From the Department of Biochemistry, Meharry Medical
College, Nashville, Tennessee 37208, the § Department of
Cancer Biology, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232, the
Department of Pathology, National Institute
of Infectious Diseases, Tokyo 162-8640, Japan, and the
** Department of Medicine, Duke University Medical Center,
Durham, North Carolina 27710
Received for publication, November 18, 2002, and in revised form, February 7, 2003
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ABSTRACT |
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Human immunodeficiency virus type 1 (HIV-1) entry
into CD4+ cells requires the chemokine receptors CCR5
or CXCR4 as co-fusion receptors. We have previously demonstrated that
chemokine receptors are capable of cross-regulating the functions of
each other and, thus, affecting cellular responsiveness at the site of
infection. To investigate the effects of chemokine receptor
cross-regulation in HIV-1 infection, monocytes and MAGIC5 and rat
basophilic leukemia (RBL-2H3) cell lines co-expressing the
interleukin-8 (IL-8 or CXCL8) receptor CXCR1 and either CCR5 (ACCR5) or
CXCR4 (ACXCR4) were generated. IL-8 activation of CXCR1, but not the
IL-8 receptor CXCR2, cross-phosphorylated CCR5 and CXCR4 and
cross-desensitized their responsiveness to RANTES (regulated on
activation normal T cell expressed and secreted) (CCL5) and stromal
derived factor (SDF-1 or CXCL12), respectively. CXCR1 activation
internalized CCR5 but not CXCR4 despite cross-phosphorylation of both.
IL-8 pretreatment also inhibited CCR5- but not CXCR4-mediated
virus entry into MAGIC5 cells. A tail-deleted mutant of
CXCR1, Chemokines are a diverse gene family of chemotactic cytokines that
induce leukocyte accumulation and activation at sites of inflammation
(1-3). They also mediate tumor cell trafficking and metastasis and
participate in many acute and chronic inflammatory diseases (4, 5).
Chemokine functions are mediated via cell surface G-protein-coupled
receptors that couple predominantly to Gi (1-3, 18,
35). Chemokine receptors, most notably CCR5 and CXCR4, also serve as
co-receptors for human immunodeficiency virus type 1 (HIV-1)1 entry into
CD4+ cells (6, 7). To date, the relationship between the
activation of these receptors and their role in HIV-1 infection is not
well understood.
Like many members of the G-protein-coupled receptor family, CCR5 and
CXCR4 become desensitized upon agonist exposure, resulting in a loss of
cellular responsiveness to agonist followed by a decrease in the number
of cell surface receptors (8-13). Phosphorylations of the carboxyl
terminus of the receptors are responsible for the desensitization and
down-regulation (8-13). We have previously shown that chemokine
receptors cross-regulate the functions of each other (14, 35). The
interleukin-8 (IL-8 or CXCL8) receptor CXCR1 cross-phosphorylated and
cross-desensitized CCR1-mediated cellular responses to RANTES (CCL5)
(14). The formyl peptide chemoattractant receptor also
cross-desensitized CCR5-mediated cellular responses to RANTES in
monocytes and diminished the ability of RANTES to mediate HIV-1
entry and infection (15, 16).
While HIV-1 infection requires the CD4 receptor, the role of a
chemokine receptor as the fusion cofactor depends on the target cell
(17). Both macrophages and T lymphocytes express CCR5 and CXCR4 (18).
Macrophages, however, utilize CCR5 for HIV-1 entry (M-tropism), whereas
CD4+ T lymphocytes use CXCR4 (T-tropism) (18). In addition
to CCR5 and CXCR4, macrophages and CD4+ T lymphocytes
express other chemokine receptors including the IL-8 receptors CXCR1
and CXCR2 (1-8 × 106 receptors/cell) (1, 18-24). In
the present study we sought to determine the role of cross-regulation
by IL-8 receptors in CCR5- and CXCR4-mediated cellular activation and
HIV-1 infection. For this purpose, monocytes and MAGIC5 and RBL-2H3
cells stably expressing different combination of receptors were used to
study the mechanisms of cross-regulation among IL-8, CCR5, and CXCR4. The results demonstrate that IL-8 led to the cross-phosphorylation and
cross-desensitization of both CCR5 and CXCR4. However, IL-8 down-regulated and inhibited HIV-1 infection to CCR5 but not CXCR4. Since CCR5 is a target for the entry of primary viruses in monocytes these results suggest a selective role for IL-8 in limiting HIV-1 infection through this receptor.
