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Prepublished online as a Blood First Edition Paper on September 5, 2002; DOI 10.1182/blood-2002-03-0978.
CHEMOKINES
From the Laboratoire Infections Rétrovirales et
Signalisation Cellulaire CNRS UMR 5121, Institut de Biologie,
Montpellier, France; St Bartholomew's and The Royal
London School of Medicine and Dentistry, London, United
Kingdom.
The CXCR4 chemokine receptor is a Gi protein-coupled
receptor that triggers multiple intracellular signals in response to stromal cell-derived factor 1 (SDF-1), including calcium mobilization and p44/42 extracellular signal-regulated kinases (ERK1/2). Transduced signals lead to cell chemotaxis and are terminated through receptor internalization depending on phosphorylation of the C terminus part of
CXCR4. Receptor endocytosis is also required for some receptors to
stimulate ERK1/2 and to migrate through a chemokine gradient. In this
study, we explored the role played by the 3 intracellular loops
(ICL1-3) and the C terminus domain of CXCR4 in SDF-1-mediated
signaling by using human embryonic kidney (HEK)-293 cells stably
expressing wild-type or mutated forms of CXCR4. ICL3 of CXCR4 is
specifically involved in Gi-dependent signals such as
calcium mobilization and ERK activation, but does not trigger CXCR4
internalization after SDF-1 binding, indicating that ERK phosphorylation is independent of CXCR4 endocytosis. Surprisingly, ICL2, with or without the aspartic acid, arginine, and tyrosine (DRY) motif, is dispensable for Gi signaling. However, ICL2
and ICL3, as well as the C terminus part of CXCR4, are needed to
transduce SDF-1-mediated chemotaxis, suggesting that this event
involves multiple activation pathways and/or cooperation of several
cytoplasmic domains of CXCR4.
(Blood. 2003;101:399-406) The chemokine receptor CXCR4 is a member of the
large family of 7-transmembrane domain receptors coupled to
heterotrimeric Gi proteins.1,2 This receptor
is also a fusion coreceptor for T-cell tropic and dual-tropic HIV-1
strains.3 Its ligand, the CXC chemokine stromal
cell-derived factor 1 (SDF-1), also named CXCL12, activates multiple
signal transduction pathways. This chemokine was first described as a
powerful chemoattractant for peripheral blood
lymphocytes,4 CD34+ progenitor
cells,5 and pre- and pro-B-cell lines.6
SDF-1 stimulation of cells expressing CXCR4 results in the increased phosphorylation of focal adhesion components, such as proline-rich tyrosine kinase 2 (Pyk-2), p130Cas, focal adhesion kinase (FAK), paxillin, Crk and Crk-L,7,8 extracellular-signal regulated kinases 1 and 2 (ERK-1 and -2), phospholipase C- SDF-1 also triggers CXCR4 internalization, involving G protein-coupled
receptor kinases (GRKs), followed by binding of
Despite the wide range of molecules activated after SDF-1 binding to
CXCR4, there is scant information on the link between the role of the
intracellular domains of CXCR4 and the activation of signal
transduction pathways. For this purpose, we constructed stable clones
by using the well-established human embryonic kidney 293 (HEK-293) cell
system already expressing a truncated form of CD4 incapable of
transducing a signal on its own. This cell line, which is widely used
for GPCR signaling studies, has also been useful for analysis of
chemotaxis14,20,24,28,29 and Materials
Construction of the CXCR4 mutants
Cell culture and transfection HEK-293 cells expressing a truncated form of CD4 lacking the cytoplasmic domain (CD4.403)32 were cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 1% penicillin-streptomycin, 1 mg/mL G418, 1% glutamax, and 10% fetal calf serum (Gibco-Life Technologies, Cergy Pontoise, France), and 107 cells were transfected with 10 µg pcDNA3 Zeo containing the CXCR4 mutants (CXCR4.ICL1m, CXCR4.ICL2m, CXCR4.ICL2mDRY, and CXCR4.ICL3m) by using the FuGENE 6 transfection reagent (Roche Diagnostics, Meylan, France) according to the manufacturer's instructions. The presence of the external part of CD4 at the cell surface allows us to verify the capability of the CXCR4 mutants to support HIV-1 infection. Clones able to grow in the presence of 250 µg/mL Zeocin were selected by flow cytometry for receptor surface