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ABSTRACT: Surface plasmon resonance (SPR)-based biosensors are established tools for measuring biomolecular interactions between unlabeled analytes in real time, and are thus an ideal method to evaluate G protein-coupled receptor (GPCR) binding interactions. Using as a vehicle lentiviral particles bearing the chemokine receptor CXCR4 in its native plasma membrane context, SPR analysis can be performed using the particles as specific receptors to monitor the CXCR4 interaction with its ligand, CXCL12. The method shows linear correlation in the 5-40nM range, with low intra- and inter-assay variation, a relative standard deviation <10%, chip-to-chip variation <12%, with stability of the sensor response for more than 150 measurements in the same chip over a four-week period. Our objective was to develop a method for rapid detection and quantification of analytes such as CXCL12 in biological samples, with no need for pretreatment. As a proof of concept, we tested for CXCL12 in urine samples from rheumatoid arthritis patients, who have elevated levels of this chemokine in plasma and synovial fluid. The biosensor method allowed sensitive, reproducible CXCL12 detection in the physiological range, suggesting its value for the diagnosis of autoimmune disorders.
Talanta 05/2013; 109:209-15. · 3.79 Impact Factor
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Borja L Holgado,
Laura Martínez-Muñoz,
Juan Antonio Sánchez-Alcañiz,
Pilar Lucas,
Vicente Pérez-García,
Gema Pérez, José Miguel Rodríguez-Frade,
Marta Nieto,
Oscar Marín,
Yolanda R Carrasco,
Ana C Carrera,
Manuel Alvarez-Dolado,
Mario Mellado
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ABSTRACT: The migratory route of neural progenitor/precursor cells (NPC) has a central role in central nervous system development. Although the role of the chemokine CXCL12 in NPC migration has been described, the intracellular signaling cascade involved remains largely unclear. Here we studied the molecular mechanisms that promote murine NPC migration in response to CXCL12, in vitro and ex vivo. Migration was highly dependent on signaling by the CXCL12 receptor, CXCR4. Although the JAK/STAT pathway was activated following CXCL12 stimulation of NPC, JAK activity was not necessary for NPC migration in vitro. Whereas CXCL12 activated the PI3K catalytic subunits p110α and p110β in NPC, only p110β participated in CXCL12-mediated NPC migration. Ex vivo experiments using organotypic slice cultures showed that p110β blockade impaired NPC exit from the medial ganglionic eminence. In vivo experiments using in utero electroporation nonetheless showed that p110β is dispensable for radial migration of pyramidal neurons. We conclude that PI3K p110β is activated in NPC in response to CXCL12, and its activity is necessary for immature interneuron migration to the cerebral cortex.
Molecular Neurobiology 04/2013; · 5.74 Impact Factor
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Gema Pérez-Rivero,
Graciela Cascio,
Silvia Fernández Soriano,
Alvaro Gil Sanz,
Julia Sáez de Guinoa, José Miguel Rodríguez-Frade,
Rosa P Gomariz,
Borja L Holgado,
Carlos Cabañas,
Yolanda R Carrasco,
Jens V Stein,
Mario Mellado
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ABSTRACT: Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, non-activated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ERM (ezrin/radixin/moesin) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated antigen-1 and very late antigen-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.
European Journal of Immunology 03/2013; · 5.10 Impact Factor
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ABSTRACT: A broad array of biological responses including cell polarization, movement, immune and inflammatory responses, as well as prevention of HIV-1 infection, are triggered by the chemokines, a family of secreted and structurally related chemoattractant proteins that bind to class A-specific seven-transmembrane receptors linked to G proteins. Chemokines and their receptors should not be considered isolated entities, as they act in complex networks. Chemokines bind as oligomers, or oligomerize after binding to glycosaminoglycans on endothelial cells, and are then presented to their receptors on target cells, facilitating the generation of chemoattractant gradients. The chemokine receptors form homo- and heterodimers, as well as higher order structures at the cell surface. These structures are dynamic and are regulated by receptor expression and ligand levels. Complexity is even greater, as in addition to regulation by cytokines and decoy receptors, chemokine and receptor levels are affected by proteolytic cleavage and other protein modifications. This complex scenario should be considered when analyzing chemokine biology and the ability of their antagonists to act in vivo. Strategies based on blocking or stabilizing ligand and receptor dimers could be alternative approaches that might have broad therapeutic potential.
