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ABSTRACT: RasGRP1, a Ras guanine-nucleotide exchange factor, critically mediates T cell development and function and controls immunodeficiency and autoimmunity. In this study, we describe a unique mechanism of mobilization and activation of RasGRP1 in response to SDF-1, a chemokine that signals via the G protein-coupled receptor CXCR4. Depletion of RasGRP1 impaired SDF-1-stimulated human T cell migration, expression of the activation marker CD69, and activation of the ERK MAPK pathway, indicating that RasGRP1 mediates SDF-1 functions. SDF-1 treatment caused RasGRP1 to localize to the plasma membrane to activate K-Ras and to the Golgi to activate N-Ras. These events were required for cellular migration and for ERK activation that mediates downstream transcriptional events in response to SDF-1. SDF-1-dependent localization of RasGRP1 did not require its diacylglycerol-binding domain, even though diacyglycerol was previously shown to mediate localization of RasGRP1 in response to Ag stimulation. This domain was, however, required for activity of RasGRP1 after its localization. Intriguingly, SDF-1 treatment of T cells induced the formation of a novel molecular signaling complex containing RasGRP1, Gαi2, and ZAP-70. Moreover, SDF-1-mediated signaling by both Gi proteins and ZAP-70 was required for RasGRP1 mobilization. In addition, RasGRP1 mobilization and activation in response to SDF-1 was dependent on TCR expression, suggesting that CXCR4 heterodimerizes with the TCR to couple to ZAP-70 and mobilize RasGRP1. These results increase understanding of the molecular mechanisms that mediate SDF-1 effects on T cells and reveal a novel mechanism of RasGRP1 regulation. Other G protein-coupled receptors may similarly contribute to regulation of RasGRP1.
The Journal of Immunology 08/2011; 187(6):3177-85. · 5.79 Impact Factor
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Courtney L Erskine,
Christopher J Krco, Karen E Hedin,
Nancy D Borson,
Kimberly R Kalli,
Marshall D Behrens,
Sabrina M Heman-Ackah,
Eric von Hofe,
Peter J Wettstein,
Mansour Mohamadzadeh,
Keith L Knutson
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ABSTRACT: CD4 Th cells are critical to the development of coordinated immune responses to infections and tumors. Th cells are activated through interactions of the TCR with MHC class II complexed with peptide. T cell activation is dependent on the density of MHC peptide complexes as well as the duration of interaction of the TCR with APCs. In this study, we sought to determine whether MHC class II peptides could be modified with amino acid sequences that facilitated uptake and presentation with the goal of improving Th cell activation in vitro and in vivo. A model epitope derived from the murine folate receptor α, a self- and tumor Ag, was modified at its carboxyl terminus with the invariant chain-derived Ii-Key peptide and at its N terminus with a peptide that enhances uptake of Ag by APC. Modification of a peptide resulted in enhanced generation of high-avidity murine folate receptor α T cells that persisted in vivo and homed to sites of Ag deposition. The nesting approach was epitope and species independent and specifically excluded expansion of CD4 regulatory T cells. The resulting Th cells were therapeutic, enhanced in vivo helper activity and had an increased ability to resist tolerizing immune microenvironments. In addition to improved immunoadjuvants, this epitope modification strategy may be useful for enhancing ex vivo and in vivo generation of Th cells for preventing and treating diseases.
The Journal of Immunology 07/2011; 187(1):316-24. · 5.79 Impact Factor
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ABSTRACT: The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-β3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-β3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-β3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.
The Journal of Immunology 06/2011; 187(3):1440-7. · 5.79 Impact Factor
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ABSTRACT: CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and, thereby, lead to gene upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11(+) vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11(+) compartment. Trafficking of CXCR4 into the Rab11(+), TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11(+) endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11(+), recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4-TCR heterodimers. To our knowledge, this is the first report of Gα13 as a mediator of receptor trafficking.
