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Anne O Watts,
Folkert Verkaar,
Miranda M C van der Lee,
Claudia A W Timmerman,
Martien Kuijer,
Jody van Offenbeek,
Lambertus H C J van Lith, Martine J Smit,
Rob Leurs,
Guido J R Zaman,
Henry F Vischer
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ABSTRACT: Chemokine receptors form a large subfamily of G protein-coupled receptors that predominantly activate heterotrimeric G(i) proteins and are involved in immune cell migration. CCX-CKR is an atypical chemokine receptor with high affinity for CCL19, CCL21 and CCL25 chemokines, but is not known to activate intracellular signaling pathways. However, CCX-CKR acts as decoy receptor and efficiently internalizes these chemokines, thereby preventing their interaction with other chemokine receptors, like CCR7 and CCR9. Internalization of fluorescently labeled CCL19 correlated with β-arrestin2-GFP translocation. Moreover, recruitment of β-arrestins to CCX-CKR in response to CCL19, CCL21, and CCL25 was demonstrated using enzyme-fragment complementation and bioluminescence resonance energy transfer methods. To unravel why CCX-CKR is unable to activate G(i) signaling, CCX-CKR chimeras were constructed by substituting its intracellular loops with the corresponding CCR7 or CCR9 domains. The signaling properties of chimeric CCX-CKR receptors were characterized using a cAMP-responsive element (CRE)-driven reporter gene assay. Unexpectedly, wild type CCX-CKR and a subset of the chimeras induced an increase in CRE activity in response to CCL19, CCL21 and CCL25 in the presence of the G(i) inhibitor pertussis toxin. CCX-CKR signaling to CRE required an intact DRY motif. These data suggest that inactive G(i) proteins impair CCX-CKR signaling most likely by hindering the interaction of this receptor with pertussis toxin-insensitive G proteins that transduce signaling to CRE. On the other hand, recruitment of the putative signaling scaffold β-arrestin to CCX-CKR in response to chemokines might allow activation of yet to be identified signal transduction pathways.
Journal of Biological Chemistry 01/2013; · 4.77 Impact Factor
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Maikel Wijtmans,
Danny J Scholten,
Luc Roumen,
Meritxell Canals,
Hans Custers,
Marjolein Glas,
Marlies C A Vreeker,
Frans J J de Kanter,
Chris de Graaf, Martine J Smit,
Iwan J P de Esch,
Rob Leurs
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ABSTRACT: The G protein-coupled chemokine receptor CXCR3 plays a role in numerous inflammatory events. The endogenous ligands for the chemokine receptors are peptides, but in this study we disclose small-molecule ligands that are able to activate CXCR3. A class of biaryl-type compounds that is assembled by convenient synthetic routes is described as a new class of CXCR3 agonists. Intriguingly, structure-activity relationship and structure-function relationship studies reveal that subtle chemical modifications on the outer aryl ring (e.g., either the size or position of a halogen atom) result in a full spectrum of agonist efficacies on CXCR3. Quantum mechanics calculations and nuclear Overhauser effect spectroscopy NMR studies suggest that the biaryl dihedral angle and the electronic nature of ortho-substituents play an important role in determining agonist efficacies. Compounds 38 (VUF11222) and 39 (VUF11418) are the first reported nonpeptidomimetic agonists on CXCR3, rendering them highly useful chemical tools for detailed assessment of CXCR3 activation as well as for studying downstream CXCR3 signaling.
Journal of Medicinal Chemistry 11/2012; · 4.80 Impact Factor
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Gerold Bongers,
Luciana R Muniz,
Michelle E Pacer,
Alina C Iuga,
Nanthakumar Thirunarayanan,
Erik Slinger, Martine J Smit,
E Premkumar Reddy,
Lloyd Mayer,
Glaucia C Furtado,
Noam Harpaz,
Sergio A Lira
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ABSTRACT: Epithelial cancers can be initiated by activating mutations in components of the mitogen-activated protein kinase signaling pathway such as v-raf murine sarcoma viral oncogene homolog B1 (BRAF), v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR). Human intestinal serrated polyps are a heterogeneous group of benign lesions, but some progress to colorectal cancer. Tumors that arise from these polyps frequently contain activating mutations in BRAF or KRAS, but little is known about the role of EGFR activation in their development.