expression.HIV-1 infection Cells (1 × 105) were infected for 4 days with 100 µL suspension of R7-GFP HIV-1 in which Nef-coding sequences were replaced by a modified form of green fluorescent protein (GFP),33 and GFP+ cells were analyzed by flow cytometry. The culture supernatant used to infect the different transfected HEK cell lines was collected after infection for 7 days of the CEM lymphoblastoid cell line. At this time, 21% of CEM cells were GFP+ and reverse transcriptase (RT) activity was 170 000 cpm/mL. GFP+ cells were analyzed by flow cytometry.Flow cytometry Cells (1 × 105) were incubated for 1 hour at 4°C with 50 µL phosphate-buffered saline containing 0.2% bovine serum albumin (PBS-BSA) or PBS-BSA supplemented with the appropriate mAb. After 3 washes with PBS-BSA, bound mAb was revealed by addition of 50 µL of a 1/50 dilution of fluorescein-conjugated (FITC) secondary immunoglobulin. After 30 minutes' staining, cells were washed with PBS-BSA, and fluorescence intensity at 543 nm was measured on an EPICS XL4-C cytofluorometer (Beckman-Coulter France, Villepinte, France). To study the direct binding of SDF-1 to CXCR4
expressed on transfected HEK/CD4.403 cells, 1 × 106
cells were incubated in 30 µL PBS for 1 hour at 4°C to prevent subsequent internalization, and 30 µL SDF-1 solution (100, 200, or
400 nM) was then incubated for 20 minutes at 4°C after cell centrifugation. After washing with PBS-BSA, 30 µL anti-SDF-1 antibody at 10 µg/mL was added for 30 minutes, and bound antibody was
revealed as described previously.
To study CXCR4 internalization on transfected HEK/CD4.403 cells,
1 × 106 cells were incubated at 37°C for 30 minutes
with SDF-1 Chemotaxis assay in adherent cells The technique used was adapted from Goya et al.34 Briefly, tissue culture inserts (Nunc) pore size 8 µm, 10 mm in diameter, were coated with 20 µg/mL collagen type I (Sigma) for 2 hours at 37°C. Cells were starved of serum overnight, trypsinized, washed 3 times in migration buffer (DMEM, 0.25% BSA), counted, and diluted to 5 × 105 cells/mL. A volume of 100 µL of cells was placed in the upper chamber, whereas a solution of SDF-1
(75, 125, or 250 nM) in migration buffer or migration buffer alone was
in the lower chamber. Migration was allowed to proceed for 6 hours at
37°C, 5% CO2. After this time, the cells were removed
from the upper chamber by washing in PBS and scraping. The migrated
cells were visualized by fixing and staining in 0.5% crystal violet in
20% methanol followed by repeated washing in H2O. The
number of migrating cells in 5 fields (×20) was counted by 2 investigators. Results are presented as a migration index calculated as
follows: number of migrating cells with SDF-1/number of migrating
cells in migration buffer alone.
Calcium signaling Cells (1 × 107) were resuspended in Hanks solution (Gibco-Life Technologies) and loaded with Fluo-3-AM at 2 µM for 20 minutes at room temperature. After 3 washes with Earle balanced salt solution (EBSS) containing 0.1% BSA (EBSS-BSA), 106 cells/mL were stimulated with buffer alone (EBSS-BSA) or SDF-1 at 250 nM.
Ionomycine at 10 6 M was then added to verify the
capability of the cells to induce a calcium influx. Ratio of
fluorescence of bound to free Fluo-3 was analyzed each 10 seconds on an
EPICS XL4-C cytofluorometer.
ERK phosphorylation induced after SDF-1 at 125 nM for 2 minutes, lysed in Tris (tris(hydroxymethyl)aminomethane)-HCl 50 mM (pH 8), Triton X-100 1%, NaCl 100 mM, MgCl2 1 mM,
benzamidine 2 mM, leupeptine 2 µg/mL, phenylmethylsulfonyl
fluoride (PMSF) 150 µM containing NaF,
Na3VO4,  glycerophosphate, and
para-nitrophenylphosphate (PNPP).35 Cell
lysates were subjected to electrophoresis through 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
electrotransferred to polyvinylidene difluoride (PVDF)
membranes (Millipore). Membranes were then blocked in Tris-buffered saline (TBS), 0.05% Tween 20, and 10% milk for 1 hour at 20°C. Blots were incubated overnight at 4°C with the primary antibody diluted 1/2500 in TBS-Tween-5% milk. After 30 minutes of washing in 6 changes of TBS-Tween, the blots were incubated for 1 hour at 20°C
with peroxidase-coupled antiserum diluted 1/2000 in TBS-Tween-5% milk.
