Control of CXCR2 activity through its ubiquitination on K327 residue
© Leclair et al.; licensee BioMed Central Ltd. 2014
Received: 26 June 2014
Accepted: 9 October 2014
Published: 22 October 2014
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.
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.
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.
CXCR2 is a seven transmembrane G-protein coupled receptor (GPCR) for the ELR + CXCL8 (IL-8) chemokine that transduces pro-angiogenic and pro-inflammatory responses in endothelial and immune cells -. Indeed, IL-8/CXCR2 signaling axis plays multiple functions in the course of physiological and pathological neo-vessel formation. Likewise, IL-8 signaling is instrumental in leukocyte migration . Notably, it does so by stimulating endothelial cell proliferation, permeability, and migration, and by attracting lymphocytes, macrophages, and neutrophils to perivascular regions. For instance, we recently demonstrated that IL-8 operates through CXCR2 and phosphoinositide 3-kinase γ (PI3Kγ) to promote angiogenesis and macrophage accumulation in retina, while curbing the endothelial barrier function . Importantly, interfering with this pathway quelled laser-induced vascular dysfunctions in mouse retina. Similar observations were done in the context of cancers, including malignant brain tumors, prostate tumors, pancreatic ductal adenocarcinoma, chemically induced-skin tumors, Ras-mediated tumors and inflammation-driven tumors ,-. These data place CXCR2 as a promising pharmacological target in many human diseases and pathological conditions . In that view, targeting GPCR constitutes the primary option for pharmacological intervention and drug development . However, many of these compounds act as direct antagonists leaving uncertain their ability to target pathways that are aberrantly and/or constitutively active.
Physiologically, GPCR signaling can be either twisted or interrupted by intracellular cascades implying endocytosis and ubiquitination . Similarly to the CXCL12 (also known as stromal derived factor 1α, SDF1α) chemokine receptor CXCR4, ligand-activated CXCR2 undergoes endocytosis in clathrin-coated vesicles ,. In that view, the viral CXCR2 homolog vGPCR from the Kaposi Sarcoma Herpes Virus, harbors a consensus adaptin 2 (AP2) binding motif in its C-terminal tail, which drives its constitutive shuttling in clathrin-coated vesicles, and further adjusts its signaling activity . Moreover, CXCR2 internalization might involve scaffold proteins, including β-arrestins, AP2, actin-binding proteins and yet to be determined PDZ ligand motif binding proteins ,,-. However, how exactly CXCR2 signaling, intracellular sorting and ubiquitination are coordinated is not fully elucidated. This prompted us to explore the molecular contribution of lysine residues, operating as ubiquitin acceptors, to CXCR2 activity.
Results and discussion
We report here that the conserved K327 residue on CXCR2 is a key ubiquitin acceptor upon IL-8 challenge in ectopic cell models. Of note, interfering with this post-translational modification abrogates CXCR2 trafficking and signaling. In this scenario, CXCR2 poly-ubiquitination, by a yet to be determined ligase, precedes β-arrestin2 recruitment and CXCR2 internalization, and is therefore placed upstream in the IL-8-mediated intracellular signaling cascade. Our data thus highlight an original option to specifically and finely tune CXCR2 signaling in pathological situations, where aberrantly elevated, such as tumor angiogenesis and inflammation.
Cell culture and transfections
HEK-293T cells (ATCC) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and antibiotics. Previously described protocols for transfection were used .
Reagents and antibodies
Recombinant human interleukin 8 (IL-8) was purchased from Peprotech, CXCR2 antagonist SB225002 and MG132 were from Calbiochem. The following antibodies were used: mouse and rabbit anti-CXCR2, rabbit anti-ERK2, mouse anti-tubulin, mouse anti-poly-ubiquitinated chains, clone P4D1 (Santa Cruz); rabbit anti-phopho-ERK1/2, rabbit anti-β-arrestin1/2 (Cell Signaling); mouse anti-phospho-tyr (4G10 clone, Millipore), and mouse anti-AU5 (Eurogentec). AlexaFluor-conjugated antibodies were from Invitrogen.
Full length (FL) K327R (R1) and K337R (R2) CXCR2 mutants were prepared from human wild type (WT) CXCR2, cloned in frame in pCEFL-AU5 expression vector . pGFP-β-arrestin2 , EGFP-β2-adaptin (AP2)  and pYFP-Rab7 were described previously .
CXCR2 protein sequence was introduced to the LOMETS software  and the predicted secondary structure with the lowest Z-score was chosen for modeling, using the Swiss-PDB viewer 36 (http://www.expasy.org/spdbv/). Mutations were introduced using the mutation tool, according to the manufacturer's instruction.