Polyp samples were obtained from adults during screening colonoscopies at Mount Sinai Hospital in New York. We measured levels of EGFR protein and phosphorylation in human serrated polyps by immunohistochemical and immunoblot analyses. We generated transgenic mice that express the ligand for EGFR, Heparin-binding EGF-like growth factor (HB-EGF), in the intestine.
EGFR and the extracellular-regulated kinases (ERK)1/2 were phosphorylated in serrated areas of human hyperplastic polyps (HPPs), sessile serrated adenomas, and traditional serrated adenomas. EGFR and ERK1/2 were phosphorylated in the absence of KRAS or BRAF activating mutations in a subset of HPP. Transgenic expression of the EGFR ligand HB-EGF in the intestines of mice promoted development of small cecal serrated polyps. Mice that expressed a combination of HB-EGF and US28 (a constitutively active, G-protein-coupled receptor that increases processing of HB-EGF from the membrane) rapidly developed large cecal serrated polyps. These polyps were similar to HPPs and had increased phosphorylation of EGFR and ERK1/2 within the serrated epithelium. Administration of pharmacologic inhibitors of EGFR or MAPK to these transgenic mice significantly reduced polyp development.
Activation of EGFR signaling in the intestine of mice promotes development of serrated polyps. EGFR signaling also is activated in human HPPs, sessile serrated adenomas, and traditional serrated adenomas.
Gastroenterology 05/2012; 143(3):730-40. · 11.68 Impact Factor
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Maikel Wijtmans,
David Maussang,
Francesco Sirci,
Danny J Scholten,
Meritxell Canals,
Azra Mujić-Delić,
Milagros Chong,
Kristell L S Chatalic,
Hans Custers,
Elwin Janssen,
Chris de Graaf, Martine J Smit,
Iwan J P de Esch,
Rob Leurs
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ABSTRACT: The chemokine receptor CXCR7 is an atypical G protein-coupled receptor as it preferentially signals through the β-arrestin pathway rather than through G proteins. CXCR7 is thought to be of importance in cancer and the development of CXCR7-targeting ligands is of huge importance to further elucidate the pharmacology and the therapeutic potential of CXCR7. In the present study, we synthesized 24 derivatives based on a compound scaffold patented by Chemocentryx and obtained CXCR7 ligands with pK(i) values ranging from 5.3 to 8.1. SAR studies were supported by computational 3D Fingerprint studies, revealing several important affinity descriptors. Two key compounds (29 and 30, VUF11207 and VUF11403) were found to be high-potency ligands that induce recruitment of β-arrestin2 and subsequent internalization of CXCR7, making them important tool compounds in future CXCR7 research.
European journal of medicinal chemistry 02/2012; 51:184-92. · 3.27 Impact Factor
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ABSTRACT: The chemokine receptor CXCR7 binds CXCL11 and CXCL12 with high affinity, chemokines that were previously thought to bind exclusively to CXCR4 and CXCR3, respectively. Expression of CXCR7 has been associated with cardiac development as well as with tumor growth and progression. Despite having all the canonical features of G protein-coupled receptors (GPCRs), the signalling pathways following CXCR7 activation remain controversial, since unlike typical chemokine receptors, CXCR7 fails to activate Gα(i)-proteins. CXCR7 has recently been shown to interact with β-arrestins and such interaction has been suggested to be responsible for G protein-independent signals through ERK-1/2 phosphorylation. Signal transduction by CXCR7 is controlled at the membrane by the process of GPCR trafficking. In the present study we investigated the regulatory processes triggered by CXCR7 activation as well as the molecular interactions that participate in such processes. We show that, CXCR7 internalizes and recycles back to the cell surface after agonist exposure, and that internalization is not only β-arrestin-mediated but also dependent on the Serine/Threonine residues at the C-terminus of the receptor. Furthermore we describe, for the first time, the constitutive ubiquitination of CXCR7. Such ubiquitination is a key modification responsible for the correct trafficking of CXCR7 from and to the plasma membrane. Moreover, we found that CXCR7 is reversibly de-ubiquitinated upon treatment with CXCL12. Finally, we have also identified the Lysine residues at the C-terminus of CXCR7 to be essential for receptor cell surface delivery. Together these data demonstrate the differential regulation of CXCR7 compared to the related CXCR3 and CXCR4 receptors, and highlight the importance of understanding the molecular determinants responsible for this process.
