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*
Department of Immunology and Oncology, Centro Nacional de Biotecnología, and
Departamento de Biología Molecular, Centro de Biología Molecular "Severo Ochoa," Consejo Superior de Investigaciones Científica (CSIC)-Universidad Autónoma de Madrid, Campus de Cantoblanco, Madrid, Spain
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chemokines are a growing family of low m.w., 70- to
80-residue proinflammatory cytokines, characterized by their ability to
induce migration and activation of specific leukocyte population
subsets (2, 3, 4, 5, 6). The two main branches of the family are distinguished
by overall sequence homology, chromosomal location, and leukocyte
population specificity (7). The CXC (
) chemokines act on neutrophils
and nonhemopoietic cells involved in wound healing, whereas the CC
(ß) chemokines act on monocytes, eosinophils, basophils, NK cells,
and different lymphocyte subpopulations (5, 6). New chemokine families
have been identified recently, which maintain overall sequence homology
but lack the typical cysteine distribution (C, or 
, and
CX3C, or 
, chemokines) (8, 9).
The CC chemokine monocyte chemoattractant protein 1
(MCP-1)4 was originally
described as a potent chemoattractant for monocytes (10), produced by
different cell types in response to a variety of mediators including
PDGF (platelet-derived growth factor), TNF-, LPS, and oxidized low
density lipoproteins (11, 12). Since then, many other activities have
been assigned to MCP-1, including induction of T cell migration (13),
suppression of tumor growth in animal models (14), and neutralization
of HIV-1 (15). MCP-1 has also been associated with several disease
states in which monocyte infiltration plays a role, such as
granulomatous disease, host responses to bacteria, rheumatoid
arthritis, inflammatory heart disease, bone trauma, asthma, and
sepsis (16, 17).
Chemokines activate their functions through interaction with
single-chain, seven-helix membrane-spanning receptors coupled to G
proteins (GPCR) (3). To date, nine receptors have been defined for the
CC chemokines (18) and four for the CXC family, together with several
putative CC or CXC receptors for which the ligands remain to be
determined. They have an N terminus and three extracellular loops that
act in concert to bind the chemokine ligand (19, 20), while the three
intracellular loops and the C terminus are involved in transduction of
the chemokine signal. The activation signals following chemokine
stimulation are not well defined, and various signal transduction
pathways have been implicated through the association of the chemokine
receptors with guanine nucleotide-binding protein (G proteins) (21). In
all cases, a pertussis toxin (PTX)-sensitive G protein was found,
although there are also examples of PTX-insensitive G proteins, as is
the case for the C5a receptor, which also couples to G16 (22). The
biochemical signaling pathways include changes in intracellular cAMP
levels (23), phospholipase activation (24), increases in tyrosine
phosphorylation including the Src substrates focal adhesion kinase
(p125FAK) and ZAP-70 (25), increased
association of Src family kinases with p21ras
(26), and activation of the MAPK (mitogen-activating protein kinase)
cascade (27).
The cellular response resulting from the activation of a given chemokine receptor can vary depending on the cell in which it is expressed. This is presumably due to the availability of specific G protein subunits and other downstream effector molecules. It has recently been demonstrated that the association of chemokine receptors to different G proteins depends on the receptor and the cell line studied (28).
Biochemical analysis of chemokine signaling has been hampered by the low levels of receptor expression and by the promiscuity of individual receptors with various ligands. Using the CCR2 chemokine receptor as a model, we have analyzed early signaling events activated by MCP-1 binding in a human monocytic cell line (Mono Mac 1). MCP-1 triggers tyrosine phosphorylation and activation of the JAK2/STAT3 pathway in a PTX-independent manner. This MCP-1-initiated phosphorylation and association to JAK2 is also observed in CCR2B-transfected HEK293 cells. In contrast, when a CCR2B Tyr139Phe mutant is expressed in HEK293 cells, it is not phosphorylated in tyrosine and triggers neither JAK2/STAT3 activation nor Ca2+ mobilization in response to MCP-1. Altogether, these data indicate that within seconds of activation, JAK2 phosphorylates CCR2 at the Tyr139 position and promotes JAK2/STAT3 complex association to the receptor. The blockage of MCP-1-induced Ca2+ mobilization and cell migration in Mono Mac 1 cells treated with the specific JAK2 kinase inhibitor, tyrphostin B42 (29), demonstrates that JAK2 activation is one of the first events following ligand binding and assigns JAK2 a critical upstream role for all other signaling events, including the association between the receptor and the G proteins.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mono Mac 1 (DSM ACC252) and HEK293 cells (ATCC TIB202) were
obtained from the German Collection of Microorganisms and Cell Cultures
(Braunschweig, Germany) and the American Type Culture Collection (ATCC,
Manassas, VA), respectively. Abs used include monoclonal anti-PTyr
(4G10) and rabbit anti-JAK2 (Upstate Biotechnology, Lake
Placid, NY); anti-Gi, anti-STAT3, and
anti-STAT5 (Santa Cruz Biotechnology, Santa Cruz, CA);
anti-PTyr (PY20) (Transduction Laboratories, Lexington, KY);
anti-CD4 (HP2.6; kindly donated by Dr. A. C. Carrera), and
anti-MHC class I (W6/32, ATCC). Anti-CCR2 mAb MCP-1R03 and MCP-1R05
were generated in our laboratory (15, 30). Epidermal growth factor
(EGF)-stimulated A-431 cell lysates were from Upstate Biotechnology and
MCP-1 from Peprotech (London, U.K.).