After further washing, the immune complexes were revealed by enhanced
chemiluminescence (ECL; NEN) and subjected to autoradiography. Quantification of ERK1/2 phosphorylation was performed by using the
Scion program (Frederick, MD) after autoradiography scanning.
Statistical analysis Variance analysis was performed after arc sine transformation of the data36: *P < .05; **P < .01; ***P < .001.
Construction and expression of the CXCR4 mutants To investigate the role of the intracellular domains of CXCR4 in signal transduction after SDF-1 binding, CXCR4 mutants were generated and stably transfected in HEK cells expressing the CD4 molecule deleted of its cytoplasmic tail. The amino acids of each loop
were replaced by those of ICL1, and the amino acids Ser, Thr, and Tyr
(putative sites of phosphorylation) were changed to alanine (Ala).
Furthermore, the sequence of ICL1 was completely modified by
interchanging the amino acid order. In this way, the global charge of
the loop was respected, but the putative binding sites to cytoplasmic
molecules were destroyed. The mutated ICL sequences are described in
Figure 1.
Cell surface expression of the CXCR4.ICL mutants was measured by flow
cytometry and compared with expression of the CXCR4 wild-type molecule
and the CXCR4.7TM mutant on HEK/CD4.403 cells (Figure
2). CXCR4 and CXCR4.7TM are very well
expressed, at an almost identical level, at the cell surface.
CXCR4.ICL3m is well expressed on HEK/CD4.403 cells, but at a lower
level to that of CXCR4 and CXCR4.7TM. CXCR4.ICL1m and the 2 CXCR4.ICL2
mutants, with or without the DRY sequence, are weakly expressed at the cell surface.
CXCR4 mutants expressed on HEK/CD4.403 cells are functional To confirm that the CXCR4.ICL1-3m and CXCR4.7TM molecules are still able to bind SDF-1 when expressed on HEK/CD4.403 cells, SDF-1 at 100, 200, or 400 nM was incubated with the 4 HEK/CD4.403/CXCR4.ICL mutants, HEK/CD4.403/CXCR4.7TM,
HEK/CD4.403/CXCR4, and HEK/CD4.403 cells. SDF-1 binding to these
cell lines was revealed by an anti-SDF-1 antibody. As shown in
Figure 3A, SDF-1 binds to CXCR4.ICL
mutants and CXCR4.7TM, and the level of SDF-1 binding is parallel with that of CXCR4 expression. These CXCR4 mutants are also capable of
serving as a coreceptor for HIV-1 entry (Figure 3B). As for SDF-1
binding, the degree of infection is strictly dependent on the level of
cell surface expression.
SDF-1 -Arrestin plays an essential role in GPCR desensitization and
has been described to bind in vitro to both the third intracellular loop and the C terminus part of CXCR4. Downmodulation of all the CXCR4
mutants expressed on HEK/CD4.403 cells was analyzed after incubation of
SDF-1 at 200nM. On stimulation with SDF-1, surface expression of CXCR4.ICL mutants is reduced at a level comparable to
that obtained with wild-type CXCR4 (Figure
4). Conversely, SDF-1 binding to the
CXCR4 molecule with C terminal truncation does not trigger endocytosis.
CXCR4 expression at the cell surface is thus differentially regulated
after SDF-1 binding, depending on the intracellular interaction
sites involved in receptor internalization. To verify that no
competition occurs in the binding of SDF-1 and the anti-CXCR4
antibody (MAB173) to CXCR4, sequential incubation of these 2 molecules
was performed with HEK/CD4.403/CXCR4 cells. The binding sites of
SDF-1 and MAB173 on CXCR4 are different because binding of one of
these molecules does not hamper binding of the other (data not
shown).
