Chemokine receptor homo- or heterodimerization activates distinct signaling pathways
2001; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.1093/emboj/20.10.2497
ISSN1460-2075
Autores Tópico(s)T-cell and B-cell Immunology
ResumoArticle15 May 2001free access Chemokine receptor homo- or heterodimerization activates distinct signaling pathways Mario Mellado Mario Mellado Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author José Miguel Rodríguez-Frade José Miguel Rodríguez-Frade Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Antonio J. Vila-Coro Antonio J. Vila-Coro Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Silvia Fernández Silvia Fernández Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Ana Martín de Ana Ana Martín de Ana Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author David R. Jones David R. Jones Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author José L. Torán José L. Torán Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Carlos Martínez-A Corresponding Author Carlos Martínez-A Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Mario Mellado Mario Mellado Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author José Miguel Rodríguez-Frade José Miguel Rodríguez-Frade Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Antonio J. Vila-Coro Antonio J. Vila-Coro Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Silvia Fernández Silvia Fernández Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Ana Martín de Ana Ana Martín de Ana Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author David R. Jones David R. Jones Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author José L. Torán José L. Torán Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Carlos Martínez-A Corresponding Author Carlos Martínez-A Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Search for more papers by this author Author Information Mario Mellado1, José Miguel Rodríguez-Frade1, Antonio J. Vila-Coro1, Silvia Fernández1, Ana Martín de Ana1, David R. Jones1, José L. Torán1 and Carlos Martínez-A 1 1Department of Immunology and Oncology, Centro Nacional de Biotecnología/CSIC, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2497-2507https://doi.org/10.1093/emboj/20.10.2497 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Introduction The chemokines are a family of pro-inflammatory cytokines that attract and activate specific leukocyte types (Baggiolini, 1998). Based on the position of the first two canonical cysteine residues and the chromosomal location of the corresponding genes, two main chemokine families have been identified: CC and CXC (Rollins, 1997; Baggiolini, 1998). They act on monocytes, lymphocytes, natural killer (NK) cells, basophils, eosinophils and neutrophils (Rossi and Zlotnick, 2000). Chemokines mediate their effects via interactions with seven-transmembrane-domain glycoprotein receptors coupled to a G protein signaling pathway (G-protein-coupled receptors or GPCRs). This type of receptor consists of a single polypeptide chain with an extracellular N-terminal domain and a cytoplasmic C-terminal domain. The N-terminus and the extracellular domains have been implicated in receptor–ligand interaction, whereas the C-terminus and the intracellular domains cooperate to bind and activate the G proteins (Bockaert and Pin, 1999). After binding to their specific receptors, and as occurs for other GPCRs (Hebert et al., 1996; Romano et al., 1996; Cvejic et al., 1997; Bai et al., 1998; Zeng et al., 1999), chemokines induce receptor homodimerization and subsequently activate the receptor-associated JAK kinase, possibly by transphosphorylation on tyrosine residues (Mellado et al., 2001). This may create Src homology 2 (SH2) docking sites, leading to the recruitment of STAT (signal transducers and activators of transcription) transcription factors. The highly conserved Tyr present in the DRY motif is a primary target for chemokine receptor phosphorylation; a Tyr-to-Phe mutation impairs Gi-mediated Ca2+ flux triggered by chemokine binding, as well as Gi association with the chemokine receptor (Mellado et al., 1998). This mutant form of the receptor behaves as a dominant negative, since co-transfection with wild-type receptor impairs its function (Rodríguez-Frade et al., 1999b). In another member of the seven-transmembrane-domain receptor family, it has been demonstrated that the response to