Post on 12-Sep-2021
Intracellular Domains of CXCR3 that Mediate CXCL9, CXCL10, and CXCL11 Function
Richard A. Colvin, Gabriele S.V. Campanella, Jieti Sun, and Andrew D. Luster
Center for Immunology and Inflammatory Diseases Division of Rheumatology, Allergy, and ImmunologyMassachusetts General HospitalBoston, MA 02129
Running title: CXCR3 functional domains
Please address all correspondence to: Andrew D. Luster, MD, PhDCenter for Immunology and Inflammatory DiseasesMassachusetts General Hospital 149 Thirteenth Street room 8031Charlestown, MA 02129Phone: (617) 726-5710Fax: (617) 726-5651E-mail: aluster@partners.org
Keywords: CXCR3, chemokine, chemotaxis, GPCR, internalization, signal transduction, CXCL10, CXCL9, CXCL11, IP-10, MIG, I-TAC, dynamin, arrestin
JBC Papers in Press. Published on May 17, 2004 as Manuscript M403595200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Abbreviations: PBS – phosphate buffered saline; BSA – bovine serum albumin; ERK –extracellular signal-related protein kinase; GPCR – G protein-coupled receptor; PCR –polymerase chain reaction; ANOVA – analysis of variance
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Summary
The chemokine receptor CXCR3 is a G protein-coupled receptor found predominantly
on T cells that is activated by 3 ligands: CXCL9 (Mig), CXCL10 (IP-10), and CXCL11
(I-TAC). Previously, we have found that of the three ligands, CXCL11 is the most potent
inducer of CXCR3 internalization and is the physiologic inducer of CXCR3
internalization after T cell contact with activated endothelial cells. We have therefore
hypothesized that these three ligands transduce different signals to CXCR3. In light of
this hypothesis, we sought to determine if regions of CXCR3 are differentially required
for CXCL9, CXCL10, and CXCL11 function. Here we identified two distinct domains
that contributed to CXCR3 internalization. The carboxyl-terminal domain and ß-
arrestin1 were predominantly required by CXCL9 and CXCL10, and the third
intracellular loop was predominantly required by CXCL11. Chemotaxis and calcium
mobilization induced by all three CXCR3 ligands were dependent on the CXCR3
carboxyl-terminus and the DRY sequence in the third trans-membrane domain. Our
findings demonstrate that distinct domains of CXCR3 mediate its functions and suggest
that the differential requirement of these domains contributes to the complexity of the
chemokine system.
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Introduction
Chemokines, or chemoattractant cytokines, are a family of small (8-10 kDa)
proteins that play an important role in the recruitment and activation of leukocytes (1).
By inducing the migration of leukocytes, they play a critical role in innate immunity as
well as the development of an adaptive immune response and the maintenance of chronic
inflammation. Chemokines induce their biological effects by binding to seven trans-
membrane-spanning G protein-coupled receptors (GPCRs) (1). Approximately 50
chemokines have been described that interact with approximately 16 GPCR chemokine
receptors, implying that there is redundancy in the chemokine system. However, this
apparent redundancy has not been supported by in vivo studies that have instead
demonstrated that individual chemokines that activate the same receptor can have unique
functions in vivo (2). This may be related to differential chemokine expression in vivo
and/or differential receptor activation by different chemokine ligands (3).
CXCR3 is expressed on the surface of a number of cell types, including activated
T cells and NK cells, and subsets of inflammatory dendritic cells, macrophages, and B
cells (4-6). CXCR3 is a pertussis toxin sensitive G protein-coupled receptor, indicating
that it is coupled to the Gi class of heterotrimeric G proteins (7). Activation of
chemokine receptors has been reported to induce a number of signaling pathways as well
as the activation of integrins, G protein related kinases (GRKs), and the binding of β↑
arrestin (8-12).
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The down regulation of a receptor’s response is important in its overall function.
Chemokine receptors are down regulated in at least two ways. The most rapid way is the
uncoupling of the receptor from the G protein, which prevents further activation by
ligand, a process referred to as desensitization (13). Chemokine receptors are also down
regulated by internalization, or endocytosis, a process that removes the receptor from the
cell surface resulting in a more prolonged unresponsiveness to the ligand. The
internalized receptor may be degraded in lysozomes or recycled back to the cell surface
(14). In addition, it has recently been proposed that following internalization, GPCR
complexes can transduce unique signaling information (15,16). The internalization of
many GPCRs, including at least two chemokine receptors, CXCR4 and CCR5, is
mediated by the phosphorylation of carboxyl-terminal serine and threonine residues by
the GRKs. This phosphorylation event induces ß-arrestin2 binding to the cytoplasmic
tail, which targets the receptor to clathrin coated pits in a dynamin- dependent manner
(8-10).
Previously, it has been shown that chemokine receptors can be differentially
activated by different ligands. For example, CXCL8 (IL-8) activation of CXCR2 results
in higher levels of receptor internalization than CXCL7 (NAP2) (17). It is not clear,
however, whether CXCL8 and CXCL7 induce CXCR2 internalization through different
pathways or if the effect is due to differences in ligand potency. Additionally, synthetic
derivatives of CCL5 (RANTES) can induce CCR5 internalization and can mobilize
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calcium, but cannot activate chemotaxis (18). These findings suggest that the activation
of a chemokine receptor by different ligands may lead to different signals.
Three ligands are known to activate CXCR3 – CXCL10 (IP-10), CXCL9 (Mig),
and CXCL11 (I-TAC) (4,19). Each of these ligands is induced by interferon-γ (IFN-γ)
and is produced in Th1-type immune responses (19-21). Although these three ligands
are all induced by IFN-γ, they appear to mediate distinct biological phenomena in vivo
(22-24). This may be related to differential expression of these ligands as has been seen
in cardiac and skin allograft rejection (22,24-29), atherosclerosis (30), host response to
infection (31), and inflammatory skin diseases (32). Alternatively, the different
biological outcomes may also be related to the differential activation of CXCR3 by
CXCL10, CXCL9, and CXCL11.