PLoS ONE 01/2012; 7(3):e34192. · 4.09 Impact Factor
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ABSTRACT: Chronic activation of Wnt/ß-catenin signaling is found in a variety of human malignancies including melanoma, colorectal and hepatocellular carcinomas. Interestingly, expression of the HCMV-encoded chemokine receptor US28 in intestinal epithelial cells promotes intestinal neoplasia in transgenic mice, which is associated with increased nuclear accumulation of ß-catenin. In this study we show that this viral receptor constitutively activates ß-catenin and enhances ß-catenin-dependent transcription. Our data illustrate that this viral receptor does not activate ß-catenin via the classical Wnt/Frizzled signaling pathway. Analysis of US28 mediated signaling indicates the involvement of the Rho-Rho kinase (ROCK) pathway in the activation of ß-catenin. Moreover, cells infected with HCMV show significant increases in ß-catenin stabilization and signaling, which is mediated to a large extent by expression of US28. The modulation of the ß-catenin signal transduction pathway by a viral chemokine receptor provides alternative regulation of this pathway, with potential relevance for the development of colon cancer and virus-associated diseases.
PLoS ONE 01/2012; 7(11):e48935. · 4.09 Impact Factor
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Petra de Kruijf,
Herman D Lim,
Luc Roumen,
Véronique A Renjaän,
Jiuqiao Zhao,
Maria L Webb,
Douglas S Auld,
Jac C H M Wijkmans,
Guido J R Zaman, Martine J Smit,
Chris de Graaf,
Rob Leurs
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ABSTRACT: We have shown previously that different chemical classes of small-molecule antagonists of the human chemokine CXCR2 receptor interact with distinct binding sites of the receptor. Although an intracellular binding site for diarylurea CXCR2 antagonists, such as N-(2-bromophenyl)-N'-(7-cyano-1H-benzotriazol-4-yl)urea (SB265610), and thiazolopyrimidine compounds was recently mapped by mutagenesis studies, we now report on an imidazolylpyrimidine antagonist binding pocket in the transmembrane domain of CXCR2. Using different CXCR2 orthologs, chimeric proteins, site-directed mutagenesis, and in silico modeling, we have elucidated the binding mode of this antagonist. Our in silico-guided mutagenesis studies indicate that the ligand binding cavity for imidazolylpyrimidine compounds in CXCR2 is located between transmembrane (TM) helices 3 (Phe130(3.36)), 5 (Ser217(5.44), Phe220(5.47)), and 6 (Asn268(6.52), Leu271(6.55)) and suggest that these antagonists enter CXCR2 via the TM5-TM6 interface. It is noteworthy that the same interface is postulated as the ligand entry channel in the opsin receptor and is occupied by lipid molecules in the recently solved crystal structure of the CXCR4 chemokine receptor, suggesting a general ligand entrance mechanism for nonpolar ligands to G protein-coupled receptors. The identification of a novel allosteric binding cavity in the TM domain of CXCR2, in addition to the previously identified intracellular binding site, shows the diversity in ligand recognition mechanisms by this receptor and offers new opportunities for the structure-based design of small allosteric modulators of CXCR2 in the future.
Molecular pharmacology 09/2011; 80(6):1108-18. · 4.53 Impact Factor
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ABSTRACT: The traffic of the different types of immune cells is an important aspect in the immune response. Chemokines are soluble peptides that are able to attract cells by interaction with chemokine receptors on their target cells. Several different chemokines and receptors exist enabling the specific trafficking of different immune cells. In chronic inflammatory disorders there is abundance of immune cells present at the inflammatory site. This review focuses on the role of chemokine receptors in chronic inflammatory disorders of the lungs, intestine, joints, skin and nervous system and the potential of targeting these receptors as therapeutic intervention in these disorders.