HEK-293T cells were grown onto glass coverslips, fixed (PBS-paraformaldheyde 4%), permeabilized (PBS-Triton 0.5%) and blocked (PBS-BSA 3%). Following incubations with primary and AlexaFluor-conjugated secondary antibodies for 1 h in PBS-BSA 1.5%, samples were mounted (ProLong, Invitrogen), visualized and analyzed under confocal microscopy (TCS/SP5, Leica). Pearson’s coefficient was measured using the Image J plugin on 3 to 5 samples.
HEK-293T cells were transfected with mock, CXCR2-WT or K327R (R1) cDNA plasmids. 24 hours post-transfection, cells were treated as indicated and were further lysed (25 mM Hepes pH7.4, 130 mM NaCl, 10% glycerol, 8 mM β − glycerophosphate, 0.2% Igepal, 1 mM DTT, 1 mM NaVO4, 1 mM NaF, protease inhibitors, 1% SDS and 1 μl benzonase (250 U/μl, Novagen)) on ice. After 30 minutes, SDS was diluted to reach a final concentration of 0.1% and samples were precleared with Protein G agarose (Sigma) for 30 min and then incubated with antibody to CXCR2 and Protein G agarose for 1 hour at 4°C. Ubiquitination status were analyzed by immunoblot using antibody against ubiquitin.
Protein lysates were processed for western-blots as described previously . Equal amounts of protein were loaded onto 4-12% acrylamide gradient gels (NUPAGE, Invitrogen) and transferred onto PVDF membranes (Thermo Scientific) for further analysis using the infrared scanner system (LiCOR).
Cytosolic calcium measurements
Changes in intracellular Ca2+ levels were assessed in HEK-293T as previously described , using Fluo-4 NW calcium indicators (Life Technologies), following the manufacturer's instructions. Fluorescence was read in 96-well plate format at room temperature, using appropriate settings (excitation/emission 494/516 nm, BMG microplate reader).
Firefly luciferase constructs downstream of NF-κB and AP1 consensus sites  were co-transfected with Renilla luciferase pRL-TK plasmid (Promega), together with 250 ng pCEFL-AU5-CXCR2 constructs. Following incubation with IL-8 (50 ng/ml, 6 hours), luciferase activity was analyzed as described previously .
HEK-293T cells were transfected with 0.1 μg per well of the DNA construct coding for double-brilliance β-arrestin2 , together with CXCR2 constructs. Two-days after transfection, IL-8 (50 ng/ml) and DeepBlue coelenterazine substrate (5 μm, Perkin Elmer) were added. Luminescence was measured (Mithras fluorescence-luminescence detector, Berthold) as previously described in . The BRET signal was determined by calculating the ratio of the light emitted by the fluorescent acceptor and the light emitted by Luc. The values (ΔmBRET) were corrected by subtracting the background BRET signals detected in non-stimulated cells.
All data are expressed as mean + s.e.m from three independent experiments. One-way and two-way ANOVA tests with post hoc Tukey's analysis were used to assess statistical significance (Prism 6.0 GraphPad Software), and P values are indicated on figures as follows: * for p < 0.05, ** for p < 0.01, and *** for p < 0.001.
HML: designed and executed the experiments, and interpreted the data; SMD: designed and executed the experiments, and interpreted the data; SA: designed and executed the experiments, and interpreted the data; JD: designed and executed the experiments, and interpreted the data; NB: designed and executed the experiments, and interpreted the data; JG: designed and executed the experiments, interpreted the data, and prepared the manuscript. All authors read and approved the final manuscript.
The authors are thankful to the members of JG laboratory for comments on the manuscript. We are grateful to Thibaud de Chevigny, Jagoda K Hebda and Lucas Treps (Institut Cochin) for technical assistance and discussion, and to Mark GH Scott (Institut Cochin) for advice on β-arrestin2 experiments and to Alexandre Benmerah for DNA plasmids (Institut Necker, Paris, France). This research was funded by: Agence Nationale de la Recherche programme Jeunes Chercheuses et Jeunes Chercheurs, Fondation Association pour la Recherche sur le Cancer, Fondation pour la Recherche Medicale, Institut National du Cancer INCA_6508, Ligue nationale contre le cancer comite de Paris, Ligue nationale contre le cancer comite du Val-de-Marne. SMD is supported by a doctoral fellowship from Universite Paris Sud. SA and JD are supported by post-doctoral fellowship from Fondation Association pour la Recherche sur le Cancer and Agence Nationale de Recherche sur le SIDA et les hépatites virales, respectively.
