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INTRODUCTION |
Chemokines comprise a family of between 40 and 50 members that
mediate inflammatory responses, chemotaxis, immune cell development, and leukocyte homing (1-3). Their biological functions are mediated through their interaction with their cognate receptors, which are
members of the G protein-coupled receptor
(GPCR)1 superfamily. Multiple
chemokine receptors have now been shown to act as coreceptors for entry
of the human immunodeficiency virus (HIV) into cells (3, 4). The
receptor for stromal derived factor-
(SDF-1), CXCR4, appears to
act as the primary coreceptor for entry of T-cell tropic strains of HIV
(5), whereas CCR5, which functions as the receptor for the chemokines
RANTES, MIP-1 and MIP-1
, mediates entry of macrophage or M-tropic
viruses (6). The simultaneous interaction of HIV envelope protein
(gp120) with both the CD4 receptor and a chemokine receptor is required for viral entry, after exposure of a chemokine receptor-binding determinant mediated by the initial interaction of gp120 with CD4
(7-10). However, there are examples of chemokine
receptor-dependent but CD4-independent entry of virus
(11-14).
Much effort has been directed toward characterizing the interactions
between gp120 and chemokine receptors and the role of chemokine
receptors in mediating viral tropism (3, 15-18). Entry of HIV appears
to be independent of the ability of the chemokine receptor to
internalize or signal (19-22). Thus, therapeutic intervention has
focused on the disruption of virus-coreceptor interactions, and small
molecule inhibitors of this interaction have been identified and
characterized (23-26). Recently, it has become apparent that interaction between gp120 or chemokines and chemokine receptors may
activate signaling pathways that affect the ability of HIV to replicate
(27-31). Furthermore, other molecules that activate HIV transcription
or influence cellular gene expression have been shown to modulate HIV
infectivity as well as chemokine receptor expression (32-37). In this
regard, it has been shown that modulation of chemokine receptor
expression on the cell surface correlates with HIV infectivity
(38-41). A small molecule agonist of CCR5, aminooxypentane-RANTES, has
been reported to interfere with HIV binding to the receptor and also
induces receptor internalization (40). To date, this is the only
chemokine receptor agonist that appears to be able to block HIV
infectivity both by disruption of HIV-coreceptor interaction and by
promotion of chemokine receptor internalization.
In contrast to the interaction between HIV and coreceptors that occurs
at the cell surface, far less is known regarding the mechanism of CXCR4
or CCR5 trafficking and signaling. An understanding of these mechanisms
is needed in order to design effective HIV therapeutics directed
against virus-chemokine receptor interactions or that promote clearance
of cell surface coreceptors. Importantly, it must be ensured that such
therapies do not inappropriately activate signaling pathways that
induce activation or replication of existing virus in latently infected
cells (29). Internalization and desensitization of an ever-growing
number of GPCRs has been shown to require phosphorylation of
agonist-activated receptors by a family of proteins known as G
protein-coupled receptor kinases (GRKs). This promotes binding of a
second family of proteins called arrestins, which act as adaptors
between the receptor and components of the endocytic machinery, such as
AP-2 and clathrin, both major components of clathrin-coated pits
(reviewed in Refs. 42 and 43). This process also promotes dissociation
of the receptor from heterotrimeric G proteins, thus terminating
receptor signaling. From early endosomes, receptors may then be
dephosphorylated and returned to the cell surface for another round of
activation or enter a degradative pathway.
The determinants for receptor phosphorylation and internalization of
both CCR5 and CXCR4 appear to reside in the C-terminal tail (21, 44).
The involvement of GRKs and arrestins in CCR5 internalization and
desensitization has been reported (21), and residues in the CCR5 C-tail
that are phosphorylated in response to different CCR5 agonists have
recently been identified (45). In this study, we sought to characterize
better the mechanism of CXCR4 internalization, examine the role of GRKs
and arrestins, and identify determinants in the CXCR4 C-tail that
mediate receptor phosphorylation and internalization. We found that
both phorbol ester and SDF-1 stimulation promoted CXCR4
internalization in a dynamin-dependent manner but that only
SDF-1-mediated internalization appeared to utilize an
arrestin-dependent pathway. In contrast to the 7 serine and
threonine residues in the CCR5 C-tail that may potentially be involved
in receptor shut-off, CXCR4 contains 16 serine and threonine residues
as well as a dileucine motif. Thus, we created a number of CXCR4
mutants to identify residues in the C-tail that mediate CXCR4
internalization and phosphorylation in response to either phorbol
esters or SDF-1 stimulation. This analysis revealed that CXCR4
phosphorylation and internalization is mediated by multiple residues in
the C-tail but that the dileucine motif and serines at positions 324, 325, 338, and 339 appear to be the most critical.
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EXPERIMENTAL PROCEDURES |
Plasmid Constructs--
Wild-type and mutant CXCR4 receptors
were subcloned in-frame into pCDNA3 containing a hemagglutinin (HA)
epitope tag at the 5' end constructed as described previously (46).
Wild-type CXCR4 was also expressed in the vector pSR1 (a gift from
Robert Doms, University of Pennsylvania) with equivalent results. CXCR4
mutants were constructed by oligonucleotide-directed polymerase chain reaction mutagenesis, using oligonucleotides up to 99 bases long. Polymerase chain reaction products were digested with EcoRI
or EcoRI and XhoI and ligated with pCDNA3 cut
with the same enzymes. All constructs were sequenced to confirm the
correct orientation and to ensure that no undesired mutations were
introduced. Constructs used to express arrestin-2, arrestin-3, GRK2,
GRK2-K220R, dynamin-I-K44A, arrestin-3-(1-320), and
arrestin-3-(284-409) have been previously described (47, 48).
Transient Transfections and Assay of Receptor
Internalization--
COS-1 or HEK-293 cells were grown in Dulbecco's
modified Eagle's medium (Life Technologies Inc.) at 37 °C in a
humidified atmosphere containing 95% air and 5% CO2.
Cells were grown to 70-80% confluence and transfected with FuGene-6
reagent (Roche Molecular Biochemicals) according to the manufacturer's
protocol. Receptor internalization was measured as described previously (48, 49). Briefly, cells in 60-mm dishes were transfected for 5 h
and then split into 24-well dishes coated with
poly-L-lysine (Sigma). The following day, cells were washed
once with serum-free Dulbecco's modified Eagle's medium and
stimulated with the indicated concentrations of phorbol
12-myristate-13-acetate (PMA, Alexis Corp.) or SDF-1 (Peprotech) for
the times indicated. Medium was aspirated, and cells were fixed for 10 min at room temperature with 3.7% formaldehyde in Tris-buffered saline
(TBS). Cells were washed three times with TBS and then blocked for 45 min with 1% bovine serum albumin/TBS. Cells were then incubated for
1 h with a monoclonal antibody directed against the HA epitope
(101R, Covance Biologicals) diluted 1:1000. Cells were washed three
times, reblocked for 15 min, and incubated for 1 h with goat
anti-mouse alkaline phosphatase-conjugated antibody (Sigma or Bio-Rad)
diluted 1:1000. Cells were washed three times, and antibody binding was
visualized by adding 0.25 ml of alkaline phosphatase substrate
(Bio-Rad). Development was stopped by removing 0.1 ml of the substrate
to a 96-well microtiter plate containing 0.1 ml of 0.4 M
NaOH. Plates were read at 405 nm in a microplate reader (Bio-Rad) using
Microplate Manager software.