Materials--
[32P]Orthophosphate (8500-9120
Ci/mmol), 125I-IL-8, and 125I-RANTES were
purchased from PerkinElmer Life Sciences. IL-8
(monocyte-derived), melanoma growth-stimulating activity (MGSA or
CXCL1), RANTES, MIP-1 Isolation of Monocytes--
Monocytes were isolated from
heparinized human blood on a multiple density gradient and
enriched for mononuclear cells as described previously (25, 26).
Construction of Epitope-tagged CXCR1, CXCR4, and
CCR5--
Nucleotides encoding the nine-amino acid (YPYDVPDYA)
hemagglutinin (HA) (CXCR1 and Cell Culture and Transfection--
RBL-2H3 cells were maintained
as monolayer cultures in Dulbecco's modified Eagle's medium
supplemented with 15% heat-inactivated fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), and streptomycin
(100 mg/ml) (27). RBL-2H3 cells (1 × 107 cells) were
transfected by electroporation with 20 µg of pcDNA3 containing
the receptor cDNAs, and geneticin-resistant cells were cloned into
a single cell by fluorescence-activated cell sorter analysis. Levels of
protein expression were monitored by fluorescence-activated cell sorter
analysis and Western blotting using 12CA5 (HA)- and M2 (FLAG)-specific antibodies.
Radioligand Binding Assays and Receptor
Internalization--
RBL-2H3 cells were subcultured overnight in
24-well plates (0.5 × 106 cells/well) in growth
medium. Cells were then rinsed with Dulbecco's modified Eagles medium
supplemented with 20 mM HEPES, pH 7.4, and 10 mg/ml bovine
serum albumin and incubated on ice for 2-4 h in the same medium (250 µl) containing the radiolabeled ligand (0.1 nM).
Reactions were stopped with 1 ml of ice-cold phosphate-buffered saline
containing 10 mg/ml bovine serum albumin and washed three times with
the same buffer. Then cells were solubilized with radioimmune precipitation assay buffer (200 µl) dried under vacuum, and bound radioactivity was counted. Nonspecific radioactivity bound was determined in the presence of a 500 nM concentration of the
unlabeled ligand (14, 27).
GTPase Activity--
Cells were treated with appropriate
concentrations of stimulants, and membranes were prepared as described
previously (9, 14). GTPase activity using 10-20 µg of membrane
preparations were carried out as described previously (14, 27).
Phosphoinositide Hydrolysis and Calcium Measurement--
RBL-2H3
cells were subcultured overnight in 96-well culture plates (50,000 cells/well) in inositol-free medium supplemented with 10% dialyzed
fetal bovine serum and 1 µCi/ml [3H]inositol. The
generation of inositol phosphates was determined as reported previously
(9, 14). For calcium mobilization, RBL cells (5 × 106) or monocytes (107) were washed with
HEPES-buffered saline and loaded with 1 µM Indo-1
acetoxymethyl ester for 30 min at room temperature. Then the cells were
washed and resuspended in 1.5 ml of buffer. Intracellular calcium
increase in the presence or absence of ligands was measured as
described previously (27).
Phosphorylation of Receptors--
Phosphorylation of receptors
was performed as described previously (27). RBL cells (5 × 106) expressing the receptors were incubated with
[32P]orthophosphate (150 µCi/dish) for 90 min. Then
labeled cells were stimulated with the indicated ligands for 5 min at
37 °C. Cells were then washed and solubilized in 1 ml of radioimmune precipitation assay buffer containing 50 mM Tris-HCl (pH
7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium
deoxycholate, and 0.1% SDS. Cells lysates were immunoprecipitated with
specific antibodies against the epitope tags, analyzed by SDS
electrophoresis, and visualized by autoradiography.
