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Control of CXCR2 activity through its ubiquitination on K327 residue.

Leclair HM, Dubois SM, Azzi S, Dwyer J, Bidère N, Gavard J - BMC Cell Biol. (2014)

Bottom Line: First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination.Finally, K327R mutant failed to promote the recruitment of β-arrestin2, as estimated by imagery and bioluminescence resonance transfer.Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8104, 22 rue Mechain, Paris, 75014, France. Julie.gavard@inserm.fr.

ABSTRACT

Background: The interleukin-8 chemokine (IL-8) G-protein coupled receptor CXCR2 governs pro-inflammatory and pro-angiogenic responses in leukocytes and endothelial cells. At a molecular standpoint, CXCR2 is widely reported to operate through calcium flux, phosphoinoisitide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK). While CXCR2 trafficking is suspected to be intertwined with its signaling, the exact mechanism is not fully elucidated.

Results: Here, we identified the lysine 327 within the CXCR2 C-terminal tail as a key residue for ubiquitination, internalization, and signaling. First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination. While WT CXCR2 was rapidly internalized following IL-8 administration, K327R mutant remained at the plasma membrane. Finally, K327R mutant failed to promote the recruitment of β-arrestin2, as estimated by imagery and bioluminescence resonance transfer. As a consequence, the activation of intracellular signaling, including both early events such as ERK phosphorylation and the increase in calcium flux, and the latter activation of the AP1 and NF-κB transcription factors, was blunted.

Conclusions: Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling. Thus, the inhibition of K327 ubiquitination might emerge as an effective mean to curb exacerbated CXCR2 signaling in several pathological conditions, such as inflammatory diseases and cancer.

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K327R mutation abrogates CXCR2 signaling in response to IL-8. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), containing in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. One day later, cells were starved (control) or exposed to IL-8 (50 ng/ml, 15 min, unless specified). a. Anti-phospho-Tyr (4G10), anti-CXCR2 western-blots were performed in CXCR2 immunoprecipitated (IP) fraction. b. Calcium flux was measured using the Fluo4NW probe in response to IL-8 for the indicated times. Fluorescent units were normalized to control conditions. c. Anti-AU5, anti-pERK1/2 and anti-ERK2 western-blots were performed. Pixel intensities of the pERK1/2 lanes were quantified using Image J software and normalized to total ERK2. d. Promoter activity of both NF-κB and AP1 was monitored through luciferase-based assays. Each panel is representative of three independent experiments. Means + sem are shown. **p < 0.01, ***p < 0.001.
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Figure 4: K327R mutation abrogates CXCR2 signaling in response to IL-8. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), containing in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. One day later, cells were starved (control) or exposed to IL-8 (50 ng/ml, 15 min, unless specified). a. Anti-phospho-Tyr (4G10), anti-CXCR2 western-blots were performed in CXCR2 immunoprecipitated (IP) fraction. b. Calcium flux was measured using the Fluo4NW probe in response to IL-8 for the indicated times. Fluorescent units were normalized to control conditions. c. Anti-AU5, anti-pERK1/2 and anti-ERK2 western-blots were performed. Pixel intensities of the pERK1/2 lanes were quantified using Image J software and normalized to total ERK2. d. Promoter activity of both NF-κB and AP1 was monitored through luciferase-based assays. Each panel is representative of three independent experiments. Means + sem are shown. **p < 0.01, ***p < 0.001.

Mentions: We next investigated whether K327 residue participates to CXCR2 signaling ability. To this aim, one of the most proximal events in GPCR activation, namely β-arrestin2 activation was first assessed. Interestingly, K327R single substitution prevented from IL-8-induced β-arrestin2 clustering, observed normally in cells transfected with WT and K337R CXCR2 (Figure 3a-b). Likewise, K327R CXCR2 mutant less efficiently activated β-arrestin2 in response to IL-8, as estimated through Bioluminescence Resonance Energy Transfer (BRET) technology (Figure 3c). As phosphorylation is also an important hallmark of GPCR activation upon ligation [23], we checked the status of CXCR2 tyrosine phosphorylation in immunoprecipitation experiments (Figure 4a). As expected, IL-8 drove WT CXCR2 phosphorylation. In sharp contrast, the signal was barely detectable when K327R mutant was ectopically expressed in HEK-293T (Figure 4a). Secondly, intracellular signaling, namely extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and increase in calcium influx, was evaluated in WT, K327R and K337R CXCR2 expressing HEK-293T cells (Figure 4b-c). Our data showed that IL-8 could not elicit either CXCR2 and ERK1/2 phosphorylation, or the elevation in intracellular calcium concentration in K327R CXCR2-transfected cells. Furthermore, luciferase-based reporter assays unveiled that K327 mutation abrogates AP1 and NF-κB activation downstream of CXCR2 stimulation by IL-8 (Figure 4d). Thus, our data demonstrate that the signaling function of CXCR2 is compromised when the K327 residue is not available for ubiquitination.


Control of CXCR2 activity through its ubiquitination on K327 residue.