Pharmacology [?] Therapeutics 08/2011; 133(1):1-18. · 8.56 Impact Factor
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ABSTRACT: Small-molecule ligands for the CXCR3 chemokine receptor receive considerable attention as a means to interrogate the roles of CXCR3 in vitro and in vivo and/or to potentially treat various conditions such as inflammatory diseases and cancer. We have synthesized and explored a novel class of small-molecule antagonists for CXCR3. A medium-throughput screen revealed an adamantane-guanidine as a hit. The guanidine unit was replaced by a small quaternary ammonium group, leading to ca. 5-fold increase in affinity. Substitution of the adamantane group by a myrtenyl moiety further increased affinity, while the benzyl group was decorated with an additional (substituted) aryl ring. This led to the identification of several bisaryl-based ligands with CXCR3 affinities of around 100 nM and with the ability to antagonize the functional activity of CXCL10.
Bioorganic & medicinal chemistry 06/2011; 19(11):3384-93. · 2.82 Impact Factor
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Saskia A Overbeek,
Paul A J Henricks,
Anja I Srienc,
Pim J Koelink,
Petra de Kruijf,
Herman D Lim, Martine J Smit,
Guido J R Zaman,
Johan Garssen,
Frans P Nijkamp,
Aletta D Kraneveld,
Gert Folkerts
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ABSTRACT: Chronic inflammation in lung diseases contributes to lung tissue destruction leading to the formation of chemotactic collagen fragments such as N-acetylated Proline-Glycine-Proline (N-ac-PGP). In this study, we investigated in more detail the mechanism of action of N-ac-PGP in neutrophilic inflammation. N-ac-PGP was chemotactic for human neutrophils via pertussis toxin sensitive G protein-coupled receptors in vitro and directly activated this cell type, which led to cytosolic calcium mobilization and release of CXCL8. Furthermore, using a selective CXCR2 antagonist confirmed that N-ac-PGP-induced neutrophil chemotaxis is mediated through CXCR2 activation. To determine whether N-ac-PGP was solely responsible for the migration and activation of human neutrophils in vitro and not the released CXCL8 upon stimulation with N-ac-PGP, an antibody directed against CXCL8 was used. Performing chemotaxis and calcium influx assays in the presence of this antibody did not alter the effects of N-ac-PGP whereas effects of CXCL8 were attenuated. These experiments indicate that N-ac-PGP, in addition to the direct induction of chemotaxis, also directly activates neutrophils to release CXCL8. In vivo, this may lead in the long term to a self-maintaining situation enhanced by both N-ac-PGP and CXCL8, leading to a further increase in neutrophil infiltration and chronic inflammation.
European journal of pharmacology 03/2011; 668(3):428-34. · 2.59 Impact Factor
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ABSTRACT: Viral G-protein-coupled receptors (vGPCRs) are chemokine receptor homologues encoded by the Herpes- and Capripoxviridae. They are thought to have been hijacked from the host genome during the course of evolution. These vGPCRs play different roles in the viral lifecycle and associated pathologies. Three members of the Herpesviridae, Kaposi sarcoma-associated herpesvirus (KSHV), Epstein-Barr virus (EBV) and human cytomegalovirus (HCMV) are capable of setting up persistent latent infections in humans. Two of the herpesviruses, KSHV and EBV, are associated with cancer, while HCMV may have an oncomodulary effect. The vGPCRs may contribute to the escape of immune surveillance and (constitutively) activate signaling pathways linked to proliferation and inflammation. Some vGPCRs induce activation of autocrine and paracrine signaling, resulting in secretion of growth factors and/or cytokines. As a result, vGPCRs effectively rewire cellular signaling networks. Delineating the cellular signaling networks modulated by these vGPCRs will be crucial for treatment of virus-associated pathologies.