Construction and Preparation of Viral Plasmids--
Infectious
proviral clones expressing the luciferase gene in place of
nef, pNL-Luc-ADA (CCR5-tropic) and pNL-Luc-HXB
(CXCR4-tropic), were generated as described previously (28). The
HIV-1-based expression vectors pNL-CXCR1 and pNL- Overexpression of CXCR1 and Cross-desensitization of CCR5- and CXCR4-mediated Ca2+
Mobilization in Human Monocytes--
To study the
cross-desensitization of CCR5 and CXCR4, intracellular Ca2+
mobilization in monocytes was elicited by RANTES and SDF-1 and used as
a measure of CCR5 and CXCR4 activation, respectively. As shown in Fig.
1A, IL-8, which activates both
CXCR1 and CXCR2, cross-desensitized Ca2+ responses to
RANTES and SDF-1. MGSA, which only activates CXCR2, had no effect on
RANTES or SDF-1. RANTES and SDF-1 pretreatment attenuated responses to
both MGSA and IL-8 (Fig. 1A). RANTES, SDF-1, IL-8, and MGSA
homologously desensitized (~90%) responses to a second dose of the
same ligand (Fig. 1B).
Cross-inhibition of CCR5- and CXCR4-mediated HIV-1 Infection by
IL-8--
We determined whether IL-8 inhibited HIV-1 entry to CCR5 and
CXCR4. MAGIC5 cells were infected with a lentiviral CXCR1 expression vector virus. Cells were pretreated with or without IL-8 (100 nM) and infected with NL-Luc-ADA (R5-tropic) or NL-Luc-HXB
(X4-tropic), and luciferase activity was measured. As shown in Fig.
2, IL-8 pretreatment inhibited by ~50%
CCR5-mediated R5-tropic virus infection but had no effect in X4-tropic
infection to CXCR4.
Co-expression, Characterization, and Cross-desensitization of
CXCR1, CCR5, and CXCR4 in RBL-2H3 Cells--
To study the mechanism of
cross-regulation among CXCR1, CCR5, and CXCR4, single
transfectant RBL cells expressing FLAG-tagged CCR5 (CCR5-RBL) or CXCR4
(CXCR4-RBL) were first generated and characterized. The
Kd values for RANTES binding to the CCR5 (4.3 ± 0.5 nM) and for SDF-1 binding to CXCR4 (5.7 ± 1 nM) were similar to those previously reported (9, 10).
CCR5 induced a comparable peak of intracellular Ca2+
mobilization in response to both RANTES (Fig.
3A) and MIP-1
In cells co-expressing CXCR1 and CCR5 (ACCR5-RBL) IL-8 pretreatment
cross-desensitized intracellular Ca2+ mobilization to both
RANTES (58%) and MIP-1 Cross-internalization of CCR5 and CXCR4--
ACCR5 and ACXCR4 were
treated with IL-8, RANTES, SDF-1, or PMA (100 nM), and
receptor clearance from the cell surface was assessed by specific
ligand binding. CXCR1 (Fig. 5,
A and C), CCR5 (Fig. 5B), and CXCR4
(Fig. 5D) were homologously internalized by exposure of the
cells to IL-8, RANTES, and SDF-1, respectively. IL-8 pretreatment
cross-internalized CCR5 (Fig. 5B) but not CXCR4 (Fig.
5D). PMA pretreatment caused internalization of both CCR5 and CXCR4 (Fig. 5, B and D). RANTES, SDF-1, and
PMA had no effect in CXCR1 internalization (Fig. 5, A and
C).
Cross-phosphorylation of CCR5 and CXCR4--
To assess the role of
receptor phosphorylation in cross-internalization ACCR5 and ACXCR4
cells were labeled with 32P and treated with IL-8 (100 nM), RANTES (100 nM), SDF-1 (100 nM), or PMA (100 nM). Cells were lysed,
immunoprecipitated with the M2-FLAG (CCR5 and CXCR4)- or HA
(CXCR1)-specific antibodies, and analyzed by SDS electrophoresis and
autoradiography. The identities of the phosphorylated bands for the
respective receptors (CXCR1, ~70 kDa; CCR5, ~40 kDa; and CXCR4,
~45 kDa) have been previously demonstrated (9, 10, 27). As shown in
Fig. 6, CCR5 (A, lane
2) and CXCR4 (B, lane 2) were homologously
phosphorylated by RANTES and SDF-1, respectively. CCR5 (A,
lane 4) and CXCR4 (B, lane 4) were
also cross-phosphorylated by IL-8. CXCR1 was homologously
phosphorylated by IL-8 (A and B, lanes
7) and cross-phosphorylated by both RANTES and SDF-1 (A
and B, lanes 6). PMA induced phosphorylation of
CCR5 (A, lane 3), CXCR4 (B, lane
3), and CXCR1 (A and B, lanes 8).