Immunofluorescence Microscopy--
To assess redistribution of
CXCR4 and arrestin in living cells, HEK-293 cells were transfected with
1 µg of receptor and 0.25 µg of GFP-tagged arrestin-2. Cells were
grown on poly-L-lysine-coated glass coverslips and mounted
on an imaging chamber (Warner Instrument Corp.) equipped with an inlet
port through which media and compounds could be perfused. For these
studies, the medium used did not contain phenol red or antibiotics.
Cell surface receptors were labeled at 4 °C by incubating cells with
rhodamine-conjugated antibody directed against the HA epitope (12CA5,
Roche Molecular Biochemicals). Cells were examined by microscopy on a
Nikon Eclipse E800 fluorescence microscope using a Plan Fluor 40 × 0.75 objective. Images were collected using QED Camera software and
processed with Adobe Photoshop. For confocal microscopy, cells were
prepared in the same manner, and images were obtained on a Bio-Rad
MRC-Zeiss Axiovert 100 confocal microscope (Hemmelholsteadt, UK) using
a Zeiss Plan-Apo 63 × 1.40 NA oil immersion objective.
Receptor Phosphorylation--
HEK-293 cells in 60-mm dishes were
transfected with 2.5 µg of the indicated CXCR4 construct. 24 h
later, cells were washed twice in serum and sodium phosphate-free
Dulbecco's modified Eagle's medium and then incubated in the same
medium for 2 h. Cells were then labeled with 0.2 mCi of
[32P]orthophosphate (NEN, 8500-9120 Ci/mmol) for 90 min.
Cells were then stimulated with either 1 µM PMA or 100 nM SDF-1 for 10 min. The medium was removed, and cells
were washed once with TBS and lysed in 1 ml of ice-cold lysis buffer
(50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (v/v) Nonidet P-40 (Igepal CA-630, Sigma), 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 mM sodium fluoride, 10 mM sodium pyrophosphate,
1 mM phenylmethylsulfonyl fluoride, 20 µg/ml benzamidine,
10 µg/ml pepstatin, and 10 µg/ml leupeptin). All subsequent steps
were performed on ice or at 4 °C. Cells were then sonicated for
20 s using a Fisher model 550 sonic dismembrator. Cell debris was
pelleted by centrifugation in an Eppendorf centrifuge at 13,000 × g for 10 min. The supernatant was precleared by adding 25 µl of a 50% slurry of protein A-agarose beads (Roche Molecular
Biochemicals) with gentle rocking for 1 h. The beads were removed
by centrifugation, and receptors were immunoprecipitated by adding 4 µl of anti-HA polyclonal antibody (101C, Covance Biologicals) to the
supernatant for 1-2 h. 50 µl of protein A-agarose beads were added,
and the suspension was incubated at 4 °C overnight. The following
day, immune complexes were collected by centrifugation and washed 4 times with ice-cold lysis buffer. Beads were resuspended in 25 µl of
sample buffer (62.5 mM Tris, pH 6.5, 10%
-mercaptoethanol, 10% glycerol, 5% sodium dodecyl sulfate, and 0.1 mg/ml bromphenol blue) and incubated at room temperature for 30 min.
The immunoprecipitates were resolved on 10% SDS-PAGE gels. The gels
were stained with Coomassie Blue, destained, dried, and subjected to
autoradiography at 
80 °C.
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RESULTS |
Characterization of PMA and SDF-1-mediated CXCR4 Internalization
and Role of Arrestins--
We first determined the parameters for
internalization of CXCR4 in response to both heterologous and
homologous receptor stimuli. We used both COS-1 and HEK-293 cells for
this analysis, since they have been shown to contain different amounts
of GRKs and arrestins (50) which affect the extent of receptor
internalization (50, 51). Specifically, COS-1 cells appear to contain
lower amounts of endogenous GRKs and arrestins that result in
inefficient receptor internalization (50). To quantify rapidly the
receptor internalization under a variety of conditions, we used an
ELISA that measures the level of epitope-tagged cell surface receptors. Thus, internalization is detected as a decrease in cell surface receptor levels compared with non-agonist-treated controls. We first
examined CXCR4 internalization in response to its natural agonist,
SDF-1. 100 nM SDF-1 promoted internalization of
approximately 40% of cell surface CXCR4 receptors (Fig.
1A). This amount of internalization was achieved by 30 min and did not increase upon longer
incubation times. SDF-1 promoted CXCR4 internalization in COS-1
cells with kinetics and magnitude nearly identical to that observed in
HEK-293 cells. CXCR4 is also rapidly internalized in response to
phorbol ester stimulation (52, 53). As shown in Fig. 1B, PMA
stimulation of HEK-293 cells expressing CXCR4 resulted in
internalization of 40-45% of surface receptors after 60 min.
Internalization was first detected at 10 nM PMA and was maximal at 1 µM. Time course studies revealed that
maximal receptor internalization occurred between 30 and 60 min after
PMA addition, similar to the kinetics of SDF-1-mediated
internalization (data not shown). However, PMA-mediated internalization
of CXCR4 was less efficient in COS-1 cells. Approximately 20-25% of
cell surface CXCR4 was internalized in COS-1 cells in response to PMA
(Fig. 1B). Inhibition of protein kinase C activity by
treatment of cells with bisindolylmaleimide IX completely abrogated
PMA-mediated internalization of CXCR4 but had no effect on
SDF-1-mediated internalization (Fig. 1C). No effect was
observed upon treatment of cells with bisindolylmaleimide V, an
inactive analog of bisindolylmaleimide IX. Therefore, internalization
of CXCR4 in response to PMA appears to be mediated by activation of
protein kinase C, whereas protein kinase C activity is not required for
CXCR4 internalization in response to SDF-1.
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Fig. 1.