- Addison CL, Daniel TO, Burdick MD, Liu H, Ehlert JE, Xue YY, Buechi L, Walz A, Richmond A, Strieter RM: The CXC chemokine receptor 2, CXCR2, is the putative receptor for ELR + CXC chemokine-induced angiogenic activity. J Immunol. 2000, 165 (9): 5269-5277. 10.4049/jimmunol.165.9.5269.View ArticlePubMedGoogle Scholar
- Dwyer J, Hebda JK, Le Guelte A, Galan-Moya EM, Smith SS, Azzi S, Bidere N, Gavard J: Glioblastoma cell-secreted interleukin-8 induces brain endothelial cell permeability via CXCR2. PLoS ONE. 2012, 7 (9): e45562-10.1371/journal.pone.0045562.PubMed CentralView ArticlePubMedGoogle Scholar
- Gavard J, Hou X, Qu Y, Masedunskas A, Martin D, Weigert R, Li X, Gutkind JS: A role for a CXCR2/phosphatidylinositol 3-kinase gamma signaling axis in acute and chronic vascular permeability. Mol Cell Biol. 2009, 29 (9): 2469-2480. 10.1128/MCB.01304-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Reutershan J: CXCR2–the receptor to hit?. Drug News Perspect. 2006, 19 (10): 615-623. 10.1358/dnp.2006.19.10.1068009.View ArticlePubMedGoogle Scholar
- Koch AE, Polverini PJ, Kunkel SL, Harlow LA, DiPietro LA, Elner VM, Elner SG, Strieter RM: Interleukin-8 as a macrophage-derived mediator of angiogenesis. Science. 1992, 258 (5089): 1798-1801. 10.1126/science.1281554.View ArticlePubMedGoogle Scholar
- Reutershan J, Morris MA, Burcin TL, Smith DF, Chang D, Saprito MS, Ley K: Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung. J Clin Invest. 2006, 116 (3): 695-702. 10.1172/JCI27009.PubMed CentralView ArticlePubMedGoogle Scholar
- Lazennec G, Richmond A: Chemokines and chemokine receptors: new insights into cancer-related inflammation. Trends Mol Med. 2010, 16 (3): 133-144. 10.1016/j.molmed.2010.01.003.PubMed CentralView ArticlePubMedGoogle Scholar
- Ijichi H, Chytil A, Gorska AE, Aakre ME, Bierie B, Tada M, Mohri D, Miyabayashi K, Asaoka Y, Maeda S, Ikenoue T, Tateishi K, Wright CV, Koike K, Omata M, Moses HL: Inhibiting Cxcr2 disrupts tumor-stromal interactions and improves survival in a mouse model of pancreatic ductal adenocarcinoma. J Clin Invest. 2011, 121 (10): 4106-4117. 10.1172/JCI42754.PubMed CentralView ArticlePubMedGoogle Scholar
- Waugh DJJ, Wilson C: The Interleukin-8 Pathway in Cancer. Clin Cancer Res. 2008, 14 (21): 6735-6741. 10.1158/1078-0432.CCR-07-4843.View ArticlePubMedGoogle Scholar
- Cataisson C, Ohman R, Patel G, Pearson A, Tsien M, Jay S, Wright L, Hennings H, Yuspa SH: Inducible cutaneous inflammation reveals a protumorigenic role for keratinocyte CXCR2 in skin carcinogenesis. Cancer Res. 2009, 69 (1): 319-328. 10.1158/0008-5472.CAN-08-2490.PubMed CentralView ArticlePubMedGoogle Scholar
- Dorsam RT, Gutkind JS: G-protein-coupled receptors and cancer. Nat Rev Cancer. 2007, 7 (2): 79-94. 10.1038/nrc2069.View ArticlePubMedGoogle Scholar
- Marchese A, Trejo J: Ubiquitin-dependent regulation of G protein-coupled receptor trafficking and signaling. Cell Signal. 2013, 25 (3): 707-716. 10.1016/j.cellsig.2012.11.024.PubMed CentralView ArticlePubMedGoogle Scholar
- Baugher PJ, Richmond A: The carboxyl-terminal PDZ ligand motif of chemokine receptor CXCR2 modulates post-endocytic sorting and cellular chemotaxis. J Biol Chem. 2008, 283 (45): 30868-30878. 10.1074/jbc.M804054200.PubMed CentralView ArticlePubMedGoogle Scholar
- Fan GH, Lapierre LA, Goldenring JR, Richmond A: Differential regulation of CXCR2 trafficking by Rab GTPases. Blood. 2003, 101 (6): 2115-2124. 10.1182/blood-2002-07-1965.View ArticlePubMedGoogle Scholar
- Azzi S, Smith SS, Dwyer J, Leclair HM, Alexia C, Hebda JK, Dupin N, Bidere N, Gavard J: YGLF motif in the Kaposi sarcoma herpes virus G-protein-coupled receptor adjusts NF-kappaB activation and paracrine actions. Oncogene. 2013Google Scholar