Role of Protein Kinase C in CCR5
Cross-internalization--
Pretreatment of ACCR5 with the PKC
inhibitor staurosporine (100 nM) partially inhibited
RANTES-mediated CCR5 internalization (Fig.
7A) and phosphorylation (Fig.
7B). Cross-internalization and cross-phosphorylation by IL-8
as well as heterologous internalization and phosphorylation by PMA were
totally inhibited by staurosporine (Fig. 7, A and
B).
Chemokines and chemokine receptors are redundant in that many
chemokines activate more than one chemokine receptor and many chemokine
receptors are activated by multiple chemokines (8, 34). To date, the
structural basis and biological significance of these redundancies
remain unclear. Initial studies in our laboratory, however, provided
evidence that chemokine receptors are capable of cross-regulating the
functions of each other, thus limiting cellular responsiveness to
chemokines. The IL-8 receptor CXCR1 was shown to cross-desensitize
responses to the CC receptor CCR1 at two levels: receptor/G-protein
uncoupling via receptor cross-phosphorylation and inhibition of
phospholipase C HIV-1 entry into CD4+ cells requires the presence of
chemokine receptors such as CCR5 and CXCR4 as co-fusion proteins.
Several studies have indicated that signaling by these receptors is not required for virus fusion and infection (28, 36-38). Activation of
CXCR1, however, diminished the ability of CCR5 to mediate virus entry
(Fig. 2). This suggests that while chemokine-mediated activation of
CCR5 and CXCR4 may not be required, activation of signaling through
other chemokine receptors may, through cross-internalization, prevent
the target receptors to serve as co-factors for HIV-1 infection.
Despite receptor cross-desensitization, CXCR4-mediated virus entry was
resistant to cross-inhibition by CXCR1. This may be due to its relative
resistance to cross-internalization since the stronger signaling
An interesting finding in these studies is the importance of signal
strength in cross-desensitization, cross-internalization, and
inhibition of HIV-1 infectivity. Upon activation by IL-8, CXCR2
internalizes rapidly (~90% after 2-5 min) and, as a consequence, does not mediate cross-regulatory signals (14, 29, 40). CXCR1, which is
more resistant to internalization (~50% after 20-40 min), mediated
cross-phosphorylation and cross-desensitization of both CXCR4 and CCR5
but cross-internalized and inhibited HIV-1 entry to CCR5 but not CXCR4
(Figs. 2, 4, and 5 and Table I) (14, 32, 33). CXCR1-mediated cross-desensitization and cross-internalization of CCR5
and CXCR4 as well as desensitization and internalization by PMA were
inhibited by the PKC inhibitor staurosporine (Fig. 7 and data not
shown). These results indicate that PKC may play a key regulatory role
in the modulation of HIV-1 infection.
The resistance of CXCR4 to cross-internalization by CXCR1 may be
explained in two ways. First, it could be that cross-regulation of
CXCR4 is mediated via a PKC isoform different from that of CCR5, which
requires greater second messenger production for its activation. Indeed
In summary, these data demonstrate that the IL-8 chemokine can inhibit
HIV-1 infection via CCR5 through activation of CXCR1 but not CXCR2.