CXCR4 internalization in COS-1 and HEK-293
cells. A, COS-1 ( ) or HEK-293 ( ) cells
transfected with HA-tagged CXCR4 were stimulated with 100 nM SDF-1 for the indicated times, and receptor
internalization was quantitated by ELISA. B, COS-1 or
HEK-293 cells transfected with HA-tagged CXCR4 were stimulated with the
indicated concentrations of PMA for 60 min, and receptor
internalization was quantitated by ELISA. C, HEK-293 cells
transfected with CXCR4 were stimulated for 60 min with 1 µM PMA or 100 nM SDF-1 in either the
presence or absence of 10 µM bisindolylmaleimide V or IX,
and receptor internalization was determined by ELISA. Results are
expressed relative to maximal CXCR4 internalization (regarded as 100%)
in the absence of compounds. All experiments were performed in
duplicate 3-5 times and are expressed as the mean value ± S.E.
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In contrast to CXCR4, internalization of CCR5 is not significantly
promoted by phorbol ester and is relatively inefficient in response to
stimulation by CCR5 ligands (21, 53). To compare directly CXCR4 and
CCR5 internalization, we examined internalization of CCR5 in response
to PMA or RANTES stimulation in HEK-293 cells. As shown in Fig.
2A, only 10-15% of surface
CCR5 internalized in response to RANTES. RANTES-mediated CCR5
internalization was not promoted by coexpression of arrestin-2 or
arrestin-3 and was modestly promoted by coexpression of GRK2. However,
simultaneous overexpression of both arrestin-3 and GRK2 promoted CCR5
internalization in a synergistic manner. These results are similar to
those obtained when CCR5 is stimulated by MIP-1 (21). PMA
stimulation of CCR5 also did not promote significant internalization
and overexpression of GRK2 or arrestins did not promote
internalization. However, overexpression of both GRK2 and arrestin-3
promoted a modest but significant increase in CCR5 internalization
(Fig. 2B). Taken together, our results indicate that CXCR4
and CCR5 have different requirements for internalization in the same
cell type. These differences may result from different affinities of
GRKs and arrestins for activated receptors.
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Fig. 2.
PMA- and RANTES-mediated CCR5
internalization. HEK-293 cells were transfected with 2.5 µg of
HA-tagged CCR5 and 2.5 µg of the indicated constructs and stimulated
with 100 nM RANTES (A) or 1 µM PMA
(B) for 60 min, and receptor internalization was quantitated
by ELISA. All experiments were performed in duplicate 3-5 times and
are expressed as the mean value ± S.E. *, p < 0.05 by independent t test. Arr, arrestin.
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We next addressed the question of whether CXCR4 internalization was an
arrestin- or dynamin-dependent process. We hypothesized that SDF-1-mediated activation of CXCR4 would promote receptor phosphorylation by GRKs and recruitment of arrestin. However, PMA may
mediate CXCR4 internalization via direct phosphorylation of CXCR4 by
protein kinase C or through protein kinase C-mediated activation of
GRK2 (54, 55). We cotransfected HEK-293 cells with CXCR4 and
dynamin-K44A, two different arrestin-3 dominant negative mutants and a
dominant negative mutant of GRK2. Dynamin-K44A is deficient in GTP
binding and functions to inhibit dynamin-mediated scission of
clathrin-coated vesicles from the plasma membrane (56).
Arrestin-3-(1-320) lacks the clathrin binding domain and thus appears
to act by competing with wild-type arrestin for receptor binding.
Conversely, arrestin-3-(284-409) lacks the receptor binding region and
thus appears to compete with wild-type arrestin for clathrin binding
(48). GRK2-K220R contains a mutation in the GRK2 catalytic domain but
retains the ability to bind receptor and has been shown to act as a
dominant negative GRK2 (57). As shown in Fig.
3, both arrestin-3-(284-409) and
dynamin-K44A inhibited internalization of CXCR4 in response to both PMA
(Fig. 3A) and SDF-1 (Fig. 3B). CXCR4
internalization was inhibited 75-90% by dynamin-K44A, whereas
arrestin-3-(284-409) inhibited internalization by 50-60%. The
greater efficiency of inhibition by dynamin-K44A is consistent with
results obtained with other GPCRs (51, 58). In contrast, neither
arrestin-3-(1-320) nor GRK2-K220R significantly inhibited
internalization of CXCR4 in response to either stimulus (Fig. 3,
A and B). The inability of arrestin-3-(1-320)
and GRK2-K220R to inhibit CXCR4 internalization might result from a
high affinity of endogenous GRKs and arrestins for activated CXCR4,
resulting in inefficient competition of GRK2-K220R and
arrestin-3-(1-320) for receptor binding. Alternatively, GRK2-K220R and
arrestin-3-(1-320) may not efficiently interact with CXCR4.
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Fig. 3.
Effect of dominant negative dynamin,
GRK2, and arrestin-3 on PMA and SDF-1-mediated
CXCR4 internalization. HEK-293 cells were transfected with 2.5 µg of CXCR4 and an equal amount of vector (pcDNA3),
pcDNA-dynamin-K44A, pcDNA-arrestin-3-(284-409),
pcDNA-arretin-3-(1-320), or pcDNA-GRK2-K220R. Cells were
stimulated with the indicated concentrations of PMA for 60 min
(A) or 100 nM SDF-1 for the indicated times
(B), and receptor internalization was quantitated by ELISA.
All experiments were repeated 4-8 times, and results are expressed as
the mean value ± S.E. , vector;  , dynamin-K44A; ,
arrestin-3-(284-409);  , arrestin-3-(1-320);  , GRK2-K220R.
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Immunofluorescence Analysis of CXCR4 and
Arrestins--
Redistribution of arrestins from the cytosol to the
plasma membrane upon receptor stimulation has been demonstrated for a number of GPCRs (59). Arrestins have also been shown to redistribute to
clathrin-coated vesicles and colocalize with internalized GPCRs (60,
61). In order to assess redistribution of CXCR4 and arrestins after PMA
or SDF stimulation, HEK-293 cells were transfected with CXCR4 and an
arrestin-2-green fluorescent protein (GFP) chimera. Cell surface CXCR4
receptors were labeled by incubating cells at 4 °C with a
rhodamine-conjugated antibody directed against the N-terminal HA
epitope tag. Cells were then stimulated with PMA or SDF-1, and
trafficking of receptors and arrestins was followed in living cells.
Prior to PMA or agonist stimulation, CXCR4 displayed a distinct cell
surface localization (Fig. 4, B and F), and arrestin displayed a diffuse
distribution suggestive of cytoplasmic localization (Fig. 4,
A and E). Upon stimulation with SDF-1 or PMA,
CXCR4 was observed to redistribute into distinct punctate vesicles
(Fig. 4, D and H, respectively). Arrestin-2-GFP also redistributed into punctate vesicles upon stimulation of cells
with SDF-1 (Fig. 4C). A significant amount of
arrestin-2-GFP was observed to colocalize with internalized CXCR4. In
contrast, redistribution of arrestin-2-GFP was not observed upon
stimulation with PMA (Fig. 4G). We occasionally detected
some cells in which a few puncta were observed; however, we did not
observe any cells in which redistribution of arrestin occurred to the
extent seen after SDF-1 stimulation. The redistribution of arrestin
was completely dependent upon CXCR4 expression, since no change in
arrestin-GFP localization was observed in cells treated with PMA or
SDF-1 that were not transfected with CXCR4 (data not shown). CXCR4
and arrestin-2-GFP that were localized in punctate vesicles also
exhibited a significant amount of colocalization with labeled
transferrin, suggesting that these molecules are present in early
endosomes (data not shown).