In this regard, our previous work has shown that CXCL11 is the physiologic
inducer of CXCR3 internalization following T cell contact with IFN-γ activated
endothelial cells, even though these cells produce greater amounts of CXCL10 and
CXCL9 than CXCL11 (33). We also found that CXCL11 was the most potent inducer of
CXCR3 internalization. Therefore, we speculated that these three ligands transduce
different signals to CXCR3 (33). To test this hypothesis, we mutated CXCR3 in the
intracellular domains to determine if CXCR3 is differentially activated by CXCL10,
CXCL9, and CXCL11. We specifically tested the effects of mutations on the receptor,
including ligand binding, receptor phosphorylation, and receptor internalization; effects
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of the mutations on downstream signaling pathways, including Erk phosphorylation and
calcium mobilization; and finally, the effects of the mutations on chemotaxis.
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Experimental Procedures
Reagents.
Recombinant CXCL9, CXCL10, and CXCL11 were purchased from Peprotech (Rock
Hill, NJ). The PE-conjugated anti-CXCR3 antibody 1C6 was purchased from R&D
Systems (Minneapolis, MN). The bovine-pre-prolactin-FLAG plasmid was a gift of Dr.
Israel Charo (University of California at San Francisco). The dynamin and ß-arrestin
dominant negative constructs were gifts of Dr. Marc Caron (Duke University Medical
Center, Durham, NC).
Plasmids and mutagenesis.
All receptors used in this study are derived from human CXCR3. A cDNA encoding
CXCR3 was inserted into the Kpn1 and EcoR1 restriction sites in the multi-cloning site
of pcDNA3.1 (Invitrogen, Carlsbad, CA). The Tail(-)-CXCR3 truncation was
constructed by amplifying the cDNA encoding CXCR3 with the appropriate primers.
Point mutations were introduced into CXCR3 by using the Quikchange Mutagenesis Kit
by Stratagene (LaJolla, CA) and oligonucleotides encoding the specific changes.
Chimeric genes encoding the bovine-pre-prolactin signal sequence and the FLAG tag
followed by CXCR3 were constructed by ligating the pre-prolactin-FLAG genes to
CXCR3 at a Sal1 site. All constructs were confirmatory sequenced.
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Tissue culture.
300-19 is a pre-B cell leukemia cell line known to functionally express chemokine
receptors following stable transfection (34). At baseline, 300-19 cells functionally
express CXCR4 allowing for a positive control in assays of chemokine function (34).
300-19 cells were cultured in RPMI (Cellgro and Mediatech, Herndon, VA) with 10%
fetal calf serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Mediatech), and 2 mM
L-glutamine (Mediatech) (complete RPMI). HEK293 cells were cultured in Dulbecco’s
modification of minimal essential medium (DMEM) (Mediatech), with 10% fetal calf
serum, 100 U/ml penicillin and 100 µg/ml streptomycin (Mediatech), and 2 mM L-
glutamine (Mediatech).
Stable transfection.
1x107 300-19 cells were incubated with 10 µg of linearized CXCR3/pcDNA3.1
constructs for 10 minutes on ice and electroporated using a Bio-RAD Gene Pulser II
(Bio-Rad, Hercules, CA) at 250 V and 975 microfarads in a 0.2 cm gap electrode cuvette
(Bio-Rad). Following the electroporation, the cells were grown in complete RPMI for 24
hours, and then placed into complete RPMI plus 80 µg/ml G418 (Mediatech) for
selection.
Enrichment of CXCR3 or CXCR3 mutant expressing cells.
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5 x 106 transfected cells were stained with 3 µl of CXCR3 antibody 1C6 conjugated to
PE (R&D Systems, Minneapolis, MN) in10% goat serum in phosphate buffered saline
(PBS) for 30 minutes at 4° C. The stained cells were washed and then incubated with
microbeads coupled to an anti-PE antibody and CXCR3 expressing cells were positively
selected over a MACS LS column (Miltenyi Biotec, Auburn, CA) and cultured in
complete RPMI without G418.
Cell surface expression of CXCR3 and CXCR3 mutants.
Cultured cells were resuspended in 100 µl of flow cytometry buffer (PBS without calcium
and magnesium containing 1% bovine serum albumin (BSA) and 0.1% sodium azide)
and 10% goat serum. They were incubated for 5 minutes at room temperature. The anti-
CXCR3 antibody, 1C6, conjugated to PE (R&D Systems) was added to the cells, which
were then incubated at 4°C for 30 minutes. The cells were washed twice in PBS and
subsequently fixed by resuspension in PBS with 2% paraformaldehyde. Receptor cell
surface expression was measured on a FACSCalibur flow cytometer and the data
analyzed using CellQuest (BD Biosciences, San Jose, CA) or FlowJo (San Carlos, CA).
Receptor binding assays.
Binding assays were performed as previously reported (35). Briefly, 400,000 wild-type-
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or mutant-CXCR3/300-19 cells were placed into 96-well tissue culture plates in a total
volume of 150 µl of binding buffer (0.5% BSA, 5 mM MgCl2, 1 mM CaCl2, 50 mM
HEPES pH 7.4). 0.04 nM of 125I labeled CXCL10 (New England Nuclear, Boston, MA)
or CXCL11 (Amersham Biosciences, Piscataway, NJ) and increasing amounts of
unlabeled CXCL10 or CXCL11 (Peprotech, Rocky Hill, NJ) were added to the cells and
incubated for 90 minutes at room temperature with shaking. The cells were transferred to
96-well filter plates (Millipore, Billerica, MA) pre-soaked in 0.3% polyethyleneimine
and washed three times with 200 µl binding buffer supplemented with 0.5 M NaCl. The
plates were dried and the radioactivity was measured after the addition of scintillation
fluid in a Wallac Microbeta scintillation counter (Perkin Elmer Life Sciences, Boston,
MA).