The Journal of Immunology 01/2011; 186(2):951-8. · 5.79 Impact Factor
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ABSTRACT: Multiprotein complexes play an important role in nearly all cell functions; therefore, the characterization of protein-protein interactions in living cells constitutes an important step in the analysis of cellular signaling pathways. Using fluorescence resonance energy transfer (FRET) as a "molecular ruler" is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. Here, we describe two methods that use FRET to detect a physical interaction between the T-cell antigen receptor (TCR) and the CXCR4 chemokine receptor in living T lymphocytes. These FRET approaches use two different sets of chromophores. We discuss the design strategies, control experiments, and pitfalls involved in using these FRET approaches. Although there is no perfect pair of chromophores for FRET, the two FRET methods described here provide complementary and reliable insight into the molecular interactions between these receptor molecules.
Methods in enzymology 02/2009; 460:379-97. · 1.90 Impact Factor
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Yazan Alsayed,
Hai Ngo,
Judith Runnels,
Xavier Leleu,
Ujjal K Singha,
Costas M Pitsillides,
Joel A Spencer,
Teresa Kimlinger,
Joanna M Ghobrial,
Xiaoying Jia, [......],
Daniel Côté,
Israel Veilleux, Karen E Hedin,
G David Roodman,
Thomas E Witzig,
Andrew L Kung,
Teru Hideshima,
Kenneth C Anderson,
Charles P Lin,
Irene M Ghobrial
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ABSTRACT: The mechanisms by which multiple myeloma (MM) cells migrate and home to the bone marrow are not well understood. In this study, we sought to determine the effect of the chemokine SDF-1 (CXCL12) and its receptor CXCR4 on the migration and homing of MM cells. We demonstrated that CXCR4 is differentially expressed at high levels in the peripheral blood and is down-regulated in the bone marrow in response to high levels of SDF-1. SDF-1 induced motility, internalization, and cytoskeletal rearrangement in MM cells evidenced by confocal microscopy. The specific CXCR4 inhibitor AMD3100 and the anti-CXCR4 antibody MAB171 inhibited the migration of MM cells in vitro. CXCR4 knockdown experiments demonstrated that SDF-1-dependent migration was regulated by the P13K and ERK/ MAPK pathways but not by p38 MAPK. In addition, we demonstrated that AMD3100 inhibited the homing of MM cells to the bone marrow niches using in vivo flow cytometry, in vivo confocal microscopy, and whole body bioluminescence imaging. This study, therefore, demonstrates that SDF-1/CXCR4 is a critical regulator of MM homing and that it provides the framework for inhibitors of this pathway to be used in future clinical trials to abrogate MM trafficking.
Blood 05/2007; 109(7):2708-17. · 9.90 Impact Factor
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ABSTRACT: Costimulation by the chemokine, stromal cell-derived factor-1 (SDF-1)/CXCL12, has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however, the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082, -819, and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) ("memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker, Foxp3. To address the molecular mechanisms of this response, we showed that SDF-1 costimulates the transcriptional activities in normal human T cells of reporter plasmids containing 1.1 kb of all three of the common IL-10 promoter haplotypes. IL-10 promoter activity was ablated by mutating two nonpolymorphic binding sites for the AP-1 transcription factor, and chromatin immunoprecipitation assays of primary human T cells revealed that SDF-1 costimulation enhances AP-1 binding to both of these sites. Together, these results delineate the molecular mechanisms responsible for SDF-1 costimulation of T cell IL-10 secretion. Because it is preserved among several human haplotypes and in diverse T cell populations including Foxp3(+) T cells, this pathway of IL-10 regulation may represent a key mechanism for modulating expression of this important immunoregulatory cytokine.
The Journal of Immunology 03/2007; 178(3):1581-8. · 5.79 Impact Factor
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ABSTRACT: SDF-1alpha (CXCL12) signaling via its receptor, CXCR4, stimulates T cell chemotaxis and gene expression. The ZAP-70 tyrosine kinase critically mediates SDF-1alpha-dependent migration and prolonged ERK mitogen-activated protein (MAP) kinase activation in T cells. However, the molecular mechanism by which CXCR4 or other G protein-coupled receptors activate ZAP-70 has not been characterized. Here we show that SDF-1alpha stimulates the physical association of CXCR4 and the T cell receptor (TCR) and utilizes the ZAP-70 binding ITAM domains of the TCR for signal transduction. This pathway is responsible for several of the effects of SDF-1alpha on T cells, including prolonged ERK MAP kinase activity, increased intracellular calcium ion concentrations, robust AP-1 transcriptional activity, and SDF-1alpha costimulation of cytokine secretion. These results suggest new paradigms for understanding the effects of SDF-1alpha and other chemokines on immunity.