Gi-dependent signals only depend on the third intracellular loop of CXCR4 SDF-1 binding to CXCR4, CXCR4.7TM, CXCR4.ICL1m, CXCR4.ICL2m,
and CXCR4.ICL2mDRY expressed on HEK/CD4.403 cells induced calcium mobilization from ionomycin-sensitive intracellular stores but failed
to trigger calcium response in HEK/CD4.403 and HEK/CD4.403/CXCR4.ICL3m cells. Pretreatment with PTX completely blocked SDF-1-induced calcium mobilization, indicating that ICL3 is involved in
Gi-dependent calcium influx (Figure
5). Increase in SDF-1 concentration (500 nM) did not enhance the level of calcium mobilization induced (data
not shown).
Next, we analyzed the activation of ERK, another
Gi-dependent event, triggered after SDF-1
SDF-1 was first studied. Only HEK/CD4.403/CXCR4 and
HEK/CD4.403/CXCR4.ICL1m cells underwent SDF-1-induced chemotaxis in
a dose-dependent manner (Figure 7A),
indicating that chemotaxis needs the presence of the second and the
third intracellular loops as well as the C terminus part of CXCR4. PTX
strongly, but not totally, inhibits chemotaxis induced by SDF-1
(Figure 7B). Elimination of the gradient by adding the same
concentration of the chemokine in the upper and lower chamber at the
same time completely inhibits chemotaxis, indicating that the migration
observed was not due to chemokinesis (Figure 7B).
These results highlight the complexity of SDF-1
Multiple PTX-sensitive and -insensitive signaling pathways are activated through CXCR4, but little is known of the components of CXCR4 necessary to transduce those signals. The present study was aimed at determining the role of the cytoplasmic domains of CXCR4 in different signaling events after SDF-1 stimulation. The ubiquitous structure of the GPCRs led to the assumption that the 7 transmembrane (TM) domains that confer 3 intracellular and 3 extracellular loops in addition to the N and C terminal segments at
opposite membrane surfaces are the minimum to achieve structural
stability and to trigger activation of signal molecules. The 3 intracellular loops of CXCR4 are highly basic, with 4, 6, and 8 basic
amino acids in the ICL1, ICL2, and ICL3, respectively. The replacement
of each loop by the mutated ICL1 still containing its own basic
residues allowed preservation of the general basic properties of these
loops. ICL1 is the shortest loop, composed of 11 residues, which are
sufficient to link the TM1 to TM2 and induce a functional conformation
of the entire CXCR4 molecule. We thus postulated that replacement of
each loop by the ICL1 modified to destroy the putative sites involved
in binding cytoplasmic signaling proteins but keeping its
characteristics of charge and length could be a novel and interesting
way to analyze the role of the intracellular loops of GPCRs. First, our
data demonstrate that this approach allows cell lines to be derived
that stably express CXCR4 mutants at the cell surface. Additionally,
these mutants are able to bind SDF-1 Numerous studies have shown that in most cases the second and the third ICLs of GPCRs are the major sites for the receptor to interact with G proteins, even if ICL1 of some receptors played a role in transducing G protein-dependent signals.40,41 The current study indicates that ICL1 is not essential for CXCR4 activation even if this loop is needed for CXCR4 to be well expressed at the cell surface. This result agrees with those found by Ling et al42 who demonstrated that a mutated form of CXCR4 in which the N-terminal segment is directly connected to TM3 was still able to transduce SDF-1-induced chemotaxis, calcium influx, and activation of Gi proteins. The DRY box in ICL2 is highly conserved among GPCRs, and its mutation
in different receptors such as rhodopsin, the Extensive work has shown that the third cytoplasmic loop is a major determinant of G protein coupling for several receptors.49-52 Furthermore, the CXCR4 ICL3 contains the BBXXB motif (RKALK) in which B is a basic and X a nonbasic residue, described as a structural determinant for Gi-stimulating function.53 To analyze the role of ICL3 of CXCR4 in transduction of Gi-dependent signaling, we studied several Gi-dependent activation signals after SDF-1 binding to CXCR4 in which ICL3 was replaced by a mutated form of ICL1. This mutant, which is well expressed at the cell surface and capable of binding efficiently to SDF-1, is unable to induce calcium mobilization and ERK phosphorylation, Gi-dependent events, after SDF-1 stimulation. This result emphasizes that the mutated ICL1 used to replace the other loops of CXCR4 and designed to destroy all the putative activation sites is unable by itself to transduce a signal. Calcium mobilization was already shown to be abolished after SDF-1 binding to CXCR4 that contained deletions in ICL3.38 Gi-dependent signaling through CXCR4 is thus supported by only one loop, without cooperation of other cytoplasmic domains. The third intracellular loop, as well as the C terminus part of CXCR4,
has also been involved in GPCR interaction with
arrestin.54-57 It was previously demonstrated that ICL3
and the C terminus of CXCR4 directly interact with Interestingly, ERK is phosphorylated after SDF-1 binding to CXCR4.7TM, in absence of receptor internalization. The role of clathrin-induced endocytosis in GPCR-mediated ERK activation has yielded, until now, conflicting results. Although the present work does not provide insight into the mechanism of ERK phosphorylation by SDF-1 binding to CXCR4, this result underlines the fact that CXCR4 endocytosis is not necessary for ERK activation. In several cell types, including HEK-293 cells, GPCR-stimulated ERK involves the ligand-independent transactivation of receptor tyrosine kinases, such as the EGF receptor.60-62 In our cellular model, SDF-1-mediated phosphorylation of ERK through CXCR4 is independent of the transactivation of the EGF receptor (data not shown). Chemotaxis through CXCR4 needs several distinct signaling pathways.