Receptor internalization.
Wild-type-CXCR3/300-19 or mutant-CXCR3/300-19 cells (250,000) were incubated
with various concentrations of CXCL10, CXCL11, or CXCL9 for 15 or 30 minutes as
indicated at 37°C. At the end of the incubations, ice-cold flow cytometry buffer was
added and cells were analyzed for cell surface expression of CXCR3 using the PE
conjugated CXCR3 antibody 1C6 as above.
Chemotaxis.
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Chemotaxis assays of 300-19 cells were performed in 96 well Neuroprobe chemotaxis
chambers with 5 µM pore size polycarbonate membranes (Neuroprobe, Gaithersburg,
MD). 31 µl of RPMI containing 1% BSA and chemokines were placed in the bottom
chamber of the device per the manufacturers directions. 25,000 cells were layered onto
the top of the membrane in RPMI containing 1% BSA. The chambers were then
incubated at 37°C for 5 hours. After washing the top of the filter with deionized water,
the chambers were subjected to centrifugation at 1,500 rpm for 5 minutes. The filters
were removed and media was aspirated. The chambers were then frozen at -80°C for at
least one hour. 20 µl of CyQuant dye mix (Molecular Probes, Eugene, OR) was added to
each well of the Neuroprobe chamber. Following a 2-hour incubation period, the
fluorescence was measured in a CytoFluor fluorescent plate reader (Applied Biosystems,
Foster City, CA). For each experiment, a cellular titration curve was completed to make
sure that the fluorescence reading was in the linear range of the CyQuant dye and the
background fluorescence was subtracted from the readings for each sample. The
chemotactic index was determined by dividing the fluorescence at each chemokine
concentration by the fluorescence when no chemokine was added. The CyQuant results
have been compared favorably with direct counting of the migrated cells. Chemotaxis
data was analyzed using analysis of the variance (ANOVA) allowing the comparison of
the chemotactic curves rather than individual points.
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Calcium flux.
5x106 wild-type- or mutant-CXCR3/300-19 cells were resuspended in 2 ml of RPMI
with 1% bovine serum albumin. 15 µl of Fura-2 (Molecular Probes, Eugene, OR) was
added and the cells were incubated at 37°C for 20 minutes. The cells were washed twice
in PBS and resuspended in 2 ml of calcium flux buffer (145 mM NaCl, 4 mM KCl, 1 mM
NaHPO4, 1.8 mM CaCl2, 25 mM HEPES, 0.8 mM MgCl2 and 22 mM glucose).
Fluorescence readings were measured at 37° C in a DeltaRAM fluorimeter (Photon
Technology International, Lawrenceville, NJ). Intracellular calcium concentrations were
recorded as the excitation fluorescence intensity emitted at 510 nM in response to
sequential excitation at 340 nm and 380 nm and are presented as the relative ratio of
fluorescence at 340/380.
Receptor Phosphorylation.
HEK293 cells transiently transfected with pcDNA3.1 with wild-type-CXCR3 or Tail(-
)-CXCR3 that had been constructed to include a bovine pre-prolactin signal sequence
followed by DNA sequences encoding the FLAG epitope tag. Forty-eight hours
following transfection, the media was changed to phosphate free DMEM with 1% BSA.
The cells were incubated for 10 minutes and 150 mCi of 32P inorganic phosphate (NEN
Life Sciences, Boston, MA) was added to each well. Cells were incubated for 3 hours
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followed by stimulation by 100 nM CXCL10 or CXCL11. Extracts were made and
immunoprecipitations were performed as described previously (36). Briefly, cells were
lysed in 0.15 M NaCl, 20 mM HEPES pH 7.4, 5 mM EDTA, 3 mM EGTA, 4 mg/ml
dodecyl-ß-maltoside, 0.2 mg/ml cholestoryl hemisuccinate, plus protease inhibitors and
phosphatase inhibitors. Following lysis, extracts were cleared by centrifugation, pre-
cleared using protein-G-sepharose beads, and proteins were immunoprecipitated using
M2-anti-FLAG antibody preconjugated to agarose beads (Sigma, St. Louis, MO).
Following overnight immunoprecipitation, beads were washed five times using
radioimmunoassay precipitation (RIPA) buffer (50 mM tris-HCl pH 7.4, 1% NP-40,
0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA) and proteins were resolved on a
10% SDS-PAGE gel. Gels were dried on a vacuum gel-drier and subsequently exposed
to autoradiography film.
Transient Transfection.
HEK293 cells were plated onto 6 well tissue culture plates in Dulbecco’s modified
essential medium (DMEM). When the cells were approximately 50% confluent they were
transfected with pcDNA3.1 with the indicated CXCR3 gene inserted using Fugene 6
(Roche Diagnostics, Indianapolis, IN) according to the manufacturer’s directions. Total
transfected DNA was kept constant using pcDNA3.1 without an insert.
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Results
Truncation of the carboxyl-terminus. A mutant of CXCR3, called Tail(-)-CXCR3,
was generated to determine the role of the carboxyl-terminus in CXCR3 function (Table
1). Eight amino acids of the predicted carboxyl-terminus were retained in order to ensure
surface expression, while removing any putative signaling elements. A population of
300-19 cells that stably and highly expressed Tail(-)-CXCR3 was positively selected.