Flow cytometry analysis
Cells were centrifuged (250 x g, 10 min, room temperature), plated in V-bottom 96-well plates (2.5 x 105 cells/well) and incubated with 50 µl/well biotin-labeled mAb (5 µg/ml, 60 min, 4°C). Cells were washed twice in PBS with 2% BSA and 2% FCS and centrifuged (250 x g, 5 min, 4°C). FITC-labeled streptavidin (Southern Biotechnologies, Birmingham, AL) was added, cells incubated (30 min, 4°C), and plates washed twice. Cell-bound fluorescence was determined in a Profile XL flow cytometer at 525 nm (Coulter Electronics, Miami, FL).
Calcium determination
Changes in intracellular calcium concentration were monitored using the fluorescent probe Fluo-3, AM (Calbiochem, La Jolla, CA). Cells (2.5 x 106 cells/ml), untreated or treated for 16 h with either cholera toxin (CTX; 0.4 µg/ml) or PTX (0.1 µg/ml), were resuspended in RPMI containing 10% FCS and 10 mM HEPES and incubated with 10 µl/106 cells of Fluo-3, AM (300 µM in DMSO) for 15 min at 37°C. After incubation, cells were washed and resuspended in complete medium containing 2 mM CaCl2 and maintained at 4°C until just before MCP-1 addition, to minimize membrane trafficking and to eliminate spontaneous Ca2+ entry. Calcium mobilization in response to 5 nM MCP-1 (Peprotech) was determined at 37°C in an EPICS XL flow cytometer at 525 nm (Coulter), and included background level stabilization and determination of the level of probe loaded for each sample. Only samples with a similar load, as assessed by determination of Ca2+ mobilization induced by a ionophore (ionomycin, 5 µg/ml; Sigma Chemicals, St. Louis, MO) are considered acceptable. When tyrphostin treatment was used, Mono Mac 1 cells were preincubated with 25 µM tyrphostin B42 or A1 (Calbiochem-Novabiochem, La Jolla, CA) for 12 h at 37°C as described (29).
Cell migration
Mono Mac 1 cells (0.5 x 106 cells/ml), untreated or preincubated with 25 µM tyrphostin B42 or A1 (Calbiochem) for 12 h at 37°C, 5% CO2 in RPMI 1640 containing 10% FCS, were placed (0.25 x 106 cells in 0.1 ml) in the upper well of 24-well transmigration chambers (Transwell; Costar, Cambridge, MA), and 5 nM MCP-1 (diluted in 0.6 ml RPMI containing 0.25% BSA) was added to the lower well. Plates were incubated 120 min at 37°C, 5% CO2, and the cells that had migrated to the lower chamber were counted as described (15).
Transfection
Human HEK293 embryonic kidney cells were transfected with human CCR2B and CCR2BY139F cDNA cloned in pcDNAIII by the calcium phosphate precipitation method. Stably transfected cells were selected in G-418 (Life Technologies, Gaithersburg, MD) and screened in flow cytometry analysis for receptor expression using anti-CCR2 Abs. All experiments were performed using at least four cell lines for each plasmid.