Inhibition of HIV-1 infection is not blocked by receptor
desensitization alone but requires receptor internalization. CXCR4 is
susceptible to CXCR1-mediated cross-desensitization but is resistant to
cross-internalization. CXCR1, produced greater signals upon activation
(Ca2+ mobilization and phosphoinositide hydrolysis)
and cross-internalized CXCR4, inhibiting HIV-1 entry. The protein
kinase C inhibitor staurosporine prevented phosphorylation and
internalization of the receptors by CXCR1 activation. Taken together,
these results indicate that chemokine receptor-mediated HIV-1 cell
infection is blocked by receptor internalization but not
desensitization alone. Thus, activation of chemokine receptors
unrelated to CCR5 and CXCR4 may play a cross-regulatory role in the
infection and propagation of HIV-1. Since
CXCR1, but not CXCR1,
cross-internalized and cross-inhibited HIV-1 infection to CXCR4, the
data indicate the importance of the signal strength of a receptor and,
as a consequence, protein kinase C activation in the suppression of HIV-1 infection by cross-receptor-mediated internalization.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(CCL4), and SDF-1 were purchased from
Peprotech. Geneticin (G418) and all tissue culture reagents were
purchased from Invitrogen. Monoclonal 12CA5 antibody, protein
G-agarose, and protease inhibitors were purchased from Roche Applied
Science. Anti-human IL-8RA (CXCR1) and IL-8RB (CXCR2) antibodies were
purchased from Pharmingen. Indo-1 acetoxymethyl ester and pluronic acid
were purchased from Molecular Probes. Phorbol 12-myristate 13-acetate
(PMA) and M2-FLAG antibody were purchased from Sigma. FuGENE 6 was
purchased from Roche Applied Science. The enzyme-linked immunosorbent
assay was obtained from PerkinElmer Life Sciences. All other reagents
are from commercial sources.
CXCR1) or the octapeptide (DYKDDDDK)
FLAG (CCR5 and CXCR4) epitope sequences were inserted between the
amino-terminal initiator methionine and the second amino acid of each
cDNA by polymerase chain reaction as described previously (9, 10, 27). The resulting PCR products were cloned into the eukaryotic expression vector pcDNA3, and the receptors were sequenced to confirm the intended mutations and lack of secondary mutations.
CXCR1 were
generated from pNL-Luc/Rev
(28)2 by replacing the
NotI-XhoI fragment encoding luciferase with a
PCR-amplified NotI-XhoI fragment encoding either
CXCR1 or
CXCR1 (CXCR1 minus amino acids 335-349 of the carboxyl
terminus). HIV-1 virus stocks and HIV-1-based lentivirus vectors were
prepared in 293T cells as described previously (30-32). Briefly, 293T
cells were transfected with 2 µg of the proviral expression plasmids carrying a luciferase reporter gene, pNL-Luc-HXB or pNL-Luc-ADA, by
using FuGENE 6. For HIV-1-based lentivirus, 293T cells were cotransfected with 0.5 µg of the Rev expression vector pcRev (32) and
0.5 µg of the vesicular stomatitis virus glycoprotein expression vector pHIT/G (33) with 1 µg of a lentivirus vector plasmid using
FuGENE 6. The culture medium was replaced 16 h later, and the
culture supernatants were harvested 40 h after transfection and
filtered through 0.45-µm-pore-size filters, and virus yield was
measured by enzyme-linked immunosorbent assay. Virus stocks were stored
at
80 °C until needed.
CXCR1 on Human Cells and
Luciferase Reporter Virus Assays--
MAGI (HeLa-CD4-LTR-
-Gal) and
MAGIC5 (MAGI stably expressing CCR5) (31) cells were maintained as
monolayer in Dulbecco's modified Eagle's medium supplemented
with 15% fetal bovine serum as described above. Cells (5 × 105) were transduced overnight with 10 ng of p24 antigen of
vesicular stomatitis virus glycoprotein-pseudotyped lentiviral vector
virus encoding either CXCR1,
CXCR1, or a control vector, pNL-con.
The cells were then washed with phosphate-buffered saline and cultured in fresh medium for an additional 24 h. CXCR1 and
CXCR1
expression was monitored by fluorescence-activated cell sorter
analysis. Then the transduced cells were treated with 100 nM IL-8 for 30 min and incubated with 20 ng of p24 antigen
of a luciferase reporter virus, NL-Luc-HXB or NL-Luc-ADA, for an
additional 60 min. The cells were washed extensively with
phosphate-buffered saline and cultured in fresh medium. After 48 h, the cells were lysed in 200 µl of lysis buffer, and luciferase
activities were determined with a MicroLumatPlus LB96V microplate luminometer.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
IL-8- and MGSA-mediated cross-desensitization
of CCR5 and CXCR4-mediated intracellular calcium mobilization in human
monocytes. Human monocytes (1 × 107) were loaded
with the calcium indicator Indo-1 and exposed to a first
EC100 dose (10 nM) of IL-8, MGSA, RANTES, or
SDF-1. Cells were rechallenged 3 min later with a second dose of the
same (B) or a different ligand (A) as indicated.