Immunity 09/2006; 25(2):213-24. · 21.64 Impact Factor
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ABSTRACT: To assess the clinical relevance of chemokine receptor expression on the progression of B-cell chronic lymphocytic leukemia (B-CLL).
Peripheral blood mononuclear cells from 45 patients with B-CLL were purified and compared with lymph node samples collected from 17 of these patients. Also compared were B cells obtained from peripheral blood samples from 5 healthy controls and B cells from reactive lymph nodes from 3 otherwise healthy persons. The patients were treated at the Mayo Clinic in Rochester, Minn, between January 15,1991, and February 7, 2003. Mononuclear cells were stained by a 2-color (fluorescein isothiocyanate/phycoerythrin) flow cytometric assay using antibodies to the chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CCR2, CCR4, CCR5, CCR6, and CCR7) and also to CD19.
Of the 45 patients in this study, 20 had Rai stage 0 disease, 12 had stage I disease, 3 had stage II disease, 2 had stage III disease, and 8 had stage IV disease. The mean fluorescent intensity (MFI) of the chemokine receptor expression on B-CLL cells was compared with normal controls and was not significantly different, except for an increase in the median expression of CXCR3 (P = .003) and CCR7 (P = .001) on B-CLL cells. We also found a significant increase in the expression of CXCR4 and CCR7 in B-CLL cells from patients with stage IV compared with stage 0 disease (P = .001 and P = .02, respectively). Furthermore, circulating B-CLL cells showed significantly higher expression of CXCR4 and CCR7 when compared with B lymphocytes in lymph nodes (P = .003 and P < .001, respectively).
The expression of CXCR4 and CCR7 on B-CLL cells correlates with Rai stage. Also, these chemokine receptors may be down-regulated once malignant B cells enter the lymph nodes. To our knowledge, this is the first published report that shows the strong association of Rai stage with CXCR4 and CCR7 expression levels in B-CLL cells.
Mayo Clinic Proceedings 03/2004; 79(3):318-25. · 5.70 Impact Factor
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ABSTRACT: Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1alpha is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1alpha that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1alpha stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1alpha-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1alpha to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1alpha stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.
The Journal of Immunology 08/2003; 171(1):360-7. · 5.79 Impact Factor
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Karen E Hedin
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ABSTRACT: The chemokines are a family of peptide hormones that regulate cellular adhesion, migration, proliferation, and survival. Accumulating evidence indicates critical regulatory roles for chemokines during the development of hyperplasias and metastatic tumors. Chemokines promote tumor metastasis, growth, survival, and angiogenesis. In addition, by regulating immunity, chemokines critically regulate anti-tumor immune responses and chronic inflammation such as that associated with various neoplasias. Since chemokine receptors are G-protein coupled receptors that are ideal drug targets, these discoveries presage the development of new and potent anti-cancer drugs that target chemokine receptors. Here, I review the chemokine system and chemokine regulation of neoplasias, with a special emphasis on pancreatic cancer.
International Journal of Gastrointestinal Cancer 02/2002; 31(1-3):23-9.
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ABSTRACT: Multiprotein complexes play an important role in nearly all cell functions; therefore, the characterization of protein–protein interactions in living cells constitutes an important step in the analysis of cellular signaling pathways. Using fluorescence resonance energy transfer (FRET) as a “molecular ruler” is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. Here, we describe two methods that use FRET to detect a physical interaction between the T‐cell antigen receptor (TCR) and the CXCR4 chemokine receptor in living T lymphocytes. These FRET approaches use two different sets of chromophores. We discuss the design strategies, control experiments, and pitfalls involved in using these FRET approaches. Although there is no perfect pair of chromophores for FRET, the two FRET methods described here provide complementary and reliable insight into the molecular interactions between these receptor molecules.
Methods in Enzymology.