Site-directed mutagenesis of CCR2B Tyr139
Phenylalanine was substituted for tyrosine at residue 139 in the CCR2B receptor by overlap extension. Briefly, two primary PCR with overlapping ends were performed from the gene cloned in pUC18. One fragment encompasses positions 1 through 429 and the other, positions 405 through 1080 of the CCR2B gene. The sequence includes the target Tyr codon (TAC) at position 417, which was replaced with a phenylalanine codon (TTC). Primary PCR products were gel purified and Klenow treated to remove extra 3' bases. For secondary PCR, both primary fragments were mixed with primers including BamHI and XhoI sites at the 5' ends. After 25 cycles, the assembled fragment (1080 bp) containing Phe at position 139 was gel purified, digested with BamHI and XhoI enzymes, and cloned in pCDNA3 (Invitrogen, San Diego, CA). The presence of the Y139F mutation was confirmed by DNA sequencing.
Immunoprecipitation, SDS-PAGE, and Western blot analysis
MCP-1-stimulated cells (20 x 106) were lysed in a detergent buffer (20 mM triethanolamine, pH 8.0, 300 mM NaCl, 2 mM EDTA, 20% glycerol, 1% digitonin, with 10 µM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 30 min at 4°C with continuous rocking, then centrifuged (15,000 x g, 15 min). Immunoprecipitations were performed essentially as described earlier (31). Protein extracts precleared by incubation with 20 µg of anti-mouse IgG-agarose (Sigma Chemicals) or protein A-Sepharose (60 min, 4°C) were centrifuged (15,000 x g, 1 min), immunoprecipitated with the appropriate Ab (5 µg/sample, 120 min, 4°C), followed by anti-mouse IgG-agarose or protein A-Sepharose if the first Ab was derived from rabbit serum. Immunoprecipitates or protein extracts were separated in 12.5% SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed as described (32), using 2% BSA in Tris-buffered saline (TBS) as blocking agent for the anti-PTyr analyses. When stripping was required, membranes were incubated for 60 min at 60°C with 62.5 mM Tris-HCl, pH 7.8, containing 2% SDS and 0.5% ß-mercaptoethanol. After washing with 0.1% Tween 20 in TBS for 2 h, membranes were reblocked, reprobed with the appropriate Ab, and developed as described above. In all cases, protein loading was carefully controlled by using a protein detection kit (Pierce, Rockford, IL) and, when necessary, by reprobing the membrane with the immunoprecipitating Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human monocytic cell line Mono Mac 1 expresses the MCP-1
chemokine receptor CCR2B (33). In response to MCP-1, these cells
mobilize calcium (Fig. 1
A), become
desensitized to a second MCP-1 stimulus, and undergo cell migration
(not shown). PTX treatment abrogates both calcium release and migration
in response to MCP-1, whereas no effect was observed following
incubation with CTX (Fig. 1A). This is consistent
with other studies showing that some MCP-1 downstream signals in other
monocyte lines and T cells are coupled to PTX-sensitive G proteins
(28). Using a CCR2-specific mAb, MCP-1R03 (15, 30), we analyzed the
physical association between the CCR2 receptor and the
Gi subunit of the heterotrimeric G protein. A CCR2
receptor-associated Gi protein was immunoprecipitated and
detected in Western blot using an anti-Gi-specific
Ab (Fig. 1B). This association is initiated within
30 s of MCP-1 triggering, and persists for 5 min after chemokine
binding. As occurs in other cell systems, the CCR2 receptor, therefore,
regulates calcium release and couples to the Gi protein in
Mono Mac 1 cells.
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In MCP-1-activated Mono Mac 1 cells, a 38-kDa protein
phosphorylated in tyrosine residues was initially identified as the
CCR2 receptor (not shown). Cell lysates were immunoprecipitated with
anti-PTyr and Western blots developed with anti-CCR2 receptor
Abs (Fig. 2, upper
panel) or precipitated with anti-CCR2 and
developed with anti-PTyr Ab (Fig. 2, lower
panel), while no differences were observed in the amount of
CCR2 in each lane (Fig. 2, lower panel) when the same
membrane was stripped and reblotted with anti-CCR2 Ab. The same
phosphorylated 38-kDa band was observed in both cases, confirming
phosphorylation of the CCR2 upon MCP-1 stimulation. An increase in CCR2
receptor phosphorylation is seen as early as 30 s after MCP-1
stimulation, and phosphorylation persists for 20 to 30 min, decreasing
thereafter. We have observed residual phosphorylation of CCR2 in
untreated cells (Fig. 2), the significance of which is discussed
below.