Tail(-)-CXCR3 was expressed on the cell surface of 300-19 cells at similar levels to
wild-type-CXCR3 (Table 2). This suggested that CXCR3 is unlikely to require
palmitoylation of carboxyl-terminal cysteines to achieve cell surface expression as has
been seen for CCR5 (37). In our binding assays, we could not determine an IC50 value
for Tail(-)-CXCR3/300-19 cells to CXCL10 and CXCL11 despite the detection of some
chemokine binding in the absence of competitor (Table 2). CXCL9 binding assays were
not performed as there is no commercially available 125I labeled preparation of this
chemokine.
CXCL10, CXCL9, and CXCL11 were tested for their ability to induce internalization
of wild-type-CXCR3 and Tail(-)-CXCR3 (Figure 1). The ability of CXCL10 and
CXCL9 to induce internalization of Tail(-)-CXCR3 was dramatically reduced compared
to wild-type-CXCR3 (p<0.01). In contrast, CXCL11 induced internalization of Tail(-)-
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CXCR3 as efficiently as wild-type-CXCR3. In chemotaxis assays, Tail(-)-
CXCR3/300-19 cells did not migrate to CXCL10 or CXCL9, and migration to CXCL11
was dramatically reduced (Figure 1). Upon stimulation with CXCL10, CXCL9, or
CXCL11 (10 nM) there was no evidence of calcium mobilization in Tail(-)-
CXCR3/300-19 cells (Figure 2) or Erk-phosphorylation (data not shown). These data
demonstrate that the carboxyl-terminus of CXCR3 was essential for CXCL10-,
CXCL9-, and CXCL11-induced chemotaxis, calcium mobilization, and Erk
phosphorylation, but was not essential for CXCL11-induced receptor internalization.
CXCR3 Phosphorylation. Carboxyl-terminal serines and threonines have been
implicated in regulating GPCR desensitization and internalization, including CXCR4 and
CCR5 (38-40). Upon receptor activation, these carboxyl-terminal serines and threonines
are phosphorylated by GRKs, which allows ß-arrestin binding and can lead to both G
protein uncoupling and internalization by targeting receptors to clathrin coated pits
(8,41). Wild-type-CXCR3 and Tail(-)-CXCR3 were immunoprecipitated from extracts
made from HEK293 cells transiently transfected with plasmids encoding FLAG-tagged
versions of these receptors (Figure 3). Forty-eight hours after transfection, the cells were
metabolically labeled with 32P inorganic phosphate and stimulated with 100 nM
CXCL10 or CXCL11. A low level of baseline phosphorylation was seen in 3 of 5
experiments for wild-type-CXCR3. Wild-type-CXCR3 phosphorylation was enhanced
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after five minutes of stimulation by CXCL10 and CXCL11. However, neither CXCL10
nor CXCL11 stimulation enhanced the phosphorylation of Tail(-)-CXCR3 and
constitutive phosphorylation of Tail(-)-CXCR3 was not seen. Five weeks after exposure
to film there were no bands present at the appropriate size in lanes from extracts of Tail(-
)-CXCR3/300-19 cells or untransfected 300-19 cells either before or after stimulation
with CXCL10 or CXCL11. In order to determine if the phosphorylated protein was
CXCR3, Western blots were performed using the anti-CXCR3 antibody 1C6. These
experiments revealed that the bands on western blots from CXCR3 transfected HEK293
cells migrated identically to the radiolabelled immunoprecipitated proteins on the SDS-
PAGE gels (Figure 3). Untransfected HEK293 cells revealed no signal at this location
(Figure 3). Interestingly, CXCR3 migrated at almost twice its expected size on SDS-
PAGE gels suggesting the presence of post-translational modifications. Additionally,
immunoprecipitation of extracts from CXCL10- and CXCL11-stimulated and
unstimulated wild-type-CXCR3/300-19 cells with a control anti-CCR7 antibody did
not detect bands at this size (data not shown). These data demonstrate that both CXCL10
and CXCL11 induced the phosphorylation of CXCR3, and that receptor phosphorylation
may be required for CXCL10-induced internalization.
Carboxyl-terminal serine and threonine substitution mutant. To further dissect the
role of the CXCR3 carboxyl-terminus in ligand mediated signaling, a serine/threonine
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mutant, ST(-)-CXCR3, was constructed that contained alanine substitutions of the serine
and threonine residues in the carboxyl-terminus (Table 1). A population of cells that
stably and highly expressed ST(-)-CXCR3 was positively selected. Cell surface
expression of ST(-)-CXCR3 was similar to wild-type-CXCR3 on 300-19 cells and
ST(-)-CXCR3/300-19 cells bound CXCL10 and CXCL11 similarly to wild-type-
CXCR3/300-19 cells as determined by IC50 values (Table 2).
CXCL10 and CXCL9 induced less internalization of ST(-)-CXCR3 than wild-type-
CXCR3 (p<0.01), demonstrating that the carboxyl-terminal serines and threonines
contributed to maximal CXCL10- and CXCL9-mediated internalization (Figure 4). The
alteration of the CXCR3 carboxyl-terminal serines and threonines, however, had no
effect on CXCL11-induced internalization (Figure 4). These data are consistent with the
Tail(-)-CXCR3 data and suggest that CXCL11 induced an internalization pathway that
does not require the carboxyl-terminal serine and threonine residues. Of note, wild-
type-CXCR3 receptor internalization was similar after 15 minutes and 5 hours of
stimulation with CXCL10, CXCL9, and CXCL11 (data not shown). Peak calcium
mobilization was similar for wild-type-CXCR3/300-19 cells and ST(-)-CXCR3/300-
19 cells following CXCL10, CXCL9, and CXCL11 stimulation (Figure 2). However, the
free intracellular calcium concentration remained elevated in the ST(-)-CXCR3/300-19
cells, suggesting a prolonged signal following chemokine stimulation (Figure 2). ST(-)-
CXCR3 was desensitized to repeated doses of CXCL10 and CXCL11 similarly to wild-
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type-CXCR3 (data not shown).