Molecular and Cellular Endocrinology 01/2011; 331(2):179-84. · 4.19 Impact Factor
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Sven Jähnichen,
Christophe Blanchetot,
David Maussang,
Maria Gonzalez-Pajuelo,
Ken Y Chow,
Leontien Bosch,
Sindi De Vrieze,
Benedikte Serruys,
Hans Ulrichts,
Wesly Vandevelde,
Michael Saunders,
Hans J De Haard,
Dominique Schols,
Rob Leurs,
Peter Vanlandschoot,
Theo Verrips, Martine J Smit
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ABSTRACT: The important family of G protein-coupled receptors has so far not been targeted very successfully with conventional monoclonal antibodies. Here we report the isolation and characterization of functional VHH-based immunoglobulin single variable domains (or nanobodies) against the chemokine receptor CXCR4. Two highly selective monovalent nanobodies, 238D2 and 238D4, were obtained using a time-efficient whole cell immunization, phage display, and counterselection method. The highly selective VHH-based immunoglobulin single variable domains competitively inhibited the CXCR4-mediated signaling and antagonized the chemoattractant effect of the CXCR4 ligand CXCL12. Epitope mapping showed that the two nanobodies bind to distinct but partially overlapping sites in the extracellular loops. Short peptide linkage of 238D2 with 238D4 resulted in significantly increased affinity for CXCR4 and picomolar activity in antichemotactic assays. Interestingly, the monovalent nanobodies behaved as neutral antagonists, whereas the biparatopic nanobodies acted as inverse agonists at the constitutively active CXCR4-N3.35A. The CXCR4 nanobodies displayed strong antiretroviral activity against T cell-tropic and dual-tropic HIV-1 strains. Moreover, the biparatopic nanobody effectively mobilized CD34-positive stem cells in cynomolgus monkeys. Thus, the nanobody platform may be highly effective at generating extremely potent and selective G protein-coupled receptor modulators.
Proceedings of the National Academy of Sciences 11/2010; 107(47):20565-70. · 9.68 Impact Factor
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Gerold Bongers,
David Maussang,
Luciana R Muniz,
Vanessa M Noriega,
Alberto Fraile-Ramos,
Nick Barker,
Federica Marchesi,
Nanthakumar Thirunarayanan,
Henry F Vischer,
Lihui Qin,
Lloyd Mayer,
Noam Harpaz,
Rob Leurs,
Glaucia C Furtado,
Hans Clevers,
Domenico Tortorella, Martine J Smit,
Sergio A Lira
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ABSTRACT: US28 is a constitutively active chemokine receptor encoded by CMV (also referred to as human herpesvirus 5), a highly prevalent human virus that infects a broad spectrum of cells, including intestinal epithelial cells (IECs). To study the role of US28 in vivo, we created transgenic mice (VS28 mice) in which US28 expression was targeted to IECs. Expression of US28 was detected in all IECs of the small and large intestine, including in cells expressing leucine rich repeat containing GPCR5 (Lgr5), a marker gene of intestinal epithelial stem cells. US28 expression in IECs inhibited glycogen synthase 3β (GSK-3β) function, promoted accumulation of β-catenin protein, and increased expression of Wnt target genes involved in the control of the cell proliferation. VS28 mice showed a hyperplastic intestinal epithelium and, strikingly, developed adenomas and adenocarcinomas by 40 weeks of age. When exposed to an inflammation-driven tumor model (azoxymethane/dextran sodium sulfate), VS28 mice developed a significantly higher tumor burden than control littermates. Transgenic coexpression of the US28 ligand CCL2 (an inflammatory chemokine) increased IEC proliferation as well as tumor burden, suggesting that the oncogenic activity of US28 can be modulated by inflammatory factors. Together, these results indicate that expression of US28 promotes development of intestinal dysplasia and cancer in transgenic mice and suggest that CMV infection may facilitate development of intestinal neoplasia in humans.