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To ascertain which kinase is responsible for the rapid CCR2
chemokine receptor phosphorylation, Mono Mac 1 cells were stimulated
with MCP-1, and cell lysates were immunoprecipitated with anti-CCR2
or anti-CD4 as an isotype-matched Ab control. Anti-JAK2 Abs
identified a 130-kDa protein in the anti-CCR2 immunoprecipitate
(Fig. 3A). Furthermore,
JAK2 phosphorylated in tyrosine is observed after MCP-1 stimulation
(Fig. 4A), while JAK2
levels are unchanged (Fig. 4A) as determined by
stripping and reblotting the same membrane with anti-JAK2 Ab.
JAK2 association to the CCR2 receptor takes place as early as 30 s
after MCP-1 stimulation (Fig. 3A); small amounts of
JAK2 were also found associated to the CCR2 receptor in the absence of
added MCP-1, consistent with receptor phosphorylation in the absence of
exogenous ligand in Mono Mac 1 cells. Immunoprecipitation of cell
lysates with isotype-matched control Abs to CD4 or to other membrane
proteins, such as MHC class I, did not reveal the presence of JAK2
(Fig. 3A), ruling out nonspecific protein association
to membrane components under our experimental conditions. The rapid
association of JAK2 to the CCR2 receptor suggests a role for this
tyrosine kinase in early receptor phosphorylation following ligand
stimulation. Anti-JAK1 and -JAK3 Abs were used to test for the presence
of other JAK family members associated to the CCR2 receptor with
negative results (data not shown).
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B); the association of STAT3 correlates in
time with JAK2 phosphorylation and binding. Furthermore, this STAT3 is
tyrosine phosphorylated, indicating its activation (Fig. 4C). It thus appears that CCR2 receptor activation
induces receptor and JAK2 phosphorylation and STAT3 binding. The
MCP-1-triggered tyrosine phosphorylation of the CCR2 receptor is
independent of PTX-sensitive G proteins (Fig. 3B);
PTX also had no effect on JAK2 kinase binding to the receptor (Fig. 3C). Whereas MCP-1-induced association of JAK2 to the
CCR2 receptor is not impeded by this toxin, PTX blocked JAK2 kinase
dissociation from the receptor (Fig. 3C), implying
that PTX-sensitive G protein activation is important in the recycling
of the JAK2/STAT3 receptor complex.
MCP-1-promoted Ca2+ influx and cell mobilization were
blocked when cells were pretreated with the specific JAK2 kinase
inhibitor, tyrphostin B42 (29) (Fig. 5,
A and B), indicating the importance of
this kinase in early signaling events. As expected, B42 treatment
inhibits Gi association to the receptor, in accordance
with the role of this G protein in GPCR-mediated effects (Fig. 5C). Tyrphostin treatment was not toxic to Mono Mac 1
cells, as shown by cell cycle analysis using propidium iodide
incorporation (not shown).
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The Tyr139 residue of CCR2 is critical in MCP-1-activated signaling
To further investigate the significance of MCP-1-induced CCR2
receptor tyrosine phosphorylation, the CCR2B receptor
Tyr139Phe mutant CCR2BY139F was constructed, expressed
in HEK293 cells, and its response to MCP-1 analyzed. Tyr139
is located in the second intracellular loop and forms part of the DRY
sequence, a highly conserved motif in GPCR that is essential for
agonist-mediated receptor activation (35); it is the only
intracellular tyrosine conserved in the functionally homologous CCR2A
receptor (33). Wild-type and CCR2BY139F mutant receptor expression
were identical in stably transfected HEK293 cells as assessed by
flow cytometry (Fig. 6A), and both receptor
forms bind MCP-1 equally well (not shown). MCP-1 induces
Ca2+ mobilization in CCR2B wild-type HEK293 cells, but not
in CCR2BY139F mutant or in mock-transfected HEK293 cells (Fig. 6B). Phosphorylation of the mutant receptors was
analyzed; in contrast to the wild-type receptor, the CCR2BY139F mutant
is unphosphorylated in tyrosine residues following MCP-1 binding (Fig. 7, upper panel) and
cannot trigger JAK2 phosphorylation or association to the receptor
(Fig. 7, middle panel). Finally, MCP-1 promotes
Gi association to the CCR2B receptor, but not to the
mutant CCR2BY139F receptor (Fig. 7, lower panel).