Neither CXCR4.ICL2m and CXCR4.ICL2mDRY, able to transduce both calcium and ERK signaling and to undergo internalization, nor
CXCR4.ICL3m, unable to transduce Gi-dependent signaling but undergoing internalization and CXCR4.7TM transducing ERK activation and
calcium mobilization without endocytosis, can induce this signaling
event. Indeed, only CXCR4.ICL1m can still transduce signals necessary
for chemotaxis. This mutant shows the same cell surface expression
level as CXCR4.ICL2m and CXCR4.ICL2mDRY, and these 3 mutants transduce
comparable levels of Gi-dependent signals. However, we
cannot totally exclude the fact that the weak expression of CXCR4.ICL2m
and CXCR4.ICL2mDRY may be responsible for the absence of chemotaxis
signaling. The specific signaling functions required for GPCRs to
mediate chemotaxis are complex and poorly understood. Gi
activation and release of free In summary, this study is the first that can dissociate the role of each intracellular domain of CXCR4 in transducing Gi-dependent signaling such as calcium influx and ERK activation, Gi-independent event (internalization), and cell migration. Our results indicate that the ICL3 of CXCR4 alone supports binding to Gi proteins, whereas the second, third, and C terminal intracellular domains are involved in chemotaxis. We have also dissociated ERK activation from receptor internalization.
Submitted August 6, 2002; accepted August 21, 2002.
Prepublished online as Blood First Edition Paper, September 5, 2002; DOI 10.1182/blood-2002-03-0978.
Supported by institutional funds from the Centre National de la Recherche Scientifique (CNRS) and grants from the Agence Nationale de Recherches sur le syndrome de l'immunodeficience acquise (SIDA) (ANRS) and Ensemble contre le SIDA, by an EMBO short-term fellowship (ASTF no. 9468) (B.J.M.), and by financial support from Professor A. J. Pinching (B.J.M. and K.E.N.).
J.R. and B.J.M. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Martine Biard-Piechaczyk, Laboratoire Infections Rétrovirales et Signalisation Cellulaire CNRS UMR 5121, Institut de Biologie, 4 Boulevard Henri IV, CS89508, 34060 Montpellier Cedex 34960, France; e-mail: piechacz{at}xerxes.crbm.cnrs-mop.fr.
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Top
Abstract
Introduction
(PLC-
-B (NF
) or absence (
) of PTX preincubated overnight at
100 ng/mL. Representative curves of [Ca2+]i changes after
stimulation with SDF-1
,
CXCR4
, CXCR4.ICL1m; 
, CXCR4.ICL2mDRY; 
, CXCR4.ICL3m; 
, CXCR4.7TM).
Results are expressed as means of at least 5 independent experiments;
error bars reflect SDs. (B) Inhibition of SDF-1-induced chemotaxis in
HEK/CD4.403/CXCR4 cells. Cells expressing wild-type CXCR4 were
preincubated overnight with PTX (100 ng/mL). SDF-1 was also added to
the upper and lower chemotaxis chambers (250 nM) to eliminate the
chemokine gradient. Data are expressed as means ± SDs of 3 independent experiments. Statistical analysis was performed as
described in "Materials and methods" (*P < .05, **P < .01, ***P < .001).