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Fig. 4.
Redistribution of CXCR4 and arrestin upon PMA
and SDF-1 stimulation. HEK-293 cells were
transfected with 1 µg of CXCR4 and 0.25 µg of arrestin-2-GFP and
then split onto poly-L-lysine-coated glass coverslips.
Prior to stimulation and viewing, coverslips were mounted in a chamber
as described under "Experimental Procedures." For visualization of
CXCR4, surface receptors were first labeled at 4 °C using
rhodamine-conjugated 12CA5 antibody directed against the N-terminal HA
epitope. The initial localization of arrestin-2 GFP is shown in
A and E, and the initial distribution of CXCR4 is
shown in B and F. 100 nM SDF-1
(C and D) or 1 µM PMA (G
and H) was added to cells, and redistribution of receptors
and arrestin was monitored in real time using the time-lapse
acquisition option (QED software) on a Nikon Eclipse E800 fluorescence
microscope using a Plan Fluor 40 × 0.75 objective. Images shown
were obtained at 20 min after agonist addition and were processed using
Adobe Photoshop software.
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Recently, it has been reported that arrestins are recruited to the
plasma membrane upon stimulation of cells transfected with 2-adrenergic, angiotensin 1a (AT1a), dopamine D1a,
endothelin type A, and neurotensin receptors. However, only in the case
of the AT1a and neurotensin receptors was arrestin observed to
colocalize with internalized receptors (62). Therefore, we further
explored the ability of stimulated CXCR4 to recruit arrestin to the
plasma membrane by confocal microscopy. In cells cotransfected with
CXCR4 and arrestin-2-GFP, SDF-1 stimulation promoted some
redistribution of arrestin to the plasma membrane (Fig.
5, A and B). In
contrast, we did not observe any redistribution of arrestin in response to PMA stimulation (Fig. 5, C and D). Even
prolonged incubation times (up to 20 min) did not produce any change in
the pattern of arrestin distribution (data not shown). The results of
our immunofluorescence studies suggest that arrestin is recruited to
the plasma membrane upon SDF-1 stimulation of CXCR4 and is colocalized with internalized receptors. In contrast, PMA stimulation of CXCR4 does not appear to promote arrestin recruitment to the plasma
membrane or redistribution of arrestin with internalized receptor.
Taken together, our results suggest that arrestins play an important
role in mediating SDF-1-promoted CXCR4 internalization. However, the
ability of arrestin-3-(284-409) to attenuate PMA-mediated CXCR4
internalization does not appear to be due to direct interference with
an arrestin-dependent pathway.
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Fig. 5.
Arrestin recruitment by
SDF-1 and PMA-stimulated CXCR4. HEK-293
cells were transfected with CXCR4 and arrestin-2-GFP and prepared for
microscopy as described in Fig. 4. Redistribution of arrestin-2 GFP to
the plasma membrane was assessed by confocal immunofluorescence
microscopy before and after exposure to 100 nM SDF-1
(A and B, respectively) or 1 µM PMA
(C and D, respectively). Images shown were
obtained at 120 s after agonist addition and were processed using
Adobe Photoshop software.
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Mutagenesis of CXCR4 and Internalization of Mutant
Receptors--
The C-tail of CXCR4 has been shown to be required for
agonist and PMA-mediated CXCR4 internalization (44). A recent study using mink lung epithelial cells (Mv-1-Lu) showed that Ser-324 and
Ser-325 and the dileucine motif at amino acids 328 and 329 appear to be
involved in phorbol ester- but not SDF-1-mediated CXCR4
internalization (53). However, a detailed analysis of the residues
required for SDF-1- or phorbol ester-mediated internalization has
not been reported. We therefore introduced multiple mutations into the
CXCR4 C-tail, focusing on the serine and threonine residues as well as
the dileucine motif. The C-tail mutations are depicted schematically in
Fig. 6A, and their relative
expression levels in HEK-293 cells are shown in Fig. 6B. The
expression level and mobility of most of the CXCR4 mutants was
comparable to wild-type CXCR4, although the IL3289A CXCR4 receptor
migrated with slightly faster mobility compared with wild-type CXCR4
(Fig. 6B). This may be due either to the alteration of two
hydrophobic residues or proteolytic degradation. In addition, we did
not detect expression of either the H350stop or S34678A CXCR4 mutants
by either ELISA or Western blot analysis, except when these constructs
were expressed in the presence of dynamin-K44A (data not shown). A
possible explanation for this result is that these receptors are poorly
expressed but that coexpression of dynamin-K44A inhibits tonic receptor
internalization and permits accumulation of detectable steady-state
levels of receptor. To circumvent this problem and analyze the role of
these residues in receptor internalization, we introduced a stop codon after the glutamic acid residue at position 345 that was expressed comparably to wild-type CXCR4 (Fig. 6B). All CXCR4 receptors
used in this study were capable of mediating viral entry into
CD4-expressing HEK-293
cells.2
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Fig. 6.
Mutagenesis of the CXCR4 C-tail.
A, schematic of CXCR4 C-tail and mutations. The indicated
serine and threonine residues and the dileucine motif were changed to
alanine (underlined). The C-terminal two
(350stop) or seven (345stop) residues were
deleted, and the location of the introduced stop codon is indicated
with an asterisk. B, expression of all HA-tagged
CXCR4 mutants in HEK-293 cells was assessed by immunoblot of total cell
lysates with a monoclonal antibody directed against the HA
epitope.
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We then determined the ability of the mutant CXCR4 receptors, expressed
in either COS-1 or HEK-293 cells, to internalize in response to PMA or
SDF-1. The results of experiments that assessed PMA-mediated
internalization of wild-type and mutant CXCR4 receptors in both COS-1
and HEK-293 cells are shown in Fig.
