Mario Mellado

The chemokine SDF-1α triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway

The chemokine stromal cell‐derived factor (SDF‐1α), the ligand for the CXCR4 receptor, induces a wide variety of effects that include calcium mobilization, chemotactic responses, bone marrow myelopoiesis, neuronal patterning, and prevention of HIV‐1 infection. Nonetheless, little is known of the biochemical pathways required to achieve this variety of responses triggered after receptor—chemokine interaction. We developed a set of monoclonal antibodies that specifically recognize the CXCR4 receptor and used them to identify the signaling pathway activated after SDF‐1α binding in human T cell lines. Here we demonstrate that SDF‐1α activation promotes the physical association of Gαi with the CXCR4. Furthermore, within seconds of SDF‐1α activation, the CXCR4 receptor becomes tyrosine phosphorylated through the activation and association with the receptor of JAK2 and JAK3 kinases. After SDF‐1α binding, JAK2 and JAK3 associate with CXCR4 and are activated, probably by transphosphorylation, in a Gαi‐independent manner. This activation enables the recruitment and tyrosine phosphorylation of several members of the STAT family of transcription factors. Finally, we have also observed SDF‐1α‐induced activation and association of the tyrosine phosphatase Shpl with the CXCR4 in a Gαi‐dependent manner. As occurs with the cytokine receptors in response to cytokines, the CXCR4 undergoes receptor dimerization after SDF‐1α binding and is a critical step in triggering biological responses. We present compelling evidence that the chemokines signal through mechanisms similar to those activated by cytokines.—Vila‐Coro, A. J., Rodríguez‐Frade, J. M., Martín de Ana, A., Moreno‐Ortíz, M. C., Martínez‐A., C., Mellado, M. The chemokine SDF‐1 α triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway. FASEB J. 13, 1699–1710 (1999)

Disrupted cardiac development but normal hematopoiesis in mice deficient in the second CXCL12/SDF-1 receptor, CXCR7

Chemotactic cytokines (chemokines) attract immune cells, although their original evolutionary role may relate more closely with embryonic development. We noted differential expression of the chemokine receptor CXCR7 (RDC-1) on marginal zone B cells, a cell type associated with autoimmune diseases. We generated Cxcr7−/− mice but found that CXCR7 deficiency had little effect on B cell composition. However, most Cxcr7−/− mice died at birth with ventricular septal defects and semilunar heart valve malformation. Conditional deletion of Cxcr7 in endothelium, using Tie2-Cre transgenic mice, recapitulated this phenotype. Gene profiling of Cxcr7−/− heart valve leaflets revealed a defect in the expression of factors essential for valve formation, vessel protection, or endothelial cell growth and survival. We confirmed that the principal chemokine ligand for CXCR7 was CXCL12/SDF-1, which also binds CXCR4. CXCL12 did not induce signaling through CXCR7; however, CXCR7 formed functional heterodimers with CXCR4 and enhanced CXCL12-induced signaling. Our results reveal a specialized role for CXCR7 in endothelial biology and valve development and highlight the distinct developmental role of evolutionary conserved chemokine receptors such as CXCR7 and CXCR4.

Chemokine receptor homo‐ or heterodimerization activates distinct signaling pathways

Chemokine receptors of both the CC and CXC families have been demonstrated to undergo a ligand‐mediated homodimerization process required for Ca2+ flux and chemotaxis. We show that, in the chemokine response, heterodimerization is also permitted between given receptor pairs, specifically between CCR2 and CCR5. This has functional consequences, as the CCR2 and CCR5 ligands monocyte chemotactic protein‐1 (MCP‐1) and RANTES (regulated upon activation, normal T cell‐expressed and secreted) cooperate to trigger calcium responses at concentrations 10‐ to 100‐fold lower than the threshold for either chemokine alone. Heterodimerization results in recruitment of each receptor‐associated signaling complex, but also recruits dissimilar signaling path ways such as Gq/11 association, and delays activation of phosphatidyl inositol 3‐kinase. The consequences are a pertussis toxin‐resistant Ca2+ flux and trig gering of cell adhesion rather than chemotaxis. These results show the effect of heterodimer formation on increasing the sensitivity and dynamic range of the chemokine response, and may aid in understanding the dynamics of leukocytes at limiting chemokine concentrations in vivo.