γ-aminobutyrate (GABA) requires heterodimerization of the GABA receptor type 1 (GBR1) and GBR2 receptors (Kaupmann et al., 1998; White et al., 1998; Kuner et al., 1999), as physical interaction between GBR1 and GBR2 appears to be essential for the activation of potassium channels. Another group of GPCRs, the opioid receptors, undergoes heterodimerization (Jordan and Devi, 1999). In this case, there is clear biochemical and pharmacological evidence for the heterodimeriza tion of two functional opioid receptors, κ and δ. Heterodimerization of these receptors causes synergistic agonist binding and potentiates the biological signal, yet there are no biochemical data to explain this phenomenon. A polymorphism reported for the CCR2 receptor, in which Val64 is replaced by Ile (CCR2V64I) and which occurs at an allelic frequency of 10–25%, is associated with a 2–4 year delay in progression to acquired immunodeficiency sydrome (AIDS) (Lee et al., 1998). Relatively few viral strains are reported to use CCR2 in conjunction with CD4 to infect cells (Premack and Schall, 1996; Berger et al., 1999); we have shown that the mechanism underlying this protective effect may be the ability of the CCR2V64I mutant receptor to heterodimerize with CCR5 and/or CXCR4 (Mellado et al., 1999). The majority of chemokine receptors bind more than one chemokine; in addition, most cell types express multiple chemokine receptors, so that if one ligand or receptor is defective, an alternative set of chemokines and their receptors can carry out the biological function (Wuyts et al., 1997; Wolf et al., 1998; Johnston et al., 1999). Although in vitro studies show overlapping functions for several chemokines, their in vivo expression and function appear to be finely controlled. Three mechanisms can be conceived to participate in this control: (i) chemo kine or chemokine receptor availability; (ii) ligand– receptor interaction; and (iii) the signal transduction mechanism activated by the chemokine receptor. Here we examine the dynamic interactions between chemokines and cell surface chemokine receptors, and analyze how the presence of several chemokine receptors regulates the response to a specific chemokine. Our results provide biochemical and functional evidence for CCR2 and CCR5 receptor heterodimerization. These heterodimers are more efficient at inducing biological responses, illustrated by the 10- to 100-fold lower chemokine concentration required to trigger these responses. This increase occurs via the synergistic interaction of several signaling complexes recruited by each individual receptor. Furthermore, receptor heterodimerization associates specific signaling pathways, such as recruitment of Gq/11, a G protein insensitive to pertussis toxin (PTx). Heterodimeric chemokine receptor interaction may have implications in understanding the in vivo processes that hinder leukocyte rolling on blood vessels and induce leukocyte parking in tissues during inflammatory responses. Results The simultaneous presence of chemokines triggers a synergistic response mediated by heterodimerization of their receptors Using human embryonic kidney (HEK)-293 cells co-transfected with CCR2b and CCR5 receptors, we evaluated the potential of these chemokine receptors to induce functional responses following stimulation with a combination of chemokine ligands. The expression levels of the two receptors were quantified by flow cytometric analysis (Figure 1A) (Poncelet and Lavabre-Bertrand, 1993) and by their ability to respond in chemotaxis and in Ca2+ flux experiments to monocyte chemotactic protein-1 (MCP-1) or RANTES (regulated upon activation, normal T cell-expressed and secreted) (Figure 1B). In these cells, MCP-1 and RANTES sensitized responses to the homologous, but not to the heterologous chemokine. When MCP-1 and RANTES were added simultaneously to CCR2- and CCR5-co-transfected HEK-293 cells, Ca2+ flux was triggered at a concentration much lower than that required to induce a response by either chemokine alone (0.1 nM versus 1 nM; Figure 1C), indicating a cooperative effect when the two receptors bind their ligands simultaneously. Figure 1.Simultaneous MCP-1 and RANTES co-activation of CCR2- and CCR5-expressing cells increases sensitivity of chemokine responses and promotes their heterodimerization. (A) CCR2b/CCR5 double-transfected HEK-293 cells were incubated with biotin-labeled mAbs