7A. In HEK-293 cells, the
PMA-mediated internalization of the S330A CXCR4 receptor was as
efficient as wild-type CXCR4, and internalization of E345stop was
modestly decreased. The remaining mutant CXCR4 receptors internalized only 40-50% as well as wild-type CXCR4. Notably, no single mutation reduced PMA-mediated internalization greater than 60% compared with
wild-type CXCR4, suggesting that multiple residues in the C-tail
mediate internalization in response to PMA. This analysis was repeated
in COS-1 cells, in which PMA-mediated CXCR4 internalization is less
efficient compared with HEK-293 (Fig. 1B). Mutation of Ser-330 had no effect, and the ST3412A, S344A, and E345stop CXCR4 receptors internalized 40-50% less efficiently than wild-type CXCR4
(Fig. 7A). In contrast, PMA-mediated internalization of the
S3389A CXCR4 mutant was almost completely undetectable, whereas internalization of S3245A and IL3289A mutant CXCR4 receptors was significantly reduced. It is possible that since PMA-mediated internalization of wild-type CXCR4 is less efficient in COS-1 cells,
mutations in the CXCR4 C-tail have a more profound effect on CXCR4
internalization when expressed in COS-1 cells than in HEK-293
cells.
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Fig. 7.
Effect of CXCR4 C-tail mutations on PMA and
SDF -mediated internalization. The indicated wild-type or mutant
CXCR4 receptors were expressed by transient transfection in HEK-293 or
COS-1 cells and challenged with 1 µM PMA (A)
or 100 nM SDF-1 (B) for 60 min.
Internalization of receptors was quantitated by ELISA. The results are
expressed as the percent internalization relative to wild-type CXCR4,
which was regarded as 100%. All experiments were performed in
triplicate, and results are expressed as the mean value ± S.E. of
6-16 separate experiments.  , HEK-293;  , COS-1.
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We performed a similar analysis of the ability of the different CXCR4
receptors to internalize in response to SDF-1. The results of these
experiments in both COS-1 and HEK-293 cells are shown in Fig.
7B. The S330A mutation resulted in an approximately 40%
decrease in receptor internalization. In contrast, internalization of
the S3245A, IL3289A, and S3389A mutants was reduced between 65 and
90%. Internalization of CXCR4 receptors containing mutations in the
distal portion of the C-tail (ST3412A, S344A, and E345stop) was reduced
by 50-60%. SDF-1-mediated internalization of the mutant CXCR4
receptors in COS-1 cells appeared to closely resemble that observed in
HEK-293 cells (Fig. 7B). The S330A mutation modestly decreased internalization, and internalization of ST3412A, S344A, and
E345stop was reduced by 40-50%. However, mutation of the dileucine motif or the serines at positions 324, 325, 338, and 339 profoundly decreased internalization. Taken together, the dileucine motif and the
serines at positions 324, 325, 338, and 339 appear to be critical for
both PMA- and SDF-1-mediated receptor internalization in both COS-1
and HEK-293 cells.
Effect of GRK2 and Arrestin Overexpression on Internalization of
Mutant CXCR4 Receptors--
Since multiple residues in the C-tail
appear to mediate CXCR4 internalization, we attempted to define the
most critical residues by coexpression of CXCR4 mutants with GRK2 and
arrestins. We reasoned that overexpression of GRK2 and/or arrestins
would promote internalization of CXCR4 receptors containing mutations
in critical residues less efficiently compared with CXCR4 receptors
containing mutations in residues that were less important for
internalization or could be compensated for by the presence of
wild-type residues at other positions. The result of this analysis is
depicted in Fig. 8 for selected CXCR4
receptors and is summarized in Table I.
Three distinct patterns of internalization were observed. First,
overexpression of GRK2, arrestin-2, or arrestin-3 alone had little
effect on SDF-1-mediated internalization of wild-type CXCR4.
However, coexpression of GRK2 and arrestin-3 promoted a significant and
synergistic increase in receptor internalization in response to
SDF-1 (Fig. 8A and Table I). Overexpression of
arrestin-2, arrestin-3, or GRK2 alone modestly increased PMA-mediated
internalization, and coexpression of GRK2 and arrestin-3 did not
significantly enhance internalization compared with expression of
either of these proteins alone (Fig. 8A and Table I). A
similar effect of GRK2 and arrestin expression was observed with regard
to SDF-mediated internalization of CXCR4 receptors containing mutations
in the distal portion of the C-tail (ST3412A, S344A, and E345stop).
Expression of GRK2, arrestin-2, or arrestin-3 alone had little or no
effect on SDF-1-mediated internalization of these receptors, whereas
coexpression of GRK2 and arrestin-3 promoted a synergistic increase in
receptor internalization that was nearly equivalent to that of
wild-type CXCR4 alone (Fig. 8B and Table I). Similar to
wild-type CXCR4, expression of arrestin-2, arrestin-3, or GRK2 alone
promoted a modest increase in PMA-mediated internalization of these
receptors. In contrast to wild-type CXCR4, coexpression of GRK2 and
arrestin-3 synergistically promoted internalization of these receptors
in response to PMA. The exception to this pattern was E345stop, which
may be related to the observation that PMA-mediated internalization of
this receptor is less impaired compared with ST3412A or S344A.
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Fig. 8.
GRK and arrestin coexpression differentially
promotes internalization of CXCR4 mutants. HEK-293 cells were
transfected with 2.5 µg of wild-type CXCR4 (A),
CXCR4(ST3412A) (B), CXCR4(IL3289A) (C), and
CXCR4(S3245A) (D) together with 2.5 µg of pcDNA3
(vector), GRK2, arrestin-2, arrestin-3 constructs, or 1.25 µg each of
GRK2 and arrestin-3. Cells were stimulated with 100 nM
SDF-1 or 1 µM PMA for 60 min, and receptor
internalization was quantitated by ELISA. Experiments were performed in
duplicate 4-8 times, and the results are expressed as the mean
value ± S.E.
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Table I
Promotion of mutant CXCR4 receptor internalization by coexpressed
arrestins and GRK2
HEK-293 cells were transfected with 2.5 µg of the indicated wild-type
or mutant CXCR4 receptor and 2.5 µg of vector, arrestin-2,
arrestin-3, (Arr-3), GRK2, or 1.25 µg each of GRK2 and arrestin-3.
24 h after transfection, cells were challenged with 1 µM PMA or 100 nM SDF-1 for 60 min, and
receptor internalization was quantitated by ELISA. Values are expressed
as percentages relative to cells transfected with wild-type CXCR4 alone
and treated with PMA or SDF-1 for 60 min. The percent internalization
of CXCR4 in cells treated with PMA was 39.6 ± 0.6 and in cells
treated with SDF-1 was 35.5 ± 2.4. These values were then
regarded as 100% internalization. The mean values and S.E. are shown
and represent 4-8 independent experiments performed in duplicate.
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The effect of GRK2 and/or arrestin overexpression on internalization of
the IL3289A and S3389A CXCR4 receptors was dramatically different.