Traces are representative of three experiments.
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Fig. 2.
HIV-1 infection of MAGIC5 cells. MAGIC5
cells were transduced overnight with 10 ng of p24 antigen of vesicular
stomatitis virus glycoprotein-pseudotyped lentiviral vector virus
encoding CXCR1. The cells were treated with 100 nM IL-8 for
30 min and incubated with 20 ng of p24 antigen of a luciferase reporter
virus, NL-Luc-HXB (R5-tropic) or NL-Luc-ADA (X4-tropic), for 60 min.
Luciferase activities in cell lysates were determined 48 h
postinfection. The data are represented as percent infection
efficiency, which is the total relative light units measured in control
or untreated cells. Data are the means ± S.E. of three different
experiments.
(data not
shown) but not IL-8, MGSA, or SDF-1 (Fig. 3A). CXCR4 was
also specific for SDF-1-mediated Ca2+ mobilization (Fig.
3B). Cells expressing CXCR1 were specific for IL-8 (data not
shown).
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Fig. 3.
Functional characterization of CCR5 and CXCR4
expressed in RBL-2H3 cells. RBL cells (5 × 106)
stably expressing CCR5 (A) or CXCR4 (B) were
loaded with Indo-1 and tested for either IL-8, MGSA, RANTES, or SDF-1
(10 nM)-stimulated Ca2+ mobilization.
Representative tracings of five experiments are shown.
(56%) (Table
I). IL-8 also inhibited SDF-1
(35%)-mediated Ca2+ response in cells co-expressing CXCR1
and CXCR4 (ACXCR4-RBL). Inhibition of responses to CCR5 was greater
than that of CXCR4 (56-58 versus 35%,
respectively). IL-8-mediated Ca2+ mobilization was also
attenuated by pretreatment of the cells with RANTES, MIP-1
, or
SDF-1 (Table I). IL-8, RANTES, MIP-1
, and SDF-1 also homologously
desensitized (~90%) the response to a second dose of the same ligand
(Table I). Pretreatment of ACCR5 (Fig.
4A) or ACXCR4 (Fig.
4B) cells with IL-8 (100 nM) also
cross-desensitized RANTES (~55%)- and SDF-1 (~40%)-induced GTPase
activity in membranes (Fig. 4). Both RANTES and SDF-1 cross-inhibited
IL-8-mediated GTPase activity by ~35 and ~45%, respectively. PMA
(100 nM) heterologously desensitized the GTPase response to
IL-8 (~35%), RANTES (~60%), and SDF-1 (~50%).
Cross-desensitization among CCR5, CXCR4, and CXCR1 in transfected
RBL cells
, SDF-1, or IL-8 (10 nM)-mediated Ca2+ mobilization was measured. Cells
were rechallenged 3 min later with a second dose (10 nM) of
the indicated ligand, and peak intracellular Ca2+ mobilization
was determined. Data are the means ± S.E. of three different
experiments.
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Fig. 4.
Cross-desensitization of CCR5- and
CXCR4-mediated GTPase activity. Double transfected RBL-2H3 cells
expressing CXCR1 and either CCR5 (ACCR5) (A) or CXCR4
(ACXCR4) (B) were treated with IL-8 (100 nM),
RANTES (100 nM), SDF-1 (100 nM), or PMA (100 nM) for 5 min. Membranes were prepared and assayed for
agonist-stimulated GTP hydrolysis. The data are presented as percentage
of control, which is the net maximal stimulation, obtained with
untreated cells. Data shown are representative of one of three
experiments performed in triplicate.
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Fig. 5.
Cross-internalization of CCR5 and CXCR4.
ACCR5 (A and B) and ACXCR4 (C and
D) RBL cells (0.5 × 106 cells/well) were
treated with a 100 nM concentration of either IL-8, RANTES,
SDF-1, or PMA at different times. Cells were then washed and assayed
for 125I-IL-8 (A and C),
125I-RANTES (B), or 125I-SDF-1
(D) binding. The values are presented as percentage of
total, which is defined as the total amount of 125I-ligand
bound to control (untreated) cells. The experiment was repeated four
times with similar results.