against CCR2 and CCR5 or their respective isotype-matched control mAbs, followed by isothiocyanate-labeled streptavidin. (B) Ca2+ mobilization was induced by treatment with 10 nM MCP-1 or 10 nM RANTES in Fluo-3-loaded CCR2/CCR5-co-transfected HEK-293 cells. Results are expressed as a percentage of the chemokine-induced calcium response. Five experiments were performed; the figure depicts one representative experiment. Arrows indicate addition of stimulus. (C) Ca2+ mobilization was determined as in (B), following stimulation with different concentrations of MCP-1 or RANTES as indicated, added separately or simultaneously. Results are expressed as a percentage of the maximum chemokine-induced calcium response. The mean ± SD of four independent experiments is shown. (D) CCR2/CCR5-co-transfected HEK-293 cells were stimulated with chemokines (10 nM for 5 min at 37°C) and, where indicated, cross-linked with 1 mM DSS. Cell lysates were immunoprecipitated with anti-CCR2 antibody, electrophoresed and transferred to nitrocellulose membranes. The western blot was analyzed with anti-CCR5 antibody (left); as a positive control, unstimulated CCR2/CCR5-co-transfected HEK-293 cells were immunoprecipitated with anti-CCR5 antibody (lane 6). The membrane was stripped and reprobed with anti-CCR2 antibody as a control for protein loading (right). Arrows indicate the position to which monomers and dimers migrated. Download figure Download PowerPoint We have shown that the initiation of chemokine signaling through the CCR2, CCR5 and CXCR4 chemokine receptors involves ligand-triggered receptor homodimerization (Rodríguez-Frade et al., 1999a,b; Vila-Coro et al., 1999). In an attempt to understand the mechanisms underlying the RANTES- and MCP-1-promoted synergistic response in Ca2+ mobilization, and based on the observed heterodimerization of other GPCRs, we tested whether chemokine binding also triggered receptor heterodimerization. CCR2b/CCR5-co-transfected HEK- 293 cells were stimulated with MCP-1, RANTES or equimolar concentrations of both. Cells were then cross-linked using disuccinimidyl suberate (DSS), lysed and immunoprecipitated with an anti-CCR2 antibody, and the western blot was developed with anti-CCR5 (Figure 1D, left) or anti-CCR2 antibodies (Figure 1D, right). In accordance with previous results (Rodríguez-Frade et al., 1999b), stimulation with MCP-1 alone induced dimerization of the CCR2 receptor (Figure 1D, right), but not of the CCR5 receptor (Figure 1D, left), as determined by the presence of higher molecular weight complexes. In the converse experiment, RANTES induced homodimerization of the CCR5 receptor, but not of the CCR2 receptor (not shown). The simultaneous presence of MCP-1 and RANTES promoted the formation of CCR2 homodimers (Figure 1D, right), CCR5 homodimers (not shown) and, interestingly, CCR2–CCR5 heterodimers (Figure 1D, left). The same results were obtained when double-transfectant HEK-293 cells were stimulated simultaneously with MCP-1 and RANTES, lysed and immunoprecipitated with anti-CCR5 antibody, and the western blot developed with anti-CCR2 antibody (not shown). We conclude, therefore, that the CCR2 and CCR5 receptors can form heterodimers following simultaneous stimulation with the ligands of both receptors. Neither synergistic chemokine responses nor heterodimerization were observed in cells expressing CCR2 and CXCR4 after stimulation with MCP-1 and SDF-1α (not shown). Chemokine receptor heterodimerization regulates chemokine responses We showed previously that the CCR2bY139F mutant under goes receptor homodimerization in response to MCP-1 and heterodimerization with the CCR2b wild-type receptor, blocking the response to MCP-1 (Rodríguez-Frade et al., 1999b). To evaluate the functional consequences of CCR5 and CCR2 heterodimerization, CCR5 and the dominant-negative CCR2bY139F mutant receptor (Rodríguez-Frade et al., 1999b) were co-transfected into HEK-293 cells. Receptor expression in these cells was quantified by flow cytometry using specific antibodies (Figure 2A). CCR2bY139F expression in HEK-293 cells resulted in an impaired response to MCP-1 (Figure 2B), as would be predicted based on previous results. Co-expression of CCR5 and CCR2bY139F did not affect the RANTES Ca2+ mobilization response, but co-addition of MCP-1 and RANTES blocked this response (Figure 2B). In control experiments, CCR2bY139F did not block the