Internalization of the IL3289A CXCR4 receptor mutant in response to PMA
was modestly promoted by GRK2 or arrestin-3 expression, but
coexpression of these proteins did not increase internalization further
(Fig. 8C and Table I). In contrast, expression of arrestins
had no effect on SDF-1-mediated internalization of IL3289A, whereas
GRK2 and GRK2/arrestin-3 modestly promoted internalization (Fig.
8C and Table I). The maximal amount of internalization of
IL3289A was significantly less than wild-type CXCR4 in the absence of
coexpressed GRK2 and arrestins, suggesting that the dileucine motif is
critical for both PMA- and SDF-1-mediated CXCR4 internalization. A
similar effect of GRK2 and arrestin expression was observed with the
S3389A CXCR4 receptor mutant, suggesting that these residues are also
critical for CXCR4 internalization (Table I). However, the
internalization of this mutant receptor in response to SDF-1 was
approximately half that for PMA, suggesting that these residues are
slightly more important for SDF-1-mediated internalization.
The S3245A CXCR4 receptor behaved in a manner intermediate between
these two patterns. PMA-mediated internalization of S3245A was modestly
increased upon coexpression of arrestin-2 and arrestin-3, unaffected by
GRK2 expression, and significantly promoted by simultaneous expression
of GRK2 and arrestin-3 (Fig. 8D and Table I). In contrast, expression of arrestins or GRK2 had little or no effect on
SDF-1-mediated internalization of S32435A, but coexpression of GRK2
and arrestin-3 promoted internalization, albeit to a lesser extent than
with PMA. Therefore, these residues may also be more critical for
SDF-1 compared with PMA-mediated internalization. Taken together,
the results of this analysis suggest that the serine residues at
positions 324, 325, 338, and 339 and the dileucine motif are most
critical for CXCR4 internalization. The presence of wild-type residues at other positions in the C-tail did not compensate as effectively for
mutations at these positions, and overexpression of GRK2 and arrestins
did not promote internalization of these mutants as efficiently.
Analysis of Phosphorylation of Wild-type and Mutant CXCR4
Receptors--
For many GPCRs, agonist activation results in
phosphorylation of receptors by GRKs (42). However, involvement of
arrestins in GPCR internalization in the absence of receptor
phosphorylation has been reported (63, 64). In the case of CXCR4,
phosphorylation could occur by several mechanisms, such as
SDF-1-mediated receptor activation and phosphorylation by GRKs,
direct phosphorylation of receptor by protein kinase C as a result of
phorbol ester stimulation, or phosphorylation by GRK2 as a result of
its activation via phorbol ester-mediated activation of protein kinase
C (54, 55). The serines at positions 344, 346, 347, and 348 are
potentially good candidates for phosphorylation by GRK2, since they are
C-terminal to an acidic residue (65). In contrast, the serines at
positions 324, 325, 338, and 339 conform more closely to potential
protein kinase C phosphorylation sites (66). Therefore, we analyzed wild-type and mutant CXCR4 receptors to determine the extent of receptor phosphorylation and to identify residues that were important for SDF-1 or PMA-mediated CXCR4 phosphorylation. To do this, HEK-293
cells were transfected with the different CXCR4 receptors, metabolically labeled with [32P]orthophosphate, and
stimulated with PMA or SDF-1 for 10 min. HA-tagged receptors were
immunoprecipitated, and phosphorylation of receptors was visualized by
autoradiography. Upon PMA stimulation, phosphorylation of wild-type and
all mutant CXCR4 receptors was increased by 2-3-fold (Fig.
9A). However, the CXCR4
receptors IL3289A and E345stop were phosphorylated to a lesser extent
compared with wild-type CXCR4, whereas receptors containing the S330A, ST3412A, and S344A mutations were phosphorylated to a somewhat greater
extent. Thus, receptor phosphorylation in response to PMA stimulation
does not appear to closely correlate with the extent of
internalization. The extent of receptor phosphorylation in response to
SDF-1 stimulation is shown in Fig. 9B. Stimulation of
wild-type CXCR4 by SDF-1 resulted in an approximately 2-fold increase in receptor phosphorylation after 10 min. However, the IL3289A, S3389A, and E345stop CXCR4 receptors did not appear to be
significantly phosphorylated upon SDF-1 stimulation (Fig. 9B). The remaining CXCR4 mutants were phosphorylated to an
equal or greater extent compared with wild-type CXCR4. These results suggest that the integrity of the dileucine motif and the serines at
positions 338 and 339 are critical for SDF-1-mediated CXCR4 phosphorylation, even in the context of an otherwise wild-type C-tail.
This also correlates with the observation that promotion of
internalization of these receptors by coexpressed GRK2 and arrestins is
less efficient (Table I). The lack of phosphorylation of E345stop may
be the result of the removal of a putative site of GRK2
phosphorylation, although the data presented in Table I suggests that
overexpression of GRK2 and arrestin-3 can overcome this defect. These
results suggest that SDF-1-mediated CXCR4 internalization is at
least partially dependent upon receptor phosphorylation but that the
integrity of the primary sequence or structure conferred by multiple
residues in the C-tail is needed for efficient CXCR4 phosphorylation
and internalization.
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Fig. 9.
Phosphorylation of CXCR4 and CXCR4 C-tail
mutants in response to PMA or SDF-1. The
indicated constructs were transfected into HEK-293 cells. 24 h
later, cells were washed and incubated for 2 h in serum and
phosphate-free medium. The cells were metabolically labeled with
[32P]orthophosphate (0.2 mCi/ml) for 90 min and then
stimulated with 100 nM SDF-1 or 1 µM PMA
(B) for 10 min. Cells were lysed, and HA-tagged CXCR4
receptors were immunoprecipitated and separated by SDS-PAGE as
described under "Experimental Procedures." The dried gel was then
subjected to autoradiography at 70 °C. The autoradiograms shown
are representative of three independent experiments.
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DISCUSSION |
The chemokine receptor CXCR4 plays an important role in B-cell
development as well as inflammation and cell migration (3, 67). In
addition, this chemokine receptor is the major coreceptor for entry of
T-cell tropic human immunodeficiency viruses. In this study, we have
characterized the trafficking of CXCR4 in two different cell types.
Internalization of CXCR4 in response to PMA or SDF-1 stimulation was
inhibited by dominant negative forms of both dynamin-I and arrestin-3.
However, only SDF-1 appeared to promote CXCR4 internalization via an
arrestin-dependent pathway, as assessed by
immunofluorescence analysis of arrestin recruitment and redistribution
after CXCR4 activation. In addition, we have identified residues in the
C-tail of CXCR4 that mediate both receptor phosphorylation and
internalization. The integrity of the dileucine motif and serines 324, 325, 338, and 339 appeared to be the most critical. Mutations at other
positions in the C-tail also appeared to play a role in receptor
internalization, but reduced internalization as a result of mutation of
other serine and threonine residues could largely be compensated for by
overexpression of GRKs and arrestins.