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Fig. 6.
Cross-phosphorylation of CCR5 and CXCR4.
32P-Labeled ACCR5 (A) and ACXCR4 (B)
RBL cells (5 × 106/60-mm plate) were incubated for 5 min with or without stimulants as shown. Cells were lysed,
immunoprecipitated first with an anti-FLAG (CCR5 and CXCR4) and second
with anti-HA (CXCR1) antibodies specific for the M2 and HA epitope
tags, respectively, expressed at the amino terminus of the receptors,
and then analyzed by SDS-PAGE and autoradiography. The results are from
a representative experiment that was repeated three times.
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Fig. 7.
Effect of staurosporine on CCR5
cross-internalization and cross-phosphorylation. A,
ACCR5 cells (0.5 × 106 cells/well) were incubated
with and without staurosporine and treated with a 100 nM
concentration of either IL-8, RANTES, or PMA for 60 min. Cells were
then washed and assayed 125I-RANTES binding as indicated in
the legend of Fig. 5. The values are presented as percentage of total,
which is defined as the total amount of 125I-RANTES bound
to control (untreated) cells. The experiment was repeated twice with
similar results. B, 32P-labeled ACCR5 cells were
incubated with and without staurosporine for 5 min and then stimulated
with a 100 nM concentration of either RANTES (lanes
3 and 4), IL-8 (lanes 5 and 6),
or PMA (lanes 7 and 8). Cells were lysed,
immunoprecipitated with anti-FLAG antibody, electrophoresed into a 10%
SDS-polyacrylamide gel, and autoradiographed. Two other experiments
yielded similar results. Cont, control; Stau,
staurosporine.
CXCR1-mediated Cross-internalization and Cross-inhibition of
CXCR4--
The role of IL-8 in CXCR4 cross-internalization was further
assessed by co-expressing a carboxyl terminus-deficient
mutant of CXCR1,
CXCR1, along with CXCR4 (
ACXCR4). The
Kd and Bmax of
CXCR1
(2.1 ± 1.10 nM and 6898 ± 523 receptors/cell, respectively) were similar to those of CXCR1 (1.7 ± 0.33 nM and 7013 ± 311 receptors/cell, respectively).
CXCR1 mediated greater phosphoinositide hydrolysis (Fig.
8A), G-protein activation,
secretion of
-hexosaminidase, and sustained Ca2+
response relative CXCR1.2 IL-8 pretreatment of
ACXCR4
but not ACXCR1 cells resulted in cross-internalization of CXCR4 (Fig.
8B). In contrast to CXCR1,
CXCR1 activation also
cross-inhibited CXCR4-mediated virus entry into MAGI cells (Fig.
8C).
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Fig. 8.
CXCR1-mediated phosphoinositide
hydrolysis, cross-internalization, and cross-inhibition of CXCR4.
A, for the generation of [3H]inositol
phosphates, RBL cells (50,000 cells/well) co-expressing CXCR4
and either CXCR1 (ACXCR4) or
CXCR1 (
ACXCR4) were
cultured overnight in the presence of [3H]inositol (1 µCi/ml). Cells were preincubated (10 min at 37 °C) with
HEPES-buffered saline containing 10 mM LiCl in a total
volume of 200 µl and stimulated with 100 nM IL-8 or SDF-1
for 10 min. Supernatant was used to determine the release of
[3H]inositol phosphates. Data are represented as
-fold stimulation over basal. The experiment was repeated three
times with similar results. B, ACXCR4 and
ACXCR4 RBL
cells (0.5 × 106 cells/well) were treated with a 100 nM concentration of IL-8 at different times and assayed for
125I-SDF-1 binding as described in the legend of Fig. 5.