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.

Identification of amino acid residues crucial for chemokine receptor dimerization.

Chemokines coordinate leukocyte trafficking by promoting oligomerization and signaling by G protein–coupled receptors; however, it is not known which amino acid residues of the receptors participate in this process. Bioinformatic analysis predicted that Ile52 in transmembrane region-1 (TM1) and Val150 in TM4 of the chemokine receptor CCR5 are key residues in the interaction surface between CCR5 molecules. Mutation of these residues generated nonfunctional receptors that could not dimerize or trigger signaling. In vitro and in vivo studies in human cell lines and primary T cells showed that synthetic peptides containing these residues blocked responses induced by the CCR5 ligand CCL5. Fluorescence resonance energy transfer showed the presence of preformed, ligand-stabilized chemokine receptor oligomers. This is the first description of the residues involved in chemokine receptor dimerization, and indicates a potential target for the modification of chemokine responses.

CXCR3 Chemokine Receptor Distribution in Normal and Inflamed Tissues: Expression on Activated Lymphocytes, Endothelial Cells, and Dendritic Cells

Using new human CXCR3 chemokine receptor–specific monoclonal antibodies, we studied human CXCR3 tissue distribution in lymphoid and nonlymphoid organs, as well as in inflammatory conditions, including rheumatoid arthritis, Hashimotos thyroiditis, and dermal vasculitis. CXCR3 was expressed by certain dendritic cell subsets, specifically myeloid-derived CD11c positive cells, not only in those present in normal lymphoid organs, but also in germinal centers generated in inflammatory conditions. CXCR3 expression was also detected in some lymphocyte subsets such as intraepithelial lymphocytes of secondary lymphoid organs and infiltrating lymphocytes in inflammatory conditions. In addition, CXCR3 was constitutively expressed by endothelial cells (EC) of vessels of medium and large caliber but not in small vessels from different organs. Finally, enhanced CXCR3 expression was found in EC and in infiltrating lymphocytes with an activated phenotype in inflammatory diseases. The CXCR3 chemokine receptor may play a role in the regulation of leukocyte migration to inflammatory sites.

Chemokine control of HIV-1 infection

Chemokines are proinflammatory cytokines that attract and activate specific types of leukocyte. There are two main chemokine families, based on the position of the first two cysteine residues: the CC and the CXC chemokines. Chemokines mediate their effects through interactions with seven-transmembrane-spanning glyco-protein receptors coupled to a G-protein signalling pathway. Chemokine receptors normally undergo a ligand-mediated homodimerization process, which is required for Ca2+ flux and chemotaxis. Here we show that in the chemokine response it is possible for heterodimerization, rather than homodimerization, to occur between a mutant form of the CCR2 receptor (the CCR2V64I receptor), which helps to delay the development of AIDS in HIV-1-infected individuals, and the CCR5 or CXCR4 chemokine receptor, which are used by HIV to gain entry into cells. These results may explain why AIDS takes longer to develop in HIV-1-infected individuals carrying the CCR2V64I mutation.

Ligand stabilization of CXCR4/δ-opioid receptor heterodimers reveals a mechanism for immune response regulation

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.

Chemokine receptor dimerization: two are better than one

The chemokines participate in an exceptional range of physiological and pathological processes, including the control of lymphocyte trafficking, tumor growth, wound healing, allograft rejection, regulation of T-cell differentiation, asthma, infection with HIV and atherosclerosis. This vast array of activities is triggered by the interaction of nearly 50 different chemokines with a relatively modest number of 20 G-protein-coupled receptors. The asymmetry between the number of receptors and ligands suggests an underlying, shared control mechanism activated at a very early stage of the response. One of the first events triggered by the binding of chemokines is the homo- and hetero-dimerization of their receptors; here, we outline these events and their consequences in chemokine signaling.

CCL2 Shapes Macrophage Polarization by GM-CSF and M-CSF: Identification of CCL2/CCR2-Dependent Gene Expression Profile

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF– and M-CSF–polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF–polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF–polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarization-associated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow–derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-α, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarization-associated genes and downmodulating proinflammatory cytokine production.