Stimulation of ST(-)-CXCR3/300-19 cells resulted in enhanced chemotaxis to
CXCL10, CXCL9, and CXCL11 when compared to wild-type-CXCR3/300-19 cells
(Figure 4). The dose-response for chemokine-induced migration was similar for both
ST(-)-CXCR3/300-19 cells and wild-type-CXCR3/300-19 cells. However, the number
of migrating cells was significantly greater for ST(-)-CXCR3/300-19 cells in response
to CXCL10, CXCL9, and CXCL11, but not to the control CXCL12 (Figure 4 and data
not shown). These differences were statistically significant (p<0.01). Although
statistically significant, the differential migration to CXCL11 was less pronounced than
that to CXCL10 or CXCL9 (Figure 4). Of note, 300-19 cells expressing CXCR3 with
subsets of the serine and threonine substitutions, (e.g. S349AS350AS351AS355AS356A,
S349AS350AS351AS364A, or S349AS350AS351AS358AT360AS361A), also
demonstrated significantly higher chemotactic activity compared to wild-type-CXCR3
(data not shown).
Effect of dynamin and ß-arrestin on CXCR3 internalization. The requirement of the
carboxyl-terminus serines and threonine for CXCL10- and CXCL9-induced CXCR3
internalization suggested that ß-arrestin might play a role in CXCR3 internalization.
Dynamin is a small GTPase that is important for ß-arrestin-dependent internalization
(15). In order to evaluate whether both CXCL10- and CXCL11-induced CXCR3
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internalization required dynamin and ß-arrestin1 or ß-arrestin2, plasmids encoding
dominant negative dynamin (K44A), ß-arrestin1 (V53D), or ß-arrestin2 (V54D) (42)
were transiently co-transfected with CXCR3 into HEK293 cells. Forty-eight hours after
transfection, the cells were harvested and the ability of CXCL10 and CXCL11 to induce
CXCR3 internalization was tested (Figure 5). CXCR3 internalization was always about
50% less in HEK293 cells than in 300-19 cells or primary lymphocytes; however, the
dynamin K44A and ß-arrestin1 V53D dominant negative mutants significantly reduced
CXCL10-induced CXCR3 internalization (p<0.01 and p<0.05), but had little effect on
CXCL11-induced CXCR3 internalization. The ß-arrestin2 V54D dominant negative had
no significant effect on CXCL10- or CXCL11-induced internalization. These results
suggest that CXCL10 required dynamin and ß-arrestin1 to induce CXCR3 internalization
in HEK293 cells, while CXCL11-induced CXCR3 internalization proceeded in a
dynamin- and ß-arrestin-independent manner.
CXCR3 intracellular loop 3. In order to define a region of CXCR3 that mediated
CXCL11-induced internalization, the third intracellular loop of CXCR3 was replaced
with the third intracellular loop of CXCR1 and called i3-CXCR1-CXCR3. i3-CXCR1-
CXCR3 surface expression on 300-19 cells was slightly elevated compared to wild-type
CXCR3, although CXCL10 and CXCL11 binding to i3-CXCR1-CXCR3/300-19 cells
was similar compared to wild-type-CXCR3/300-19 cells (Table 2). CXCL11-induced
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internalization of i3-CXCR1-CXCR3 was significantly reduced in 300-19 cells
compared to wild-type-CXCR3, while CXCL10- and CXCL9-induced internalization
was not significantly different (Figure 6). These data suggest that the third intracellular
loop of CXCR3 was required for maximal internalization induced by CXCL11 but not
CXCL9 or CXCL10. CXCL10-, CXCL9-, and CXCL11-induced i3-CXCR1-
CXCR3/300-19 chemotaxis was slightly diminished compared to wild-type-
CXCR3/300-19 (Figure 6). Calcium mobilization following stimulation with CXCL10,
CXCL9, and CXCL11 was slightly reduced compared to wild-type-CXCR3 (Figure 2).
Additionally, following stimulation with CXCL11, i3-CXCR1-CXCR3/300-19 cells
were desensitized to a second dose of CXCL11 similarly to wild-type-CXCR3/300-19
cells (data not shown).
DRY site mutation: R149N. It has previously been shown that Gαi dependent
chemokine receptors require an aspartate-arginine-tyrosine (DRY) sequence in the third
trans-membrane domain for inducing chemotaxis (43). The DRY sequence has also been
shown to be important for the internalization of some G-protein coupled receptors
following activation (44).
In order to further define the role of the CXCR3 DRY sequence in ligand-
induced internalization, chemotaxis, and calcium mobilization, a receptor mutant was
generated that changed the DRY sequence to aspartate-asparagine-tyrosine (DNY).
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(Table 1).
R149N-CXCR3 was expressed at slightly reduced levels compared to wild-type
CXCR3 on 300-19 cells (Table 2). R149N-CXCR3/300-19 cells bound CXCL10 with a
similar IC50 value compared to wild-type-CXCR3/300-19 cells (Table 2).
Internalization of this receptor following stimulation with CXCL10, CXCL9, or CXCL11
was similar to internalization of wild-type-CXCR3, suggesting that this sequence played
no role in CXCL10-, CXCL9-, or CXCL11-induced CXCR3 internalization (Figure 7).
However, CXCL10-, CXCL9-, and CXCL11-induced chemotaxis was reduced to
background levels, suggesting that this sequence was essential for CXCR3 mediated
chemotaxis (Figure 7). Similar to chemotaxis, CXCL10-, CXCL9-, and CXCL11-
induced calcium mobilization and Erk-phosphorylation to were abolished by this
substitution (Figure 2 and data not shown).