These results clearly demonstrate the relevance of JAK2
phosphorylation, as well as of JAK2 and Gi association
in the functional response to MCP-1.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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MCP-1 was first described as a potent chemoattractant for monocytes and
binds the CCR2 and the CCR4 chemokine receptors (36). In
CCR2-expressing cells, MCP-1 binding promotes Ca2+
mobilization and transmigration, processes that are blocked by
PTX treatment but not by CTX (21). Using a human monocytic cell
line, Mono Mac 1, which also responds to MCP-1 and CCR2-specific mAb
(15, 30), we have demonstrated that Gi associates rapidly
(within 30 s) to CCR2 after MCP-1 activation. This confirms
previous data indicating that other chemokine receptors, such as IL-8R,
bind Gi in response to IL-8 (37) and extends the
observation to members of the CC family of chemokine receptors.
Despite Gi binding to the CCR2 receptor and its implications in signaling events subsequent to MCP-1 binding, we cannot exclude the participation of other G proteins in the signaling activated by this chemokine. It is reasonable to consider that the cellular response of a given chemokine receptor varies depending on the cell in which it is expressed. This is presumably due to the availability of specific G protein subunits and other downstream effector molecules. It has thus recently been demonstrated that the association of chemokine receptors to different G proteins depends on the receptor and the cell line studied (28).
Chemokines also promote an increase in tyrosine kinase activity; it has recently been shown that RANTES induces activation and assembly of macromolecular focal adhesion complexes and provokes T lymphocyte homotypic adhesion by phosphorylation and association of p125FAK and ZAP-70 (25).
Even more interesting is the identification of the phosphorylated CCR2 receptor itself. Several assays performed to identify the kinase responsible for CCR2 receptor phosphorylation led to the conclusion that JAK2 causes early receptor activation. Both JAK2 association and CCR2 tyrosine phosphorylation occur even in the presence of PTX, indicating no Gi participation in this process; JAK2 dissociation was not observed under these conditions, suggesting an active participation of Gi pathways in uncoupling JAK2 from the receptor. In response to MCP-1, this kinase is phosphorylated as soon as 30 s after binding, indicating that JAK2 activation is simultaneous with its association to CCR2.
Furthermore, STAT3, but not STAT5, is associated to the CCR2 following MCP-1 activation in accordance with the role assigned to the JAK tyrosine kinases in transducing signals from hemopoietic growth factor receptors (38). In these receptors, the activation and association of JAK kinase to the receptor creates docking sites for SH2-containing proteins such as STAT, leading to their phosphorylation and activation of gene transcription.
Neither MCP-1-induced, PTX-sensitive G protein-mediated physiologic
effects nor Gi association to CCR2 were observed after
treatment of Mono Mac 1 cells with the JAK2-specific inhibitor
tyrphostin B42. This is not the case when cells are treated with other
tyrphostins, indicating that inhibition of JAK2 kinase activity
abolishes the association and activation of the G proteins responsible
for this response. This result and the JAK2 association to the CCR2
receptor in PTX-treated cells imply that the first event after MCP-1
binding to the CCR2 in Mono Mac 1 cells is association of the JAK2
kinase. The conformational changes promoted by both ligand interaction
and tyrosine kinase association induce Gi protein
association to its binding site, probably located in the second
intracellular loop, as is the case for the IL-8R (37).
To determine which CCR2B receptor Tyr residue is involved in JAK2 kinase association, and as Tyr139 is the only intracellular tyrosine conserved in the functionally homologous CCR2A receptor (33), we generated a CCR2BY139F receptor by point mutation. Although this mutated receptor binds MCP-1 as well as does the wild-type receptor, it does not elicit MCP-1-triggered functional responses such as Ca2+ mobilization, cell migration, or CCR2B tyrosine phosphorylation. This lack of response is due to abolition of the association to the receptor by the JAK2 kinase and then of the Gi protein. The tyrosine at position 139 is part of the conserved DRY motif present in most chemokine receptors. Earlier studies characterized the critical roles of arginine and aspartic acid in the DRY motif, implicated in G protein-mediated signaling in other GPCR (35). The importance of the tyrosine in G protein activation is demonstrated here, as the Tyr139Phe mutation also impaired Ca2+ mobilization triggered by MCP-1 binding and Gi association to the CCR2B receptor.
These data fit a model in which Gi association to the chemokine is a consequence of conformational changes promoted by both ligand interaction and JAK2 activation. If this is the case, a mutant form with a constitutively associated Gi protein should activate signaling in response to the chemokine. This occurs, in fact, in another chemotactic peptide receptor belonging to the GPCR family, the C5a receptor (39), in which phenylalanine replaces the tyrosine in the DRY motif, while its capacity to activate Gi signaling remains intact.