Leclair HM, Dubois SM, Azzi S, Dwyer J, Bidère N, Gavard J - BMC Cell Biol. (2014)

K327R mutation abrogates CXCR2 signaling in response to IL-8. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), containing in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. One day later, cells were starved (control) or exposed to IL-8 (50 ng/ml, 15 min, unless specified). a. Anti-phospho-Tyr (4G10), anti-CXCR2 western-blots were performed in CXCR2 immunoprecipitated (IP) fraction. b. Calcium flux was measured using the Fluo4NW probe in response to IL-8 for the indicated times. Fluorescent units were normalized to control conditions. c. Anti-AU5, anti-pERK1/2 and anti-ERK2 western-blots were performed. Pixel intensities of the pERK1/2 lanes were quantified using Image J software and normalized to total ERK2. d. Promoter activity of both NF-κB and AP1 was monitored through luciferase-based assays. Each panel is representative of three independent experiments. Means + sem are shown. **p < 0.01, ***p < 0.001.
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Figure 4: K327R mutation abrogates CXCR2 signaling in response to IL-8. HEK-293T cells were transfected with pCEFL-AU5 plasmid (mock), containing in frame wild type (WT), K327R (R1), and K337R (R2) mutant forms of FL CXCR2. One day later, cells were starved (control) or exposed to IL-8 (50 ng/ml, 15 min, unless specified). a. Anti-phospho-Tyr (4G10), anti-CXCR2 western-blots were performed in CXCR2 immunoprecipitated (IP) fraction. b. Calcium flux was measured using the Fluo4NW probe in response to IL-8 for the indicated times. Fluorescent units were normalized to control conditions. c. Anti-AU5, anti-pERK1/2 and anti-ERK2 western-blots were performed. Pixel intensities of the pERK1/2 lanes were quantified using Image J software and normalized to total ERK2. d. Promoter activity of both NF-κB and AP1 was monitored through luciferase-based assays. Each panel is representative of three independent experiments. Means + sem are shown. **p < 0.01, ***p < 0.001.
Mentions: We next investigated whether K327 residue participates to CXCR2 signaling ability. To this aim, one of the most proximal events in GPCR activation, namely β-arrestin2 activation was first assessed. Interestingly, K327R single substitution prevented from IL-8-induced β-arrestin2 clustering, observed normally in cells transfected with WT and K337R CXCR2 (Figure 3a-b). Likewise, K327R CXCR2 mutant less efficiently activated β-arrestin2 in response to IL-8, as estimated through Bioluminescence Resonance Energy Transfer (BRET) technology (Figure 3c). As phosphorylation is also an important hallmark of GPCR activation upon ligation [23], we checked the status of CXCR2 tyrosine phosphorylation in immunoprecipitation experiments (Figure 4a). As expected, IL-8 drove WT CXCR2 phosphorylation. In sharp contrast, the signal was barely detectable when K327R mutant was ectopically expressed in HEK-293T (Figure 4a). Secondly, intracellular signaling, namely extracellular regulated kinase 1/2 (ERK1/2) phosphorylation and increase in calcium influx, was evaluated in WT, K327R and K337R CXCR2 expressing HEK-293T cells (Figure 4b-c). Our data showed that IL-8 could not elicit either CXCR2 and ERK1/2 phosphorylation, or the elevation in intracellular calcium concentration in K327R CXCR2-transfected cells. Furthermore, luciferase-based reporter assays unveiled that K327 mutation abrogates AP1 and NF-κB activation downstream of CXCR2 stimulation by IL-8 (Figure 4d). Thus, our data demonstrate that the signaling function of CXCR2 is compromised when the K327 residue is not available for ubiquitination.

Bottom Line: First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination.Finally, K327R mutant failed to promote the recruitment of β-arrestin2, as estimated by imagery and bioluminescence resonance transfer.Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: CNRS, UMR8104, 22 rue Mechain, Paris, 75014, France. Julie.gavard@inserm.fr.

ABSTRACT

Background: The interleukin-8 chemokine (IL-8) G-protein coupled receptor CXCR2 governs pro-inflammatory and pro-angiogenic responses in leukocytes and endothelial cells. At a molecular standpoint, CXCR2 is widely reported to operate through calcium flux, phosphoinoisitide 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK). While CXCR2 trafficking is suspected to be intertwined with its signaling, the exact mechanism is not fully elucidated.

Results: Here, we identified the lysine 327 within the CXCR2 C-terminal tail as a key residue for ubiquitination, internalization, and signaling. First, the substitution to an arginine of K327 mutation was associated with a reduction in CXCR2 poly-ubiquitination. While WT CXCR2 was rapidly internalized following IL-8 administration, K327R mutant remained at the plasma membrane. Finally, K327R mutant failed to promote the recruitment of β-arrestin2, as estimated by imagery and bioluminescence resonance transfer. As a consequence, the activation of intracellular signaling, including both early events such as ERK phosphorylation and the increase in calcium flux, and the latter activation of the AP1 and NF-κB transcription factors, was blunted.

Conclusions: Overall, our results demonstrate that CXCR2 ubiquitination on K327 residue modulates agonist-activated CXCR2 cell sorting and intracellular signaling. Thus, the inhibition of K327 ubiquitination might emerge as an effective mean to curb exacerbated CXCR2 signaling in several pathological conditions, such as inflammatory diseases and cancer.

Show MeSH
Related in: MedlinePlus