Additional intracellular domain mutants. Additional intracellular loop mutants
were constructed to determine the role of the first intracellular loop of CXCR3 in
chemotaxis and receptor internalization following ligand stimulation. Alanines were
substituted for the serines and threonines in the first intracellular loop using site-directed
mutagenesis. S80A-CXCR3, T83A-CXCR3, and T90A-CXCR3, had no effect on
receptor expression, induction of chemotaxis, or internalization by CXCL10 and
CXCL11 (data not shown).
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The sequence LLLRL332-336 in the carboxyl-terminus is similar to the adaptin-
2 binding site that has been demonstrated to be important for CXCR2 internalization (45).
Alanines were substituted for leucines 332-334. LLL332-334AAA-CXCR3 was
expressed at levels comparable to wild-type-CXCR3 in 300-19 cells (Table 2).
LLL332-334AAA-CXCR3/300-19 cells bound CXCL10 and CXCL11 similarly to
wild-type-CXCR3/300-19 cells (Table 2). Additionally, CXCL10-, CXCL9-, and
CXCL11-induced receptor internalization (Figure 7) and calcium mobilization for
LLL332-334AAA-CXCR3/300-19 cells was similar to wild-type-CXCR3/300-19
cells (Figure 2). However, CXCL10-, CXCL9-, and CXCL11-induced chemotaxis was
reduced in LLL332-334AAA-CXCR3/300-19 cells compared to wild-type-
CXCR3/300-19 cells (Figure 7). These data demonstrate that the LLLRL motif in the
carboxyl terminus of CXCR3 participates in regulating chemotaxis but not receptor
internalization or calcium mobilization.
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Discussion
One of the complexities of the chemokine system is that different ligands for the
same receptor can induce different biological effects. In this study, we have explored the
structural basis for these effects in the CXCR3 receptor ligand system.
We have now shown that different domains of CXCR3 are required for CXCL10-
, CXCL9-, and CXCL11-induced function. In summary, the DRY site was essential for
CXCL10-, CXCL9-, and CXCL11-induced chemotaxis, calcium mobilization, and Erk
phosphorylation but not for CXCR3 internalization; the CXCR3 carboxyl-terminus was
essential for CXCL10-, CXCL9-, and CXCL11-induced chemotaxis, calcium
mobilization, and Erk phosphorylation; the CXCR3 carboxyl-terminus and ß-arrestin1
were required for CXCL10-induced receptor internalization, while the more effective
CXCL11-induced internalization was independent of the carboxyl-terminus and ß-
arrestin1; the third intracytoplasmic loop was required for maximal CXCL11-induced
internalization; and residues LLL332-334 played a role in mediating CXCR3 ligand-
induced chemotaxis (Figure 8).
Carboxyl-terminus. CXCR3 truncated at the carboxyl-terminus clearly showed
that this region was essential for maximal chemokine binding, chemotaxis, calcium
mobilization, and MAPK activation, but was not important for receptor expression.
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Although ligand binding to Tail(-)-CXCR3 was severely diminished, signaling through
this receptor was not completely abrogated, as demonstrated by the ability of CXCL11 to
induce efficient internalization of this receptor. CXCL10 and CXCL9 induced only
minimal internalization of Tail(-)-CXCR3, demonstrating that these chemokines
differentially activate CXCR3 leading to distinct biological outcomes. One possibility is
that CXCL9 and CXCL10 did not bind Tail(-)-CXCR3 while CXCL11 did. It is possible
that Tail(-)-CXCR3 is predominantly in the uncoupled conformation that has been
shown to bind CXCL11 but not CXCL9 or CXCL10 (46). Binding data were not helpful
to differentiate these possibilities, as there was only minimal binding of CXCL10 and
CXCL11 to Tail(-)-CXCR3. Previously it had been shown that chemokine receptors can
be activated by ligands that show no detectable binding (47,48). It has been speculated
that this results from a chemokine-chemokine receptor interaction with an off rate that is
faster than the sensitivity of competitive binding assays (47).
Carboxyl-terminal serines and threonine. Mutation of the CXCR3 carboxyl-
terminal serines and threonine resulted in a receptor (ST(-)-CXCR3) that was
internalized less than wild-type-CXCR3 when stimulated with CXCL9 and CXCL10.
This mutant also exhibited enhanced migration to CXCL10, CXCL9, and CXCL11,
which may have resulted from the prolonged signaling following chemokine stimulation
as seen in the calcium mobilization data.
Third intracellular loop. The substitution of the third intracellular loop of CXCR3
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with that of CXCR1 (i3-CXCR1-CXCR3) resulted in a receptor that was internalized
less than wild-type-CXCR3 upon stimulation with CXCL11 but not CXCL9 and
CXCL10, suggesting that the third intracellular loop of CXCR3 was required for maximal
CXCL11-induced internalization and that this region of CXCR3 was differentially
activated by the CXCR3 ligands.
DRY motif. We found the DRY sequence in the third trans-membrane domain of
CXCR3 to be important for chemotaxis, calcium mobilization, and Erk phosphorylation,
but not for receptor internalization. This sequence has been previously shown to be
important for signaling by Gαi-coupled chemokine receptors (49). Consistent with
pertussis toxin data, our data also support that the DRY sequence was not necessary for
CXCR3 internalization (33).
LLLRL motif. It has been reported that CXCL8-induced CXCR2 internalization
can proceed through a carboxyl-terminal serine- and threonine-independent manner that
is dependent on the adaptin-2 binding motif (LLKIL) in the CXCR2 carboxyl terminus
(17,45). The CXCR3 carboxyl-terminus contains the similar sequence LLLRL. This
motif cannot be responsible for CXCL11-induced CXCR3 internalization as its absence
did not effect Tail(-)-CXCR3 internalization. Consistent with this, substitution of the
LLL332-334 residues with alanine residues had no effect on CXCL11-induced CXCR3
internalization. This substitution also had no effect on CXCL9- and CXCL10-induced
CXCR3 internalization. However, this sequence appears to play a role in CXCR3-
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mediated chemotaxis as CXCL10, CXCL9, and CXCL11-induced less chemotaxis in
LLL332-334AAA-CXCR3/300-19 cells compared to wild-type-CXCR3/300-19 cells.