Immunology letters 07/2012; 145(1-2):23-9. · 2.91 Impact Factor
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ABSTRACT: Since the first reports on chemokine function, much information has been generated on the implications of these molecules in numerous physiological and pathological processes, as well as on the signaling events activated through their binding to receptors. Despite these extensive studies, no chemokine-related drugs have yet been approved for use in patients with inflammatory or autoimmune diseases. This discrepancy between efforts and results has forced a re-evaluation of the chemokine field. We have explored chemokine receptor conformations at the cell surface and found that, as is the case for other G protein-coupled receptors, chemokine receptors are not isolated entities that are activated following ligand binding; rather, they are found as dimers and/or higher order oligomers at the cell surface, even in the absence of ligands. These complexes form organized arrays that can be modified by receptor expression and ligand levels, indicating that they are dynamic structures. The way in which these receptor complexes are stabilized modulates ligand binding, as well as their pharmacological properties and the signaling events activated. These conformations thus represent a mechanism that increases the broad variety of chemokine functions. Understanding these receptor interactions and their dynamics at the cell surface is thus critical for influencing chemokine function and could open up new possibilities for drug design.
Pharmacology [?] Therapeutics 05/2011; 131(3):351-8. · 8.56 Impact Factor
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ABSTRACT: Although homo- and heterodimerization are reported for some chemokine receptors, it remains unclear whether these functional states are in dynamic equilibrium and how receptor/ligand levels influence oligomerization. In human neutrophils and in cell lines that coexpress the chemokine receptors CXCR1 and CXCR2, we used fluorescence resonance energy transfer techniques to show that these two receptors form homo- and heterodimers. Receptor expression and ligand activation were found to regulate the balance between these complexes, adapting the response to changes in the milieu. CXCL8, a ligand for both receptors, alters heterodimeric complexes, whereas it stabilizes homodimers and promotes receptor internalization. Oligomerization of receptors, together with the regulation of their expression and desensitization, could thus contribute to the fine control of chemokine functions.
The Journal of Immunology 11/2009; 183(11):7337-46. · 5.79 Impact Factor
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ABSTRACT: The chemokines, a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins, trigger a broad array of biological responses ranging from cell polarization, movement, immune and inflammatory responses to prevention of HIV-1 infection. Chemokine-mediated cell activation was thought to be due to the binding of a monomeric chemokine to its monomeric receptor. Chemokine biology is nonetheless more complex than was initially predicted, as several studies suggest that chemokines can dimerize and that their receptors are found as dimers and/or higher order oligomers at the cell surface. There is also evidence that they cluster in arrays, rather like bundles of cigars. Here we evaluate how these arrays might be organized, the influence of ligand levels and receptor expression on them, and their influence on chemokine function.
Current Opinion in Pharmacology 10/2009; 10(1):38-43. · 6.86 Impact Factor
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ABSTRACT: Tumor dissemination is a complex process, in which certain steps resemble those in leukocyte homing. Specific chemokine/chemokine receptor pairs have important roles in both processes. CXCL12/CXCR4 is the most commonly expressed chemokine/chemokine receptor pair in human cancers, in which it regulates cell adhesion, extravasation, metastatic colonization, angiogenesis, and proliferation. All of these processes require activation of signaling pathways that include G proteins, phosphatidylinositol-3 kinase (PI3K), JAK kinases, Rho GTPases, and focal adhesion-associated proteins. We analyzed these pathways in a human melanoma cell line in response to CXCL12 stimulation, and found that PI3Kgamma regulates tumor cell adhesion through mechanisms different from those involved in cell invasion. Our data indicate that, following CXCR4 activation after CXCL12 binding, the invasion and adhesion processes are regulated differently by distinct downstream events in these signaling cascades.
