Laura Martínez Muñoz

HMGB1 promotes recruitment of inflammatory cells to damaged tissues by forming a complex with CXCL12 and signaling via CXCR4

CXCL12 forms a complex with HMGB1 that binds to the chemokine receptor CXCR4 and increases inflammatory cell migration.

Chemokine receptor oligomerization: functional considerations.

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.

Dynamic Regulation of CXCR1 and CXCR2 Homo- and Heterodimers

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.

Chemokine receptor oligomerization: a further step toward chemokine function.

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.

EBI2 regulates CXCL13-mediated responses by heterodimerization with CXCR5

B‐cell movement into lymphoid follicles depends on the expression of the chemokine receptor CXCR5 and the recently reported Epstein‐Barr virus‐induced receptor 2 (EBI2). In cooperation with CXCR5, EBI2 helps to position activated B cells in the follicle, although the mechanism is poorly understood. Using human HEK293T cells and fluorescence resonance energy transfer (FRET) techniques, we demonstrate that CXCR5 and EBI2 form homo‐ and heterodimers. EBI2 expression modulated CXCR5 homodimeric complexes, as indicated by the FRET50 value (CXCR5 homodimer, 0.9851±0.0784; CXCR5 homodimer+EBI2, 1.7320±0.4905; P<0.05). HEK293T cells expressing CXCR5/EBI2 and primary activated murine B cells both down‐modulated CXCR5‐mediated responses, such as Ca2+ flux, cell migration, and MAPK activation; this modulation did not occur when primary B cells were obtained from EBI2–/– mice. The mechanism involves a reduction in binding affinity of the ligand (CXCL13) for CXCR5 (KD: 5.05×10–8 M for CXCR5 alone vs. 1.49×10–7 M for CXCR5/EBI2) and in the efficacy (Emax) of G‐protein activation in CXCR5/EBI2‐coexpressing cells (42.33±4.3%; P<0.05). These findings identify CXCR5/EBI2 heterodimers as functional units that contribute to the plasticity of CXCL13‐mediated B‐cell responses.—Barroso, R., Muñoz, L. Martínez., Barrondo, S., Vega, B., Holgado, B. L., Lucas, P., Baíllo, A., Sallés, J., Rodríguez‐Frade J. M., Mellado, M. EBI2 regulates CXCL13‐mediated responses by heterodimerization with CXCR5. FASEB J. 26, 4841–4854 (2012). www.fasebj.org

Receptor oligomerization: a pivotal mechanism for regulating chemokine function.

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.

Technical Advance: Surface plasmon resonance-based analysis of CXCL12 binding using immobilized lentiviral particles

Use of SPR‐based biosensors is an established method for measuring molecular interactions. Their application to the study of GPCRs is nonetheless limited to detergent‐solubilized receptors that can then be reconstituted into a lipid environment. Using the chemokine receptor CXCR4 and its specific ligand CXCL12, we outline here a highly reproducible biosensor method based on receptor presentation on the surface of lentiviral particles; the approach is simple and does not require the use of antibodies to achieve correct receptor orientation on the sensorchip surface. We measured the kinetic parameters of CXCR4/CXCL12 binding in a single step and in real time and evaluated the effect of GAG presentation of chemokines on this interaction. The data indicate that at low concentrations, soluble heparin modulates CXCR4/CXCL12 interaction and at high concentrations, abrogates binding. These observations suggest that in addition to their known role in modulating local chemokine availability, GAG affect the receptor/ligand interaction, although their influence on affinity parameters is very limited. The method will also be useful for quantifying these biomarkers in biological fluids and for the development of high‐throughput screening for their antagonists.

Chemokine receptor dimerization and chemotaxis.

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.

Chapter 5. Multiple approaches to the study of chemokine receptor homo- and heterodimerization.

Chemokines belong to a family of structurally related chemoattractant proteins that bind to specific seven-transmembrane receptors linked to G proteins. They are implicated in a variety of biologic responses ranging from cell polarization, movement, immune and inflammatory responses, as well as prevention of HIV-1 infection and cancer metastasis. Recent evidence indicates that chemokine receptors can adopt several conformations at the cell membrane. Chemokine receptor homo- and heterodimers preexist on the cell surface, even in the absence of ligands. Chemokine binding stabilizes specific receptor conformations and activates distinct signaling cascades. Analysis of the conformations adopted by the receptors at the membrane and their dynamics is crucial for a complete understanding of the function of these inflammatory mediators. We focus here on conventional biochemical and genetic methods, as well as on new imaging techniques such as those based on resonance energy transfer, discussing their advantages, disadvantages, and possible complementarity in the analysis of chemokine receptor dimerization.