CXCR3 function and ß-arrestins. Many G protein-coupled receptors are
internalized after activation through a process that involves G protein-related kinase
phosphorylation of carboxyl-terminal serine and threonine residues. This leads to ß-
arrestin binding, which uncouples the receptor from the G proteins, and subsequent
dynamin- and clathrin-mediated internalization (50).
Our data demonstrated that the dynamin and ß-arrestin1 dominant negatives
impaired CXCL10-, but not CXCL11-induced internalization in transiently transfected
HEK293 cells. Although CXCR3 internalization was not as robust in HEK293 cells as in
300-19 cells, the data show a clear difference between CXCL10- and CXCL11-induced
CXCR3 internalization. These data are consistent with our findings that CXCL10 and
CXCL11 require two distinct domains of CXCR3. It is noteworthy that the ß-arrestin1
and not the ß-arrestin2 dominant negative protein affected CXCR3 internalization, as
previously it was shown that ß-arrestin2 was required for CXCL12-induced CXCR4
internalization (39,51).
In summary, our results indicate that the ligands of CXCR3 can preferentially
activate distinct internalization pathways. CXCL10 and CXCL9 predominantly induce a
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carboxyl-terminal dependent pathway, whereas CXCL11 predominantly induces a
carboxyl-terminal independent pathway. Since we have previously shown that CXCL11
is the more potent and physiological inducer of CXCR3 internalization, the tail-
independent mechanism is potentially more important (33). This is reminiscent of the
tail- and dynamin-independent process that has been described for CXCR2
internalization (52). The relative role of the internalization pathways in the in vivo
function of CXCL10, CXCL9, and CXCL11 as well as the role of the CXCR3 carboxyl-
terminus in mediating chemotaxis is of great interest in understanding CXCR3 function
in inflammatory processes.
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Acknowledgements
This work was supported by NIH grant K08 AI50147 to RAC and NIH grants PO1
DK50305 and R01 CA69212 to ADL. We would like to thank Josephine Leung for
technical assistance, Dr. William Hipkin from Schering-Plough for technical advice and
Dr. Robert Gerszten for helpful discussions.
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Figure Legends.
Figure 1. Receptor internalization and chemotaxis of Tail(-)-CXCR3. Wild-type-
CXCR3/300-19 cells and Tail(-)-CXCR3/300-19 cells were activated with the CXCR3
ligands CXCL10, CXCL9, and CXCL11. (A) Receptor internalization. Wild-type-
CXCR3/300-19 (black bars) or Tail(-)-CXCR3/300-19 cells (gray bars) were exposed
to increasing amounts of CXCL10, CXCL9, or CXCL11 for 30 minutes. Subsequently,
the cell surface expression of wild-type-CXCR3 or Tail(-)-CXCR3 was measured by
flow cytometry and compared to unstimulated cells. The X-axis represents the indicated
chemokine concentration used to stimulate the cells. The Y-axis represents the receptor
expression relative to the unstimulated receptor expression normalized to 1. The data
shown represent the mean internalization of three experiments with error bars
representing the standard error of the mean. Using two-way analysis of variance
(ANOVA), p<0.01 for CXCL10 and CXCL9-induced internalization. There was no
statistical difference for CXCL11-induced internalization. (B) Chemotaxis. The bars
represent the migration of wild-type-CXCR3/300-19 cells (black bars) or Tail(-)-
CXCR3/300-19 cells (gray bars) across a Neuroprobe membrane to CXCL10, CXCL9,
or CXCL11. The X-axis represents the chemokine concentration and the Y-axis
represents the chemotactic index. The data shown are the mean of 2 samples and the
experiment shown is representative of 5 separate experiments.
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Figure 2. Calcium mobilization assays of CXCR3 and CXCR3 mutants. The top row
contains the calcium mobilization plots for wild-type-CXCR3/300-19 cells loaded with
Fura-2 and activated with 10 nM CXCL10, CXCL9, or CXCL11 as indicated. The
curves show the ratio of the 510 nm emissions after activation at 340 nm and 380 nm.
Subsequent rows contain the calcium mobilization plots of the mutant-CXCR3/300-19
cells as indicated to the same treatments. The plots are representative of at least three
experiments and are all shown on the same scale. Lines for wild-type-CXCR3, ST(-)-
CXCR3, and LLL-CXCR3 represent the baseline.
Figure 3. Phosphorylation and Western Analysis of wild-type-CXCR3 and Tail(-)-
CXCR3. (A) Phosphorylation. An autoradiogram of a 10% SDS-PAGE gel showing
immunoprecipitations of HEK293 cell extracts from cells transfected with wild-type-
Flag-CXCR3, Tail(-)-Flag-CXCR3, and untransfected HEK-293 cells. Cells were
either unstimulated or stimulated with 100 nM CXCL10 or 100 nM CXCL11.
Immunoprecipitation was performed using M2-anti-FLAG antibody conjugated to
agarose beads. (B) Western Blotting. Extracts from the wild-type-CXCR3/300-19 and
300-19 cells were run on 10% SDS-PAGE gels, transferred to nitrocellulose
membranes, blotted with the anti-CXCR3 antibody 1C6 developed by
chemiluminescence, and detected by autoradiography.
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Figure 4. Receptor internalization and chemotaxis of ST(-)-CXCR3. (A) Receptor
internalization. Wild-type-CXCR3/300-19 cells or ST(-)-CXCR3/300-19 cells were
exposed to increasing amounts of CXCL10, CXCL9, or CXCL11 for 15 minutes.