The Journal of clinical investigation 10/2010; 120(11):3969-78. · 15.39 Impact Factor
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ABSTRACT: Cells express distinct G protein-coupled receptor (GPCR) subtypes on their surface, allowing them to react to a corresponding variety of extracellular stimuli. Cross-regulation between different ligand-GPCR pairs is essential to generate appropriate physiological responses. GPCRs can physically affect each other's functioning by forming heteromeric complexes, whereas cross-regulation between activated GPCRs also occurs through integration of shared intracellular signaling networks. Human herpesviruses utilize virally encoded GPCRs to hijack cellular signaling networks for their own benefit. Previously, we demonstrated that the Epstein-Barr virus-encoded GPCR BILF1 forms heterodimeric complexes with human chemokine receptors. Using a combination of bimolecular complementation and bioluminescence resonance energy transfer approaches, we now show the formation of hetero-oligomeric complexes between this viral GPCR and human CXCR4. BILF1 impaired CXCL12 binding to CXCR4 and, consequently, also CXCL12-induced signaling. In contrast, the G protein uncoupled mutant BILF1-K(3.50)A affected CXCL12-induced CXCR4 signaling to a much lesser extent, indicating that BILF1-mediated CXCR4 inhibition is a consequence of its constitutive activity. Co-expression of Gα(i1) with BILF1 and CXCR4 restored CXCL12-induced signaling. Likewise, BILF1 formed heteromers with the human histamine H(4) receptor (H(4)R). BILF1 inhibited histamine-induced Gα(i)-mediated signaling by H(4)R, however, without affecting histamine binding to this receptor. These data indicate that functional cross-regulation of Gα(i)-coupled GPCRs by BILF1 is at the level of G proteins, even though these GPCRs are assembled in hetero-oligomeric complexes.
Journal of Biological Chemistry 09/2010; 285(38):29632-41. · 4.77 Impact Factor
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ABSTRACT: Neutrophils transmigrate from the blood into inflamed tissue via the interaction of chemokines produced in this tissue with chemokine receptors, such as CXCR1 and CXCR2, that are expressed on the membranes of neutrophils. Subsequently, activation of neutrophils will in turn lead to increased tissue damage and thereby enhanced clinical symptoms of inflammatory diseases like chronic obstructive pulmonary disease, inflammatory bowel disease and psoriasis. Besides chemokines, also the collagen-breakdown product N-acetyl-Proline-Glycine-Proline (N-alpha-PGP) attracts neutrophils. In a recent article (Weathington et al., 2006) it was suggested that N-alpha-PGP exerts its effect via CXCR1 and CXCR2. In this study, we show that N-alpha-PGP, in contrast to CXCL8, does not directly activate or interact with CXCR1 or CXCR2. N-alpha-PGP was not able to displace the radioligand [(125)I]CXCL8 from CXCR1 and CXCR2 expressing HEK293T cells or neutrophils. In addition, N-alpha-PGP did not displace the radioligand [(3)H]-SB265610, a CXCR2 antagonist, from CXCR2 expressing cells. Furthermore, N-alpha-PGP was not able to activate G protein signalling in cells expressing CXCR1 and CXCR2. N-alpha-PGP was also not able to recruit beta-arrestin2, an intracellular scaffolding protein involved in G protein-independent signalling, in cells expressing CXCR2. These studies indicate that N-alpha-PGP is not a ligand of CXCR1 or CXCR2.
European journal of pharmacology 09/2010; 643(1):29-33. · 2.59 Impact Factor
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ABSTRACT: US28 is a viral G protein (heterotrimeric guanosine triphosphate-binding protein)-coupled receptor encoded by the human cytomegalovirus (HCMV). In addition to binding and internalizing chemokines, US28 constitutively activates signaling pathways linked to cell proliferation. Here, we show increased concentrations of vascular endothelial growth factor and interleukin-6 (IL-6) in supernatants of US28-expressing NIH 3T3 cells. Increased IL-6 was associated with increased activation of the signal transducer and activator of transcription 3 (STAT3) through upstream activation of the Janus-activated kinase JAK1. We used conditioned growth medium, IL-6-neutralizing antibodies, an inhibitor of the IL-6 receptor, and short hairpin RNA targeting IL-6 to show that US28 activates the IL-6-JAK1-STAT3 signaling axis through activation of the transcription factor nuclear factor kappaB and the consequent production of IL-6. Treatment of cells with a specific inhibitor of STAT3 inhibited US28-dependent [(3)H]thymidine incorporation and foci formation, suggesting a key role for STAT3 in the US28-mediated proliferative phenotype. US28 also elicited STAT3 activation and IL-6 secretion in HCMV-infected cells. Analyses of tumor specimens from glioblastoma patients demonstrated colocalization of US28 and phosphorylated STAT3 in the vascular niche of these tumors. Moreover, increased phospho-STAT3 abundance correlated with poor patient outcome. We propose that US28 induces proliferation in HCMV-infected tumors by establishing a positive feedback loop through activation of the IL-6-STAT3 signaling axis.