Phosphorylation of the DRY motif tyrosine in a ß2-adrenergic receptor has also recently been suggested as linking this receptor to the insulin-like growth factor (IGF)-1 receptor pathway (40). In accordance with recent reports suggesting JAK pathway modulation by other GPCR (41), as well as agonist-induced ß-adrenergic receptor dimerization (42), we suggest that receptor dimerization may be a primary event following agonist binding to G protein-coupled receptors. We thus amplify earlier interpretations by suggesting that the inability of CCR2BY139F to promote Gi protein association results from the failure of the receptor to dimerize. Since signaling by G protein-coupled receptors leads to calcium mobilization within seconds, we postulate that the ligand promotes a conformational change that stabilizes the receptor dimers.
Finally, the dimerization hypothesis may also be helpful in
understanding how chemokines prevent HIV-1 infection, as we conjecture
that receptor dimerization prevents HIV-1 interaction with the
chemokine receptor. Recent reports indicate that heterodimerization
between CCR5 and its mutant CCR5
32 is a molecular explanation for
the delayed onset of AIDS in heterozygous CCR5/ccr532 individuals
(43). Our results also extend the model of cytokine receptor signaling
to the chemokines, which are functionally related molecules although
they use structurally unrelated receptors.
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Acknowledgments |
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Footnotes |
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2 M.M., J.M.R.-F., and A.A. contributed equally to the work leading to the present manuscript.
3 Address correspondence and reprint requests to Dr. Carlos Martínez-A., Department of Immunology and Oncology, Centro Nacional de Biotecnología, Universidad Autónoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail address:
4 Abbreviations used in this paper: MCP-1, monocyte chemoattractant protein 1; CTX, cholera toxin; EGF, epidermal growth factor; GPCR, G protein-coupled receptor; JAK, Janus kinase; PTX, pertussis toxin; PTyr, phosphotyrosine.
Received for publication October 2, 1997. Accepted for publication March 18, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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receptor, confers chemokine selectivity: evidence for a two-step mechanism for MCP-1 receptor activation. J. Biol. Chem. 271:19084.32. J. Biol. Chem. 272:30602.
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 J. Guo, M. Van Eck, J. Twisk, N. Maeda, G. M. Benson, P. H.E. Groot, and T. J.C. Van Berkel Transplantation of Monocyte CC-Chemokine Receptor 2-Deficient Bone Marrow Into ApoE3-Leiden Mice Inhibits Atherogenesis Arterioscler Thromb Vasc Biol, March 1, 2003; 23(3): 447 - 453. [Abstract] [Full Text] [PDF]
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 S. Pelletier, F. Duhamel, P. Coulombe, M. R. Popoff, and S. Meloche Rho Family GTPases Are Required for Activation of Jak/STAT Signaling by G Protein-Coupled Receptors Mol. Cell. Biol., February 15, 2003; 23(4): 1316 - 1333. [Abstract] [Full Text] [PDF]
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J. V. Stein, S. F. Soriano, C. M'rini, C. Nombela-Arrieta, G. G. de Buitrago, J. M. Rodriguez-Frade, M. Mellado, J.-P. Girard, and C. Martinez-A. CCR7-mediated physiological lymphocyte homing involves activation of a tyrosine kinase pathway Blood, January 1, 2003; 101(1): 38 - 44. [Abstract] [Full Text] [PDF]
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M. Rey, M. Vicente-Manzanares, F. Viedma, M. Yanez-Mo, A. Urzainqui, O. Barreiro, J. Vazquez, and F. Sanchez-Madrid Cutting Edge: Association of the Motor Protein Nonmuscle Myosin Heavy Chain-IIA with the C Terminus of the Chemokine Receptor CXCR4 in T Lymphocytes J. Immunol., November 15, 2002; 169(10): 5410 - 5414. [Abstract] [Full Text] [PDF]