Subsequently, the cell surface expression of wild-type-CXCR3 (black bars) or ST(-)-
CXCR3 (gray bars) was measured by flow cytometry and compared to unstimulated cells.
The X axis represents the indicated chemokine concentration used to stimulate the cells.
The Y axis represents the receptor expression relative to the unstimulated receptor
expression normalized to 1. The data shown represent the mean internalization of three
experiments with error bars representing the standard error. Using ANOVA, the
differences were significant to P<0.01 for CXCL10 and CXCL9. (B) Chemotaxis.
Migration of wild-type-CXCR3/300-19 cells (black bars) or ST(-)-CXCR3/300-19
cells (gray bars) was measured in Neuroprobe chemotaxis chambers. The X-axis
represents the chemokine concentration and the Y-axis represents the chemotactic index.
The data shown are the means of 5 experiments, each done in duplicate. The differences
between wild-type-CXCR3/300-19 cells and ST(-)-CXCR3/300-19 cells using the
means of all 5 experiments were significant at p<0.01 for CXCL10, CXCL9, and
CXCL11 by ANOVA.
Figure 5. The effect of dynamin, ß-arrestin1, and ß-arrestin2 on CXCR3 internalization.
HEK293 cells were transiently co-transfected with CXCR3/pcDNA3.1 and either
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dynamin dominant negative K44A/pCDNA3.1, ß-arrestin1 dominant negative V53D, ß-
arrestin2 dominant negative V54D (gray bars), or pcDNA3.1 as control (black bars).
Wild-type-CXCR3 cell surface expression was measured by flow cytometry after
stimulation with increasing amounts of CXCL10 or CXCL11. The X-axis represents the
indicated chemokine concentration used to stimulate the cells. The Y-axis represents the
receptor expression relative to the unstimulated receptor expression normalized to 1.
p<0.01 (dynamin dominant negative) and p<0.05 (ß-arrestin1 dominant negative) for
CXCL10-induced internalization using ANOVA. There was no statistical difference for
CXCL11-induced internalization or internalization in the presence of the ß-arrestin2
dominant negative. The data shown represent the mean internalization of four
experiments with error bars representing the standard error of the mean.
Figure 6. Receptor internalization and chemotaxis of i3-CXCR1-CXCR3. (A) Receptor
internalization. Wild-type-CXCR3/300-19 cells or i3-CXCR1-CXCR3/300-19 cells
were exposed to increasing amounts of CXCL10, CXCL9, or CXCL11 for 15 minutes.
Subsequently, the cell surface expression of wild-type-CXCR3 (black bars) or i3-
CXCR1-CXCR3 (gray bars) was measured by flow cytometry and compared to
unstimulated cells. The X axis represents the indicated chemokine concentration used to
stimulate the cells. The Y axis represents the receptor expression relative to the
unstimulated receptor expression normalized to 1. The data shown represent the mean
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internalization of four experiments with error bars representing the standard error of the
mean. Using ANOVA, the differences between wild-type-CXCR3/300-19 cells and i3-
CXCR1-CXCR3/300-19 cells using the means of all four experiments were not
significant for CXCL10 and CXCL9, and significant for CXCL11 (p<0.01). (B)
Chemotaxis. Migration of wild-type-CXCR3/300-19 cells (black bars) or i3-CXCR1-
CXCR3/300-19 cells (gray bars) was measured in Neuroprobe chemotaxis chambers.
The X-axis represents the chemokine concentration and the Y-axis represents the
chemotactic index. The data shown are the mean of 2 samples and the experiment shown
is representative of 3 separate experiments. The differences in chemotactic index were
statistically significant for CXCL10 (p<0.01) and CXCL9 (p<0.01) but not for CXCL11
(p=0.058) by ANOVA.
Figure 7. Receptor internalization and chemotaxis of R149N-CXCR3 and LLL332-
334AAA-CXCR3. (A) Receptor internalization. Wild-type-CXCR3/300-19 (black
bars) or mutant-CXCR3/300-19 cells (gray bars) were exposed to increasing amounts of
CXCL10, CXCL9, or CXCL11 for 30 minutes. Subsequently, the cell surface expression
of wild-type-CXCR3 or mutant-CXCR3 was measured by flow cytometry and
compared to unstimulated. The X axis represents the indicated chemokine concentration
used to stimulate the cells. The Y axis represents the receptor expression relative to the
unstimulated receptor expression normalized to 1. The data shown represent the mean
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internalization of three experiments with error bars representing the standard error of the
mean. p values are indicated on each chart. (B) Chemotaxis. Migration of wild-type-
CXCR3/300-19 cells (black bars) or mutant-CXCR3/300-19 cells (gray bars) was
measured in Neuroprobe chemotaxis chambers. The X-axis represents the chemokine
concentration and the Y-axis represents the chemotactic index. The data shown are the
mean of 2 samples and the experiment shown is representative of 3 separate experiments.
The differences in chemotactic index were statistically significant, p<0.01 for R149N-
CXCR3/300-19 cells, and p<0.05 for LLL332-334-CXCR3/300-19 cells, by ANOVA.
Figure 8. CXCR3 schematic. Domains required for activation by CXCL10 and CXCL9
are shown on the left and CXCL11 on the right. Black filled circles represent sites of
mutation that affected CXCR3 function. Italicized functions represent functions for
domains that were required differentially by the CXCR3 ligands.
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Richard A. Colvin, Gabriele S.V. Campanella, Jieti Sun and Andrew D. Lusterfunction
Intracellular domains of CXCR3 that mediate CXCL9, CXCL10, and CXCL11
published online May 17, 2004J. Biol. Chem.
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