response to SDF-1α, even with simultaneous addition of MCP-1 and SDF-1α (Figure 2C), whose receptor, CXCR4, is constitutively expressed in HEK-293 cells. Here we demonstrate that, in the presence of RANTES and MCP-1, the CCR2bY139F mutant also dimerizes with the CCR5 receptor. Indeed, when cells were cross-linked and immunoprecipitated with anti-CCR2b antibody and the western blot developed with anti-CCR5 antibody (Figure 2D, left), we observed heterodimer formation only in the presence of both MCP-1 and RANTES (Figure 2D, left, lane 5). In anti-CCR2b antibody immunoprecipitates developed in western blotting with the same antibody, CCR2 dimers were observed after stimulation with MCP-1 or MCP-1 plus RANTES (Figure 2D, right). We conclude that these two receptors, CCR5 and CCR2bY139F, undergo heterodimerization after stimulation with MCP-1 plus RANTES, impairing the downstream responses to RANTES. Figure 2.The mutant CCR2bY139F receptor impairs the response to RANTES but to SDF-1α. (A) CCR2bY139F/CCR5 double-transfected HEK-293 cells were incubated with biotin-labeled mAbs to CCR2, CCR5 or CXCR4 or their respective isotype-matched control mAbs, followed by isothiocyanate-labeled streptavidin. (B) Ca2+ flux was triggered by 10 nM RANTES, 10 nM MCP-1, or a combination of both chemokines (0.1 nM each) as indicated using CCR2bY139F/CCR5-co-transfected HEK-293 cells. Results are expressed as a percentage of the maximum chemokine-induced Ca2+ response. The figure depicts one representative experiment of four performed. (C) Ca2+ mobilization was determined as in Figure 1B, following stimulation of CCR2b Y139F/CCR5-co-transfected HEK-293 cells with different concentrations of MCP-1 or SDF-1α, separately or simultaneously as indicated. Results are expressed as a percentage of the maximum chemokine-induced response. The mean ± SD of three independent experiments is shown. (D) HEK-293 cells co-transfected with the CCR2bY139F and CCR5 receptors were processed as in Figure 1D. Arrows indicate the monomer and the dimer. Download figure Download PowerPoint To exclude the possibility that the synergistic response observed with the combination of RANTES and MCP-1 was due to CCR5 and CCR2 receptor overexpression, we tested the effects of these two chemokines on peripheral blood mononuclear cells (PBMC) derived from a CCR5 homozygotic donor and on PBMC derived from a donor bearing the CCR5Δ32 mutation and therefore not expressing the CCR5 receptor (Benkirane, 1997) (Figure 3A). Expression of CCR2 and CCR5 was assessed by flow cytometry using specific antibodies. In CCR5 wild-type donors, 15–20% of CD3+ and 30–35% of CD14+ cells expressed both types of receptor, and very few cells were detected that expressed CCR2 or CCR5 alone. PBMC from the CCR5Δ32 donor showed similar CCR2 levels and cell distribution, and no significant expression of CCR5 (Figure 3B). When combined, RANTES and MCP-1 triggered both a chemotactic response (Figure 3C, left) and Ca2+ flux (Figure 3D, upper panel) at a concentration 10–100 times lower than that of either one alone in CCR5 homozygotic donors. This synergistic effect was not seen for the CCR5Δ32 (Figure 3D, lower panel), although we observed a RANTES-mediated response when PBMC from this donor were stimulated with the chemokine. This synergistic effect may be due to a RANTES-mediated response using a receptor other than CCR5; in fact, MIP-1β, a specific ligand for CCR5, promoted no response (not shown). To confirm that heterodimerization requires specific, simultaneous activation of both receptors, we used antagonistic anti-CCR2 and -CCR5 monoclonal antibodies (mAbs) (Mellado et al., 1998; Rodríguez-Frade et al., 1999a). Both of these mAbs completely blocked the synergistic response in PBMC from CCR5 homozygous donors (Figure 3C, right), suggesting that heterodimerization is involved in this response. Figure 3.Simultaneous MCP-1 and RANTES co-activation of PBMC increases sensitivity of chemokine responses. (A) Amplification of gene fragments corresponding to the CCR5 (245 bp) and CCR5Δ32 (213 bp) with specific primers as described in Materials and methods using genomic DNA from PBMC isolated from CCR5-homozygous and CCR5Δ32-homozygous donors. (B) PBMC from CCR5 wild-type and CCR5Δ32 donors were incubated with anti-CCR2, anti-CCR5 mAbs or their respective isotype-matched control mAbs in the presence of an excess of human