We noted differences between CXCR4 internalization and internalization
of the other major coreceptor for HIV, CCR5. Although CCR5 is
phosphorylated in response to PMA (45), this does not result in
efficient receptor internalization (53), a result confirmed in this
study. Similarly, CCR5 internalization was also inefficient in response
to RANTES, one of its endogenous ligands, in agreement with a previous
study using MIP-1 (21). However, RANTES-mediated CCR5
internalization was synergistically promoted by coexpression of both
GRK2 and arrestin-3, whereas PMA-mediated internalization was modestly
promoted. In contrast, CXCR4 internalization was efficient in the
absence of GRK2 and arrestin overexpression in both HEK-293 and COS-1
cells. COS cells contain lower levels of endogenous GRKs and arrestins,
and internalization of both - and -adrenergic receptors in COS
cells is inefficient in the absence of overexpressed arrestins (47, 50,
51, 68). However, SDF-1-mediated internalization of CXCR4 in COS
cells was equivalent to that observed in HEK-293 cells, although
PMA-mediated internalization was less efficient. Taken together, these
observations suggest that the affinity of GRKs and arrestins present in
the same cell types for agonist-activated CXCR4 may be higher than for
the CCR5 and adrenergic receptors. Biochemical studies will be needed
to understand better the determinants of GRK and arrestin affinity and
specificity at the level of both receptor and effector.
CXCR4 was internalized in response to stimulation with PMA and SDF-1
as assessed by immunofluorescence of labeled cell surface receptors.
However, we failed to observe either arrestin recruitment to the plasma
membrane or redistribution of arrestin with internalized CXCR4 after
PMA stimulation, suggesting that PMA-mediated CXCR4 internalization is
not an arrestin-dependent process. It is possible that
arrestin interaction with CXCR4 activated by PMA may be very transient
and thus was not detected under our conditions. Interestingly, both
PMA- and SDF-mediated CXCR4 internalization was inhibited by
coexpression of dynamin-K44A and dominant negative arrestin-3. Expression of dynamin-K44A, which lacks the ability to bind GTP, almost
completely ablated internalization in response to both stimuli.
Arrestin-3-(284-409), which competes for clathrin binding, inhibited
internalization by approximately 50%. Inhibition of receptor
internalization by dynamin-K44A occurs at a very early step in
endocytosis, i.e. pinching off of clathrin-coated pits. Inhibition of internalization by arrestin-3-(284-409), which
encompasses the clathrin binding domain, is predicted to occur by
competition with endogenous arrestins for clathrin binding. Thus,
expression of arrestin-3-(284-409) may sequester clathrin and
potentially inhibit endocytosis by either
arrestin-dependent or -independent pathways. It has
recently been reported that arrestin-3 may also interact with the AP-2
-adaptin subunit (69). Thus, it is conceivable that expression of
the arrestin C-tail may sequester clathrin, AP-2, or other proteins
with which they interact.
In contrast to PMA, SDF-1-mediated CXCR4 internalization does appear
to be mediated by a pathway involving arrestin. Stimulation of CXCR4 by
SDF-1 promoted redistribution of arrestin to the plasma membrane.
Arrestin also appeared to colocalize with internalized CXCR4, similar
to the colocalization of arrestin observed with internalized AT1a and
neurotensin receptors (62). Dynamin-K44A and arrestin-3-(284-409)
significantly inhibited SDF-1-mediated internalization. Importantly,
this effect was seen in concert with the endogenous cellular complement
of GRKs and arrestins. This is significant, since it has previously
been shown that internalization of the AT1a receptor in the absence of
overexpressed wild-type arrestins, which is equally efficient in both
COS-7 and HEK-293 cells, appears to be dynamin- and
arrestin-independent (70). Neither arrestin-3-(1-320) nor GRK2-K220R
had a significant effect on CXCR4 internalization. However, the
function of both of these dominant negative proteins is dependent upon
their ability to bind to receptor (48, 57). If GRKs and arrestins have
a higher affinity for activated CXCR4, inhibition of internalization by these dominant negative proteins might be inefficient. Alternatively, these mutant proteins may have an intrinsically low affinity for CXCR4.
Indeed, previous studies have shown very modest effects of dominant
negative GRK2 mutants in concert with other receptors (57, 71, 72).
Moreover, the affinity of the GRK2-K220R protein for the
2-adrenergic receptor has been reported to be 10-fold lower than wild-type GRK2 in the presence of G
subunits (57).
Our results further underscore the differences between PMA- and
SDF-1-mediated CXCR4 endocytosis. Moreover, they suggest that the
use of dominant negative arrestins, regardless of their mechanism of
action, should not be the sole criteria used to determine if GPCR
internalization is mediated by an arrestin-dependent
pathway. Studies from several groups have noted differences in CXCR4
trafficking depending on the cell type studied and the receptor
stimulus. CXCR4 internalization in response to phorbol ester appears to be mediated by clathrin-coated vesicles, and CXCR4 was reported to be
recycled to the cell surface after phorbol ester removal (52). In
contrast, SDF-1 or gp120 stimulation of CXCR4-expressing U937, HeLa,
and CEM cells appeared to be clathrin-dependent, but CXCR4
appeared to enter a degradative pathway (73). However, removal of
RANTES appears to result in significant recycling of internalized CCR5
(40). We are currently investigating the cellular localization of CXCR4
at different times after agonist addition, and we are attempting to
develop a quantitative assay for receptor recycling. This analysis
should reveal whether the differences observed in arrestin recruitment
correlate with delivery of the receptor to distinct populations of
endosomes that are involved in receptor recycling or degradation and
whether differences in the affinity of arrestins for activated
receptors play a role in this process.
Trafficking of both CXCR4 and CCR5 appears to require the
serine/threonine-rich C-tail (21, 44). GRKs mediate phosphorylation of
agonist-activated receptors on serine and threonine residues (74).
CXCR4 is unique in that it, unlike other HIV coreceptors, contains a
dileucine motif similar to that identified in the subunit of the
CD3 receptor and the CD4 receptor. The phosphorylation of one or both
of the serine residues in CD3 or CD4 is required for the dileucine
motif to function as an endocytic signal (75-77). The mutation of
individual serines, serine/serine or serine/threonine pairs, and the
dileucine motif revealed residues that are likely to be important for
CXCR4 internalization. A considerable amount of plasticity was observed
with regard to the requirements for PMA-induced internalization. In
HEK-293 cells, no single mutation resulted in greater than a 60%
decrease in CXCR4 internalization. Since PMA-induced internalization of
CXCR4 was less efficient in COS-1 cells, the effect of the C-tail
mutations in this cell line was more revealing. Mutation of the two
serines at positions 338 and 339 almost completely eliminated CXCR4
internalization, and mutation of the two serines at positions 324 and
325 or the dileucine motif severely reduced internalization. The effect
of C-tail mutations on SDF-1-mediated internalization was similar in
both HEK-293 and COS-1 cells. Internalization of CXCR4 receptors containing the mutations S3245A, S3389A, and IL3289A was significantly impaired. Mutation of the serine/threonine pair at positions 341 and
342, the serine at position 344, or deletion of the C-terminal seven
amino acids reduced SDF-1-mediated internalization by approximately 50-60%. Together, these results suggest that the dileucine motif, SSLKIL, and serines 338 and 339 are the most critical for both PMA- and
SDF-mediated CXCR4 internalization.