Science Signaling 08/2010; 3(133):ra58. · 7.50 Impact Factor
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Pia Tschische,
Elisabeth Moser,
Dawn Thompson,
Henry F Vischer,
Gerald P Parzmair,
Veronika Pommer,
Wolfgang Platzer,
Thomas Schwarzbraun,
Helmut Schaider, Martine J Smit,
Lene Martini,
Jennifer L Whistler,
Maria Waldhoer
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ABSTRACT: Human cytomegalovirus (HCMV) encodes the seven transmembrane(7TM)/G-protein coupled receptor (GPCR) US28, which signals and endocytoses in a constitutive, ligand-independent manner. Here we show that, following endocytosis, US28 is targeted to the lysosomes for degradation as a consequence of its interaction with the GPCR-associated sorting protein-1 (GASP-1). We find that GASP-1 binds to US28 in vitro and that disruption of the GASP-1/US28 interaction by either (i) overexpression of dominant negative cGASP-1 or by (ii) shRNA knock-down of endogenous GASP-1 is sufficient to inhibit the lysosomal targeting of US28 and slow its post-endocytic degradation. Furthermore, we found that GASP-1 affects US28-mediated signalling. The knock-down of endogenous GASP-1 impairs the US28-mediated Galphaq/PLC/inositol phosphate (IP) accumulation as well as the activation of the transcription factors Nuclear Factor-kappaB (NF-kappaB) and cyclic AMP responsive element binding protein (CREB). Overexpression of GASP-1 enhances both IP accumulation and transcription factor activity. Thus, GASP-1 is an important cellular determinant that not only regulates the post-endocytic trafficking of US28, but also regulates the signalling capacities of US28.
Traffic 05/2010; 11(5):660-74. · 4.92 Impact Factor
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ABSTRACT: Human cytomegalovirus (HCMV) is a widespread human pathogen, possessing onco-modulatory properties. Constitutive signaling of the HCMV-encoded chemokine receptor US28 and its ability to bind a broad spectrum of chemokines might facilitate HCMV-associated tumor progression. Novel nonpeptidergic chemotypes were identified as neutral antagonists or inverse agonists on US28, that allosterically inhibit chemokine binding to US28.
Bioorganic & medicinal chemistry 01/2010; 18(2):675-88. · 2.82 Impact Factor
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ABSTRACT: This is a conversation with Martine Smit about a Research Article published in the 3 August 2010 issue of Science Signaling.
Science Signaling 01/2010; 3(133):pc16. · 7.50 Impact Factor
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ABSTRACT: Human herpesviruses (HHVs) are widespread pathogens involved in proliferative diseases, inflammatory conditions, and cardiovascular diseases. During evolution, homologs of human chemokine receptors were integrated into the HHV genomes. In addition to binding endogenous chemokines, these viral G protein-coupled receptors (vGPCRs) have acquired the ability to signal in a constitutive manner. Ligand-induced and ligand-independent and autocrine and paracrine signaling properties of vGPCRs modify the functions of the expressing cells and lead to transformation and escape from immune surveillance. Furthermore, cross-talk or heterodimerization with endogenous chemokine receptors represent other ways for vGPCRs to modify intracellular signaling and cellular functions. As such, these viral receptors seem to play a prominent role during viral pathogenesis and life cycle and thus represent innovative antiviral therapies.
Molecular pharmacology 08/2009; 76(4):692-701. · 4.53 Impact Factor