immunoglobulins, followed by fluorescein isothiocyanate-labeled anti-mouse IgG and phycoerythrin (PE)-labeled anti-mouse IgM antibodies. The figure also shows the percentage of double-staining cells and single positives. (C) PBMC from CCR5- and CCR5Δ32-homozygous donors were allowed to migrate following stimulation with MCP-1 or RANTES, added separately or simultaneously as indicated. The migration index was calculated as described in Materials and methods. Data represent the mean of quadruplicate determinations, with the SD indicated. Migration of PBMC from CCR5-homozygous donors in response to 0.1 nM MCP-1 plus 0.1 nM RANTES was blocked by pre-treatment of the cells with antibodies against CCR2 and CCR5 (50 μg/ml for 30 min at 37°C). As a control, pre-treatment with isotype-matched antibodies is also shown. (D) Ca2+ flux was triggered by 10 nM or 0.1 nM RANTES, MCP-1, or a combination, using PBMC from CCR5- and CCR5Δ32-homozygous donors. Results are expressed as a percentage of the maximum chemokine-induced calcium response. The figure depicts one representative experiment of four performed. Download figure Download PowerPoint Receptor heterodimerization results in recruitment of both receptor-associated signaling complexes We next analyzed whether heterodimers could recruit the signaling complex associated with either receptor. In RANTES-stimulated, CCR5-transfected HEK-293 cells, we identified JAK1, but not JAK2 or JAK3, associated with the CCR5 receptor, whereas in CCR2-transfected HEK-293 cells, MCP-1 stimulation promoted JAK2 association with the receptor (Mellado et al., 1998; Rodríguez-Frade et al., 1999a). The identification of the downstream signaling pathway activated by JAK1/JAK2 kinases has also revealed phosphorylated STAT5 transcription factors in anti-CCR5 immunoprecipitates (Rodríguez-Frade et al., 1999a) and STAT3 in anti-CCR2 immunoprecipitates (Mellado et al., 1998). As predicted, when CCR2- and CCR5-co-transfected HEK- 293 cells were stimulated with MCP-1, STAT3 was observed in the immunoprecipitates obtained with the anti-CCR2 antibody (Figure 4A, left). When these cells were stimulated with RANTES, however, anti-CCR5 antibody did not precipitate STAT3, as seen in the western blot (Figure 4A, center). When the cells were stimulated with both MCP-1 and RANTES, STAT3 was found in the CCR5 receptor immunoprecipitates (Figure 4A, right). To check that equivalent amounts of protein had been loaded, membranes were stripped and probed with the immunoprecipitating antibodies (Figure 4A, lower panels). Since STAT3 does not associate with RANTES-activated CCR5, it can be inferred that the simultaneous presence of MCP-1 and RANTES triggers the formation of a CCR2–CCR5 complex, which recruits the signaling machinery associated with each of the receptors. Figure 4.Heterodimerization of chemokine receptors results in recruitment of specific signaling events. (A) Serum-starved CCR2/CCR5-transfected HEK-293 cells (10 × 106) were used alone or treated with 10 nM MCP-1, RANTES, or a combination of both for the times indicated. Cell lysates were immunoprecipitated with anti-CCR2 (left) or anti-CCR5 antibody (center and right), and western blots were developed with anti-STAT3 antibody. As a control, an unstimulated, unprecipitated transfected HEK-293 cell lysate was analyzed in a western blot with an anti-STAT3 antibody. In each case, CCR2 or CCR5 protein loading was assessed by reprobing membranes with anti-CCR2 or -CCR5 mAb. (B) Serum-starved, CCR2bY139F/CCR5-transfected HEK-293 cells were treated with 10 nM MCP-1, 10 nM RANTES, or a combination of MCP-1 and RANTES (10 nM of each). Cell lysates were immunoprecipitated with anti-CCR2 (left) or anti-CCR5 antibody (center and right) and western blots were developed with anti-STAT5b antibody. Protein loading was controlled for as in (A). (C) Ca2+ mobilization induced by MCP-1 (10 nM), RANTES (10 nM) or MCP-1 plus RANTES (0.1 nM of each) was determined in CCR2- and CCR5-co-transfected HEK-293 cells, and in PBMC from CCR5 wild-type donors untreated or pre-incubated with PTx. The figure depicts one representative experiment of three performed. (D) PBMC from CCR5 wild-type donors were used alone or pre-incubated with PTx, as indicated, and allowed to migrate following stimulation with MCP-1 or RANTES, added separately (10 nM each) or simultaneously (0.1 nM of each) as indicated. The migration