Biochemical and Biophysical Research Communications 09/2009; 388(2):199-204. · 2.48 Impact Factor
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ABSTRACT: Direct determination of four pituitary peptide hormones: human thyroid stimulating hormone (hTSH), growth hormone (hGH), follicle stimulating hormone (hFSH), and luteinizing hormone (hLH) has been carried out using a portable surface plasmon resonance (SPR) immunosensor.
A commercial SPR biosensor was employed. The immobilization of the hormones was optimized and monoclonal antibodies were selected in order to obtain the best sensor performance. Assay parameters as running buffer and regeneration solution composition or antibody concentration were adjusted to achieve a sensitive analyte detection.
The performance of the assays was assessed in buffer solution, serum and urine, showing sensitivity in the range from 1 to 6 ng/mL. The covalent attachment of the hormones ensured the stability of the SPR signal through repeated use in up to 100 consecutive assay cycles. Mean intra- and inter-day coefficients of variation were all <7%, while batch-assay variability using different sensor surfaces was <5%.
Taking account both the excellent reutilization performance and the outstanding reproducibility, this SPR immunoassay method turns on a highly reliable tool for endocrine monitoring in laboratory and point-of-care (POC) settings.
Clinica chimica acta; international journal of clinical chemistry 01/2009; 403(1-2):56-62. · 2.54 Impact Factor
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ABSTRACT: A broad array of biological responses ranging from cell polarization, movement, immune and inflammatory responses, as well as prevention of HIV-1 infection, are triggered by the chemokines, a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins. Although it was initially believed that chemokine receptors act as monomeric entities, it has now been shown that they function as oligomers. Chemokine receptor homo- and heterodimers are found on the cell membrane; binding to their ligands stabilizes specific receptor conformations and activates distinct signaling cascades. Thorough analysis of the conformations adopted by the receptors at the membrane is therefore a prerequisite for understanding the function of these inflammatory mediators. For study of the chemokine receptor conformations at the cell surface, we focus here on conventional biochemical and genetic methods, as well as on new imaging techniques such as those based on resonance energy transfer; we also evaluate in vitro and in vivo methods to determine certain chemokine receptor functions.
Methods in molecular biology (Clifton, N.J.) 01/2009; 571:179-98.
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ABSTRACT: Described in this unit are methods for measuring the cell migration process. While cell adhesion protocols allow study of migrating cell interactions with the endothelial matrix, cellular migration assays permit analysis of directed cell movement towards a chemotactic gradient, both in vivo and in vitro. An in vitro cell invasion protocol is provided for analysis of the sum of the cell adhesion, migration, and invasion activities involved in tumor cell motility.
Current protocols in pharmacology 06/2008; Chapter 12:Unit 12.11.
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ABSTRACT: The CXCR4 chemokine receptor and the delta opioid receptor (DOR) are pertussis toxin-sensitive G protein-coupled receptors (GPCR). Both are widely distributed in brain tissues and immune cells, and have key roles in inflammation processes and in pain sensation on proximal nerve endings. We show that in immune cells expressing CXCR4 and DOR, simultaneous addition of their ligands CXCL12 and [D-Pen2, D-Pen5]enkephalin does not trigger receptor function. This treatment does not affect ligand binding or receptor expression, nor does it promote heterologous desensitization. Our data indicate that CXCR4 and DOR form heterodimeric complexes that are dynamically regulated by the ligands. This is compatible with a model in which GPCR oligomerization leads to suppression of signaling, promoting a dominant negative effect. Knockdown of CXCR4 and DOR signaling by heterodimerization might have repercussions on physiological and pathological processes such as inflammation, pain sensation and HIV-1 infection.