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 S. F. Soriano, P. Hernanz-Falcon, J. M. Rodriguez-Frade, A. M. de Ana, R. Garzon, C. Carvalho-Pinto, A. J. Vila-Coro, A. Zaballos, D. Balomenos, C. Martinez-A., et al. Functional Inactivation of CXC Chemokine Receptor 4-mediated Responses through SOCS3 Up-regulation J. Exp. Med., August 5, 2002; 196(3): 311 - 321. [Abstract] [Full Text] [PDF]
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 A. Christopoulos and T. Kenakin G Protein-Coupled Receptor Allosterism and Complexing Pharmacol. Rev., June 1, 2002; 54(2): 323 - 374. [Abstract] [Full Text] [PDF]
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N. Wright, A. Hidalgo, J. M. Rodriguez-Frade, S. F. Soriano, M. Mellado, M. Parmo-Cabanas, M. J. Briskin, and J. Teixido The Chemokine Stromal Cell-Derived Factor-1{alpha} Modulates {alpha}4{beta}7 Integrin-Mediated Lymphocyte Adhesion to Mucosal Addressin Cell Adhesion Molecule-1 and Fibronectin J. Immunol., May 15, 2002; 168(10): 5268 - 5277. [Abstract] [Full Text] [PDF]
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M. Roque, W. J.H. Kim, M. Gazdoin, A. Malik, E. D. Reis, J. T. Fallon, J. J. Badimon, I. F. Charo, and M. B. Taubman CCR2 Deficiency Decreases Intimal Hyperplasia After Arterial Injury Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 554 - 559. [Abstract] [Full Text] [PDF]
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X.-F. Zhang, J.-F. Wang, E. Matczak, J. Proper, and J. E. Groopman Janus kinase 2 is involved in stromal cell-derived factor-1{alpha}-induced tyrosine phosphorylation of focal adhesion proteins and migration of hematopoietic progenitor cells Blood, June 1, 2001; 97(11): 3342 - 3348. [Abstract] [Full Text] [PDF]
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 K. Kito, K. Morishita, and K. Nishida MCP-1 receptor binding affinity is up-regulated by pre-stimulation with MCP-1 in an actin polymerization-dependent manner J. Leukoc. Biol., April 1, 2001; 69(4): 666 - 674. [Abstract] [Full Text]
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B. Cambien, M. Pomeranz, H. Schmid-Antomarchi, M.-A. Millet, V. Breittmayer, B. Rossi, and A. Schmid-Alliana Signal transduction pathways involved in soluble fractalkine-induced monocytic cell adhesion Blood, April 1, 2001; 97(7): 2031 - 2037. [Abstract] [Full Text] [PDF]
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B. Cambien, M. Pomeranz, M.-A. Millet, B. Rossi, and A. Schmid-Alliana Signal transduction involved in MCP-1-mediated monocytic transendothelial migration Blood, January 15, 2001; 97(2): 359 - 366. [Abstract] [Full Text] [PDF]
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 C. L. Bone-Larson, C. M. Hogaboam, M. L. Steinhauser, S. H. P. Oliveira, N. W. Lukacs, R. M. Strieter, and S. L. Kunkel Novel Protective Effects of Stem Cell Factor in a Murine Model of Acute Septic Peritonitis : Dependence on MCP-1 Am. J. Pathol., October 1, 2000; 157(4): 1177 - 1186. [Abstract] [Full Text] [PDF]
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C. Murdoch and A. Finn Chemokine receptors and their role in inflammation and infectious diseases Blood, May 15, 2000; 95(10): 3032 - 3043. [Abstract] [Full Text] [PDF]
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G. Venkatakrishnan, R. Salgia, and J. E. Groopman Chemokine Receptors CXCR-1/2 Activate Mitogen-activated Protein Kinase via the Epidermal Growth Factor Receptor in Ovarian Cancer Cells J. Biol. Chem., March 15, 2000; 275(10): 6868 - 6875. [Abstract] [Full Text] [PDF]
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 A. J. VILA-CORO, J. M. RODRÍGUEZ-FRADE, A. MARTÍN DE ANA, M. C. MORENO-ORTÍZ, C. MARTÍNEZ-A, and M. MELLADO The chemokine SDF-1{alpha} triggers CXCR4 receptor dimerization and activates the JAK/STAT pathway FASEB J, October 1, 1999; 13(13): 1699 - 1710. [Abstract] [Full Text]
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M. Vicente-Manzanares, M. Rey, D. R. Jones, D. Sancho, M. Mellado, J. M. Rodriguez-Frade, M. A. del Pozo, M. Yanez-Mo, A. M. de Ana, C. Martinez-A., et al. Involvement of Phosphatidylinositol 3-Kinase in Stromal Cell-Derived Factor-1{alpha}-Induced Lymphocyte Polarization and Chemotaxis J. Immunol., October 1, 1999; 163(7): 4001 - 4012. [Abstract] [Full Text] [PDF]