C, MAGI cells were transduced overnight with 10 ng of vesicular stomatitis virus glycoprotein-pseudotyped lentiviral
vector encoding either CXCR1 or
CXCR1. Cells were treated with IL-8
(100 nM) and infected with X4-tropic virus, and luciferase
activities in cell lysates were determined as described in the legend
of Fig. 2.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
activity via phosphorylation of the enzyme,
which diminishes its activation by G-protein (14, 35). The data herein
describe another level of cross-regulation among chemokines that may
have important consequences in their action: chemokine-mediated
receptor cross-internalization. Chemokine-mediated receptor
cross-internalization appears to be selective and distinct from
receptor desensitization. This contention is based on the following
observations. First, IL-8 activation of CXCR1, but not CXCR2,
cross-internalized CCR5 (Fig. 5 and data not shown). Second, while
CXCR1 cross-phosphorylated and cross-desensitized Ca2+
mobilization and GTPase activity to both CCR5 and CXCR4, receptor internalization occurred only with CCR5.
CXCR1 or PMA, which induced cross-internalization as well as
cross-desensitization of CXCR4, also inhibited HIV-1 infection through
CXCR4. Activation of the formyl peptide receptor also
cross-phosphorylated and cross-inhibited HIV-1 infection to CCR5 (16),
but these chemoattractants are less commonly present at sites of
inflammation than the chemokines. Chemokine production can be induced
by many stimuli including cytokines, lipopolysaccharides, and viral
products (39). Modulation of chemokine receptor internalization may
therefore be a useful target for therapeutic intervention against HIV-1 infection.
CXCR1, however, which
is far more resistant to internalization (~10% after 60 min) and
mediated greater cellular responses (i.e. phosphoinositide hydrolysis, exocytosis, and Ca2+
mobilization), cross-internalized CXCR4 and inhibited T4-tropic virus
entry (Fig. 8).2 This indicates a hierarchy in
receptor-mediated cross-regulation that is directly correlated with the
receptor resistance to desensitization, internalization, and, as a
consequence, signaling time. Supporting that contention is that in
monocytes isolated from mice deficient in
-arrestin-2 in which CXCR2
internalization is delayed (~25% after 5 min) MGSA
cross-desensitized Ca2+ mobilization to RANTES by ~50%
relative to control or wild type mice (~90% after 5 min).3
CXCR1, which mediated greater phosphoinositide hydrolysis and
Ca2+ mobilization, cross-internalized CXCR4. Second,
previous studies in our laboratory have shown that PMA-induced CXCR4
internalization occurs via a mechanism distinct from receptor
phosphorylation (9). It is likely that phosphorylation of (an)other
component(s) distal from the receptor/G-protein coupling is necessary
for the PKC-mediated internalization and may require a higher level of second messenger production. Orsini et al. (11) have shown
that a dileucine motif of the carboxyl terminus of the receptor that binds the adaptor protein-2 was necessary for the
phosphorylation-independent internalization of the receptor. Whether or
not adaptor protein-2 plays a role in the immunomodulation of HIV-1
infection by CXCR4 remains to be explored. Thus far our preliminary
studies have shown that, upon activation, both CXCR4 and CCR5 bind
adaptor protein-2 (data not shown).
CXCR1 and PMA, which mediated greater
cellular responses, cross-internalized and cross-inhibited CXCR4-mediated virus entry. This suggests that signaling through other
chemokine receptors with stronger signal strengths may regulate the
ability of CXCR4 as well as CCR5 to function as co-fusion proteins with
CD4+. This observation presents new targets for therapeutic
intervention against the infection and propagation of HIV-1.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants AI-38910 (to R. M. R.) and DE-03738 (to R. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, Meharry Medical College, 1005 Dr. D. B. Todd, Jr. Blvd., Nashville, TN 37208. Tel.: 615-327-6749; Fax: 615-327-6442; E-mail: mrrichardson@mmc.edu.
Published, JBC Papers in Press, February 19, 2003, DOI 10.1074/jbc.M211745200
2 Richardson, R. M., Marjoram, R. J., Barak, L. S., and Snyderman, R. (2003) J. Immunol. 70, 2904-2911.
3 R. M. Richardson and R. Marjoram, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: HIV-1, human immunodeficiency virus type 1; IL-8, interleukin-8; CXCR1, IL-8 receptor A; CXCR2, IL-8 receptor B; PMA, phorbol 12-myristate 13-acetate; RANTES, regulated on activation normal T cell expressed and secreted; SDF, stromal derived factor; RBL, rat basophilic leukemia; MGSA, melanoma growth-stimulating activity; HA, hemagglutinin; PKC, protein kinase C.
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