European Journal of Immunology 03/2008; 38(2):537-49. · 5.10 Impact Factor
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ABSTRACT: The pathophysiology of asthma is characterized by accumulation and activation of several cell types in the lung, which correlates with coordinated production of specific cytokines and chemokines. To study the effect of selective CCR2 chemokine receptor blockade on leukocyte recruitment to the lung and on bronchial function, we used a nonhuman primate model of allergic airway disease that closely resembles human asthma. Allergic cynomolgus monkeys were treated with the antagonist anti-CCR2 (CCR2-05) monoclonal antibody and then challenged with Ascaris suum antigen; the effect of antibody treatment on macrophage and eosinophil infiltration was determined. Pulmonary function was calculated by measurement of lung resistance and dynamic compliance. Local inflammatory responses were analyzed after intradermal challenge with A. suum antigen. CCL2 up-regulation in bronchoalveolar lavage (BAL) was analyzed by enzyme-linked immunosorbent assay, and in vitro CCR2-05 antagonistic activity was tested in monkey peripheral blood mononuclear cells using chemotaxis and calcium mobilization assays. The results show that neutralization of CCR2 reduces antigen-induced bronchial hyper-responsiveness and attenuates macrophage and eosinophil accumulation in the BAL of asthmatic monkeys. The results confirm that selective blockade of a single chemokine receptor involved in early stages of asthma can condition later disease stages and suggest the utility of anti-CCR2-neutralizing monoclonal antibodies in the treatment of asthma in man.
Journal of Pharmacology and Experimental Therapeutics 03/2008; 324(2):769-75. · 3.83 Impact Factor
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Oscar M Pello,
María del Carmen Moreno-Ortiz, José Miguel Rodríguez-Frade,
Laura Martínez-Muñoz,
Daniel Lucas,
Lucio Gómez,
Pilar Lucas,
Enrique Samper,
Miguel Aracil,
Carlos Martínez,
Antonio Bernad,
Mario Mellado
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ABSTRACT: The chemokine CXCL12 influences self-renewal and differentiation of hematopoietic stem cell precursors in bone marrow by directing them toward specific stromalcell components. CXCL12 up-regulates members of the SOCS family through JAK/STAT activation, a mechanism that attenuates chemokine responses. SOCS expression may thus modulate retention of hematopoietic precursors (Sca-1(+) c-Kit(+)Lin(-) cells) in bone marrow. We show that in bovine growth hormone transgenic mice and in growth hormone-treated mice, SOCS up-regulation correlated with a large number of Sca-1(+) c-Kit(+)Lin(-) cells in blood. Retroviral transduction of SOCSs blocked in vitro migration of Sca-1(+)c-Kit(+)Lin(-) cells, as well as their capacity to reconstitute lethally irradiated mice. Furthermore, in lethally irradiated mice reconstituted with bone marrow infected by a tetracycline-regulated, SOCS-expressing lentiviral vector, doxycycline treatment promoted rapid, extensive precursor mobilization to the periphery. The results indicate that by blocking CXCR4-mediated functions, SOCSs modulate hematopoietic precursor cell retention in bone marrow, and suggest the therapeutic interest of SOCS manipulation in several pathologic situations.
Blood 01/2007; 108(12):3928-37. · 9.90 Impact Factor
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ABSTRACT: Inflammatory reactions involve a network of chemical and molecular signals that initiate and maintain host response. In inflamed tissue, immune system cells generate opioid peptides that contribute to potent analgesia by acting on specific peripheral sensory neurons. In this study, we show that opioids also modulate immune cell function in vitro and in vivo. By binding to its specific receptor, the opioid receptor-specific ligand DPDPE triggers monocyte adhesion. Integrins have a key role in this process, as adhesion is abrogated in cells treated with specific neutralizing anti-alpha5beta1 integrin mAb. We found that DPDPE-triggered monocyte adhesion requires PI3Kgamma activation and involves Src kinases, the guanine nucleotide exchange factor Vav-1, and the small GTPase Rac1. DPDPE also induces adhesion of pertussis toxin-treated cells, indicating involvement of G proteins other than Gi. These data show that opioids have important implications in regulating leukocyte trafficking, adding a new function to their known effects as immune response modulators.