index was calculated as described in Materials and methods. Data represent the mean of quadruplicate determinations, with the SD indicated. (E) Serum-starved CCR2- and CCR5-transfected HEK-293 cells (10 × 106) were used alone or treated with 10 nM MCP-1, RANTES, or a combination of both ligands for the times indicated. Cell lysates were immunoprecipitated with anti-CCR2 or anti-CCR5 antibody, and western blots developed with anti-Gq/11 antibody. As a control, an unstimulated, unprecipitated transfected HEK-293 cell lysate was analyzed in a western blot with anti-Gq/11 antibody. As a protein loading control, membranes were reprobed with the immunoprecipitating antibody. Download figure Download PowerPoint Finally, we performed similar experiments in which HEK-293 cells co-expressing CCR5 and the CCR2b Y139F mutant were stimulated with MCP-1 and/or RANTES. STAT5b was detected in anti-CCR5 immunoprecipitates after stimulation with RANTES, but not after stimulation with MCP-1 and RANTES together (Figure 4B). These results and the impaired response to the combined stimulus in these co-transfected cells (Figure 2A) lead us to conclude that CCR2bY139F acts as a trans dominant-negative mutant, blocking RANTES responses by its ability to form non-productive complexes with partners containing the functional domain; this demonstrates the biological relevance of dimerization in chemokine responses. Chemokine receptor heterodimers recruit unique signaling pathways We have attempted to establish the molecular basis of this reduction in the threshold required to induce a biological response. Treatment with PTx abrogated both calcium release and migration in response to MCP-1 or RANTES (Figure 4C). However, when HEK-293 cells transfected with both CCR2b and CCR5 were stimulated simultaneously with 0.1 nM MCP-1 and 0.1 nM RANTES, PTx did not block the response (Figure 4C, left), illustrating the presence of a unique signaling pathway activated through receptor heterodimerization. Similar results were obtained when this assay was performed using PBMC derived from a normal donor, ruling out the possibility that this effect is an artifact due to the use of transfected cells (Figure 4C, right). In contrast, the synergistic migration induced by heterodimerization was sensitive to PTx (Figure 4D), suggesting that although Gαi is needed for chemotaxis, other factors are probably also required. Some studies report that the calcium response to chemokines is not completely blocked by PTx (Al-Aoukaty et al., 1996; Arai and Charo, 1996; Kuang et al., 1996), suggesting that chemokine receptors may couple to multiple G proteins, such as Gi, Gq or Gs, depending on the chemokine receptor and/or the chemokine used. A CCR2–CCR5 receptor-associated protein distinct from Gi was immunoprecipitated and detected in western blots using an anti-Gq/11-specific antibody when cells were stimulated with the MCP-1–RANTES mixture, but not when the chemokines were used individually (Figure 4E). This association was also observed after immunoprecipitation with the CCR5-03 antibody following heterodimer formation, but not in homodimers. Finally, the failure of a specific antibody to detect Gq (not shown) prompted the conclusion that heterodimeric CCR2–CCR5 receptors associate specifically with G11. We conclude, therefore, that, in chemokine responses, distinct pathways can be activated in the simultaneous presence of chemokines able to bind receptors susceptible to heterodimerization. The implication of G11 in heterodimer activation would explain the resistance of Ca2+ flux to PTx treatment and the reduction in the chemokine response threshold. Signaling through heterodimeric chemokine receptors fails to induce receptor down-regulation and triggers cell adhesion To characterize the biological consequences of the specific signaling pathways activated by the heterodimer in greater detail, we analyzed whether the simultaneous presence of MCP-1 and RANTES modified the internalization process mediated by the individual activation of CCR2 and CCR5 by their respective ligands. Surprisingly, the simultaneous presence of these chemokines at concentrations that trigger receptor heterodimerization and Ca2+ mobilization did not promote receptor down-regulation, as measured by flow cytometry (Figure 5A). Figure 5.Chemokine receptor hetero
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