for 2 h with the <i>de novo</i> protein synthesis inhibitor cycloheximide (10 µg/ml). (<b>D</b>) ELISA performed as in C on intact HEK293/CXCR7 cells treated with vehicle or 1 µM of bafilomycin A1 (Baf A1), 30 min prior to incubation with CXCL12. <b>(E) C-terminal Ser/Thr clusters determine receptor fate after internalization.</b> HEK293T cells were transiently transfected with CXCR7 wt (white bars) or with a chimeric receptor consisting of CXCR7 harboring the C-terminal sequence of CXCR3 (CXCR7-X3, filled bars). To assess the cell surface expression of the receptor, ELISA experiments were performed after 30 min or 3 hours of incubation with 10⁻⁸ M CXCL12. Data represent the mean ± SEM of 3 experiments each performed in triplicate. ***, p<0.001, **, p<0.01, and *, p<0.05 by one-way ANOVA and Bonferroni post test.</p

Model simulation and experimental validation of Wnt-pathway activation upon WNT stimulation.

<p>(A) β-catenin (referred to by its official gene name CTNNB1 in the figure) token levels predicted by our model with initial WNT token levels ranging from 0 to 5. For WNT = 0, 1 or 2, we observed a flat β-catenin response. For WNT = 3, 4 and 5 β-catenin increases from low to moderate levels. (B) Graph combining the results from panels C and D to allow easy comparison to the modeling results depicted in panel (A), showing dose- and time-dependent activation of a Wnt/β-catenin responsive TCF/LEF luciferase reporter in HEK293T^WOO cells. For all curves with black data points (corresponding to panel C), luciferase activity was plotted relative to the vehicle control (not shown), which was set at 1 for each of the three time points (3, 8 and 24 hours). For the curve with white data points (corresponding to panel D), luciferase activity was plotted relative to the vehicle control, which was set at 1 for the t = 0 hours condition. (C) Reporter assay in HEK293T^WOO cells, showing dose-dependent activation at 3, 8 and 24 hours after stimulation with purified Wnt3a (same concentrations as depicted in B). (D) Reporter assay in HEK293T^WOO cells, showing time-dependent activation upon treatment with 100 ng/ml of Wnt3a. Values were plotted relative to the vehicle control, which was set at 1 for t = 0 hours. (E) Western blot from the experiment depicted in (D), showing total and active (i.e. non-phosphorylated) β-catenin levels. Since the soluble, signaling pool of β-catenin constitutes only a minor fraction of the total pool of β-catenin, the use of antibody against active β-catenin ensures that only the pool involved in WNT signaling is visualized. Tubulin was used as a loading control. (F) Quantification of the Western blot shown in (E). Total and active β-catenin levels were normalized to tubulin. The increase in either total or active β-catenin levels was plotted relative to time point 0, for which the normalized levels were set to 1. Experiments were repeated two (C) or three (D-F) times. A representative experiment is shown.</p

Model prediction of Wnt-pathway activation upon WNT addition with AXIN2 feedback.

<p>β-catenin (referred to by its official gene name CTNNB1 in the figure) token levels predicted by our model with arc weight from t11 to AXIN varied from 0 (no feedback; solid lines) to 0.15 (high feedback, dashed lines), and initial WNT token levels at 3, 4 and 5 (top, middle and bottom panels, respectively). We observed three spectra of β-catenin stabilizations depending on initial WNT levels. The highest β-catenin stabilizations correspond to simulations without AXIN2 feedback (solid lines), whereas with high AXIN2 feedback the β-catenin stabilization was attenuated (dashed lines).</p

Illustration of Wnt/β-catenin signaling.

<p>(A) In the absence of an external WNT stimulus β-catenin (referred to by its official gene name CTNNB1 in the figure) is continuously degraded by a ‘destruction complex’ consisting of AXIN1, adenomatous polyposis coli (APC), casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). (B) Extracellular WNT interacts with the membrane-bound receptors frizzled (FZD) and lipoprotein receptor-related protein (LRP). Dishevelled (DVL) interacts with the intracellular tail of FZD and sequesters AXIN1 to the plasma membrane forming a so-called ‘signalosome’. The ensuing depletion of the cytoplasmic pool of AXIN1 inhibits the formation of the destruction complex. β-catenin thereby stabilizes and translocates to the nucleus, where it interacts with TCF/LEF transcription factors activating transcription of specific target genes, including the negative feedback regulator <i>AXIN2</i>.</p