- Fan GH, Yang W, Wang XJ, Qian Q, Richmond A: Identification of a motif in the carboxyl terminus of CXCR2 that is involved in adaptin 2 binding and receptor internalization. Biochemistry. 2001, 40 (3): 791-800. 10.1021/bi001661b.PubMed CentralView ArticlePubMedGoogle Scholar
- Neel NF, Barzik M, Raman D, Sobolik-Delmaire T, Sai J, Ham AJ, Mernaugh RL, Gertler FB, Richmond A: VASP is a CXCR2-interacting protein that regulates CXCR2-mediated polarization and chemotaxis. J Cell Sci. 2009, 122 (Pt 11): 1882-1894. 10.1242/jcs.039057.PubMed CentralView ArticlePubMedGoogle Scholar
- Raman D, Sai J, Neel NF, Chew CS, Richmond A: LIM and SH3 protein-1 modulates CXCR2-mediated cell migration. PLoS ONE. 2010, 5 (4): e10050-10.1371/journal.pone.0010050.PubMed CentralView ArticlePubMedGoogle Scholar
- Raman D, Sai J, Hawkins O, Richmond A: Adaptor protein2 (AP2) orchestrates CXCR2-mediated cell migration. Traffic. 2014, 15 (4): 451-469. 10.1111/tra.12154.PubMed CentralView ArticlePubMedGoogle Scholar
- Dubois SM, Alexia C, Wu Y, Leclair HM, Leveau C, Schol E, Fest T, Tarte K, Chen ZJ, Gavard J, Bidere N: A catalytic-independent role for the LUBAC in NF-kappaB activation upon antigen receptor engagement and in lymphoma cells. Blood. 2014, 123 (14): 2199-2203. 10.1182/blood-2013-05-504019.View ArticlePubMedGoogle Scholar
- Oeckinghaus A, Wegener E, Welteke V, Ferch U, Arslan SC, Ruland J, Scheidereit C, Krappmann D: Malt1 ubiquitination triggers NF-kappaB signaling upon T-cell activation. EMBO J. 2007, 26 (22): 4634-4645. 10.1038/sj.emboj.7601897.PubMed CentralView ArticlePubMedGoogle Scholar
- White JR, Lee JM, Young PR, Hertzberg RP, Jurewicz AJ, Chaikin MA, Widdowson K, Foley JJ, Martin LD, Griswold DE, Sarau HM: Identification of a potent, selective non-peptide CXCR2 antagonist that inhibits interleukin-8-induced neutrophil migration. J Biol Chem. 1998, 273 (17): 10095-10098. 10.1074/jbc.273.17.10095.View ArticlePubMedGoogle Scholar
- Lefkowitz RJ: Historical review: a brief history and personal retrospective of seven-transmembrane receptors. Trends Pharmacol Sci. 2004, 25 (8): 413-422. 10.1016/j.tips.2004.06.006.View ArticlePubMedGoogle Scholar
- Hebda JK, Leclair HM, Azzi S, Roussel C, Scott MG, Bidere N, Gavard J: The C-terminus region of beta-arrestin1 modulates VE-cadherin expression and endothelial cell permeability. Cell Commun Signal. 2013, 11 (1): 37-10.1186/1478-811X-11-37.PubMed CentralView ArticlePubMedGoogle Scholar
- Rappoport JZ, Taha BW, Lemeer S, Benmerah A, Simon SM: The AP-2 complex is excluded from the dynamic population of plasma membrane-associated clathrin. J Biol Chem. 2003, 278 (48): 47357-47360. 10.1074/jbc.C300390200.View ArticlePubMedGoogle Scholar
- Lakadamyali M, Rust MJ, Zhuang X: Ligands for clathrin-mediated endocytosis are differentially sorted into distinct populations of early endosomes. Cell. 2006, 124 (5): 997-1009. 10.1016/j.cell.2005.12.038.PubMed CentralView ArticlePubMedGoogle Scholar
- Wu S, Zhang Y: LOMETS: a local meta-threading-server for protein structure prediction. Nucleic Acids Res. 2007, 35 (10): 3375-3382. 10.1093/nar/gkm251.PubMed CentralView ArticlePubMedGoogle Scholar
- Gavard J, Gutkind JS: Protein kinase C-related kinase and ROCK are required for thrombin-induced endothelial cell permeability downstream from Galpha12/13 and Galpha11/q. J Biol Chem. 2008, 283 (44): 29888-29896. 10.1074/jbc.M803880200.PubMed CentralView ArticlePubMedGoogle Scholar
- Charest PG, Terrillon S, Bouvier M: Monitoring agonist-promoted conformational changes of beta-arrestin in living cells by intramolecular BRET. EMBO Rep. 2005, 6 (4): 334-340. 10.1038/sj.embor.7400373.PubMed CentralView ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.