The Journal of Immunology 03/2006; 176(3):1675-85. · 5.79 Impact Factor
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ABSTRACT: G protein-coupled receptors are involved in the regulation of many aspects of normal physiology and pathology. Recent research has broadened our view of how the cell transduces ligand binding to cellular responses. It is becoming clear that phenomena that take place both at the cell surface, such as receptor oligomerization, as well as intracellularly, such as interaction between different signaling pathways, have important roles in the response elicited by a ligand. The study of these events requires the combined use of classical biochemical techniques with novel methods that allow analysis of these mechanisms. This chapter gives an overview of both types of techniques, with an emphasis on discussing their main applications and the conclusions that can be drawn in each case.
Methods in molecular biology (Clifton, N.J.) 02/2006; 332:141-57.
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ABSTRACT: G protein-coupled receptors are involved in the regulation of many aspects of normal physiology and pathology. Recent research
has broadened our view of how the cell trans-duces ligand binding to cellular responses. It is becoming clear that phenomena
that take place both at the cell surface, such as receptor oligomerization, as well as intracellularly, such as interaction
between different signaling pathways, have important roles in the response elicited by a ligand. The study of these events
requires the combined use of classical biochemical techniques with novel methods that allow analysis of these mecha-nisms.
This chapter gives an overview of both types of techniques, with an emphasis on discussing their main applications and the
conclusions that can be drawn in each case.
Key WordsGPCR–dimerization–G protein–kinases–immunoprecipitation–West-ern blot–resonance energy transfer–fusion proteins
12/2005: pages 141-158;
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ABSTRACT: Members of the human chemokine family are considered a suitable target for therapeutic intervention, as they have a fundamental role in several important human diseases. Here we outline potential new areas of intervention based on recent findings on chemokine receptor function.
Mini Reviews in Medicinal Chemistry 10/2005; 5(9):781-9. · 2.53 Impact Factor
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ABSTRACT: Solving relevant biological problems requires answering complex questions. Addressing such questions traditionally implied the design of time-consuming experimental procedures which most of the time are not accessible to average-sized laboratories. The current trend is to move towards a multidisciplinary approach integrating both theoretical knowledge and experimental work. This combination creates a powerful tool for shedding light on biological problems. To illustrate this concept, we present here a descriptive example of where computational methods were shown to be a key aspect in detecting crucial players in an important biological problem: the dimerization of chemokine receptors. Using evolutionary based sequence analysis in combination with structural predictions two CCR5 residues were selected as important for dimerization and further validated experimentally. The experimental validation of computational procedures demonstrated here provides a wealth of valuable information not obtainable by any of the individual approaches alone.
Bioinformatics 10/2005; 21 Suppl 2:ii13-8. · 5.47 Impact Factor
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ABSTRACT: Coordinated action between cytokines and chemokines is required for effective endocrine and immune responses. Proteins of both families promote receptor oligomerization, activation of the Janus kinase (JAK)/STAT pathway, and transcription of many genes, including the suppressor of cytokine signaling (SOCS) family. In this study, we show that chemokine-mediated SOCS1 and SOCS3 up-regulation modulates the signaling and function associated to a cytokine receptor, both in vitro and in vivo. The effect is mediated by SOCS binding to JAK2 and to the cytokine receptor, which blocks subsequent signaling events. The data reinforce the premise of cytokine-chemokine cross-talk, which helps contribute to modulating individual responses and in defining the functional plasticity of the immune system.
Journal of Biological Chemistry 11/2004; 279(43):44460-6. · 4.77 Impact Factor