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A. J. Vila-Coro, M. Mellado, A. Martin de Ana, C. Martinez-A., and J. M. Rodriguez-Frade Characterization of RANTES- and Aminooxypentane-RANTES- Triggered Desensitization Signals Reveals Differences in Recruitment of the G Protein-Coupled Receptor Complex J. Immunol., September 15, 1999; 163(6): 3037 - 3044. [Abstract] [Full Text] [PDF]
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C. H. Kim, C.-K. Qu, G. Hangoc, S. Cooper, N. Anzai, G.-S. Feng, and H. E. Broxmeyer Abnormal Chemokine-Induced Responses of Immature and Mature Hematopoietic Cells from Motheaten Mice Implicate the Protein Tyrosine Phosphatase Shp-1 in Chemokine Responses J. Exp. Med., September 6, 1999; 190(5): 681 - 690. [Abstract] [Full Text] [PDF]
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K. S. C. Weber, P. J. Nelson, H.-J. Grone, and C. Weber Expression of CCR2 by Endothelial Cells : Implications for MCP-1 Mediated Wound Injury Repair and In Vivo Inflammatory Activation of Endothelium Arterioscler Thromb Vasc Biol, September 1, 1999; 19(9): 2085 - 2093. [Abstract] [Full Text] [PDF]
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C. Cicala, J. Arthos, M. Ruiz, M. Vaccarezza, A. Rubbert, A. Riva, K. Wildt, O. Cohen, and A. S. Fauci Induction of Phosphorylation and Intracellular Association of CC Chemokine Receptor 5 and Focal Adhesion Kinase in Primary Human CD4+ T Cells by Macrophage-Tropic HIV Envelope J. Immunol., July 1, 1999; 163(1): 420 - 426. [Abstract] [Full Text] [PDF]
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 J. M. Rodriguez-Frade, A. J. Vila-Coro, A. Martin de Ana, J. P. Albar, C. Martinez-A., and M. Mellado The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2 PNAS, March 30, 1999; 96(7): 3628 - 3633. [Abstract] [Full Text] [PDF]
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 J. M. Rodriguez-Frade, A. J. Vila-Coro, A. Martin, M. Nieto, F. Sanchez-Madrid, A. E.I. Proudfoot, T. N.C. Wells, C. Martinez-A, and M. Mellado Similarities and Differences in RANTES- and (AOP)-RANTES-triggered Signals: Implications for Chemotaxis J. Cell Biol., February 22, 1999; 144(4): 755 - 765. [Abstract] [Full Text] [PDF]
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M. Wong, S. Uddin, B. Majchrzak, T. Huynh, A. E. I. Proudfoot, L. C. Platanias, and E. N. Fish RANTES Activates Jak2 and Jak3 to Regulate Engagement of Multiple Signaling Pathways in T Cells J. Biol. Chem., March 30, 2001; 276(14): 11427 - 11431. [Abstract] [Full Text] [PDF]
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V. Lukashova, C. Asselin, J. J. Krolewski, M. Rola-Pleszczynski, and J. Stankova G-protein-independent Activation of Tyk2 by the Platelet-activating Factor Receptor J. Biol. Chem., June 22, 2001; 276(26): 24113 - 24121. [Abstract] [Full Text] [PDF]
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M. Roque, W. J.H. Kim, M. Gazdoin, A. Malik, E. D. Reis, J. T. Fallon, J. J. Badimon, I. F. Charo, and M. B. Taubman CCR2 Deficiency Decreases Intimal Hyperplasia After Arterial Injury Arterioscler Thromb Vasc Biol, April 1, 2002; 22(4): 554 - 559. [Abstract] [Full Text] [PDF]
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) or
incubated with 25 µM tyrphostin B42 (
) or control tyrphostin A1
(
) for 12 h and allowed to migrate in porous cell culture
inserts in the presence of 5 nM MCP-1 in the lower chamber. Cells that
migrated to the lower chamber were counted and expressed as a migration
index, calculated as the x-fold increase in the migration observed over
the negative control (PBS). Data represent the mean of triplicate
determinations, with the SD indicated. C, B42-treated Mono
Mac 1 cells were stimulated with MCP-1, lysed, and immunoprecipitated,
and Western blot was analyzed as described in the legend to Figure 1