However, it is clear that multiple residues in the C-tail play a role
in CXCR4 internalization. Therefore, we attempted to discern the
relative importance of different residues by attempting to rescue CXCR4
internalization by overexpression of GRK2 and arrestins. Thus, if the
effect of certain mutations could be compensated for by the presence of
wild-type residues at other sites, overexpression of GRKs and arrestins
might be expected to promote internalization of these receptors.
Conversely, if internalization of certain mutant CXCR4 receptors was
not promoted or was promoted inefficiently, this would indicate
residues that were potentially more important for receptor
internalization. The result of this analysis yielded three distinct
patterns of receptor internalization. Similar to wild-type CXCR4,
internalization of the CXCR4 mutants ST3412A, S344A, and E345stop was
modestly promoted by overexpression of GRK2 or arrestins alone but
promoted in a synergistic manner by simultaneous expression of GRK2 and
arrestin-3. In contrast, internalization of the CXCR4 mutant receptors
S3389A and IL3289A was modestly promoted by expression of GRK2 or
arrestin-3 alone, depending on the stimulus. However, simultaneous
expression of GRK2 and arrestin-3 had no additional effect, and the
maximal amount of receptor internalization in the presence of
overexpressed GRK2 and/or arrestin-3 was significantly less compared
with the other mutant CXCR4 receptors. The behavior of the CXCR4 mutant
S3245A was intermediate. GRK2 and arrestin-3 promoted PMA-mediated
internalization of S3245A in a synergistic manner to levels similar to
wild-type receptor alone. The coexpression of GRK2 and arrestin-3
synergistically promoted SDF-mediated internalization, albeit to a
lesser extent than with PMA. These data suggest that Ser-324 and
Ser-325 might play a greater role in CXCR4 internalization mediated by
SDF-1. Therefore, although multiple residues appear to be involved
in PMA- and SDF-mediated CXCR4 internalization, the serine residues at
positions 338 and 339, and the dileucine motif SSLKIL appear to be the
most critical.
SDF-1 and PMA both promoted phosphorylation of CXCR4 in HEK-293
cells, although the extent of phosphorylation was modest. PMA
stimulation promoted phosphorylation of all of the mutant receptors to
some extent, although phosphorylation of the IL3289A, S3389A, and
E345stop CXCR4 mutant receptors was less than wild-type CXCR4 or the
remaining mutants. This observation is possibly the result of CXCR4
phosphorylation by either protein kinase C-mediated GRK2 activation or
direct phosphorylation of the receptor by protein kinase C. However,
phosphorylation of CXCR4 by PMA stimulation, at least in HEK-293 cells,
does not appear to be a significant determinant for receptor
internalization. In contrast, SDF-1 stimulation promoted
significantly less phosphorylation of the IL3289A and S3389A CXCR4
receptors. Interestingly, expression of GRK2, but not arrestin,
modestly rescued internalization of these receptors, and coexpression
of GRK2 and arrestin-3 did not promote greater internalization than
GRK2 alone. This suggests that the IL3289A and S3389A receptors may
retain a phosphorylation site(s) for GRK2 but interact poorly with
arrestin. Thus, the ability of CXCR4 to undergo phosphorylation in
response to SDF-1 stimulation appears to at least partially
correlate with receptor internalization. Although the E345stop receptor
was not significantly phosphorylated in response to SDF-1,
internalization of E345stop was not decreased to the same extent as
IL3289 and S3389A. Thus, it is possible that internalization of the
E345stop CXCR4 receptor is less dependent upon its ability to be
phosphorylated. Moreover, internalization of E345stop was significantly
promoted by overexpression of GRK2 and arrestin-3, suggesting that
other sites in the C-tail can partially compensate for the absence of
these residues.
Our results suggest that internalization of CXCR4 is mediated by
multiple serines in the C-tail as well as the dileucine motif. This
partially contrasts with a recent study by Signoret et al. (53) who reported that the SSLKIL motif was required for phorbol ester-
but not SDF-1-mediated internalization of CXCR4. This difference
might readily be explained by differences in receptor trafficking or in
the levels of endogenous GRKs and arrestins related to the use of
different cell types (Mv-1-Lu cells compared with COS-1 and HEK-293
cells in this study) or the methods used to assess receptor
internalization. The method we use to assess receptor internalization
employs a common epitope tag rather than an antibody directed against
the receptor itself. Other groups have detected cell surface receptors
with labeled antibodies directed against CXCR4 (12G5) after acid
stripping of the SDF-1 used to stimulate receptor internalization
(52, 53). This could potentially be problematic since SDF-1 can
compete for 12G5 binding (20, 22). In the dileucine motifs present in
the CD4 and CD3 receptors, phosphorylation of a proximal serine
residue(s) is required for the motif to function as an endocytic signal
(77, 78). In this regard, it is interesting that mutation of the
isoleucine/leucine pair in CXCR4 abolishes SDF-1- and reduces
PMA-mediated phosphorylation even though the proximal serine residues
are present. The CXCR4 IL3289 mutant also appeared to run slightly
faster than wild-type CXCR4 or the other mutants in reducing SDS-PAGE
gels (Fig. 5). Therefore, these residues may confer a conformational
structure that affects receptor phosphorylation and internalization.
Dileucine motifs have also been shown to mediate interaction with the
AP-1 and AP-2 clathrin adaptor complexes (77, 78). In light of the
recent report that arrestin-3 may also bind to AP-2 (69), this suggests
that arrestin-mediated CXCR4 endocytosis may be regulated at multiple
junctures. Pitcher et al. (77) have recently reported that
phorbol esters can promote endocytosis of certain CD4 mutants lacking
phosphorylation sites in the dileucine motif. These authors speculate
that phosphorylation of adaptor complexes may regulate the association
of adaptor complexes with sorting signals. Thus, it is possible that
agonist or phorbol ester-mediated phosphorylation of adaptor complexes
as well as receptors themselves may mediate receptor endocytosis. Our
results suggest that regulation of CXCR4 endocytosis is likely to be
mediated by multiple mechanisms. However, further studies will clearly
be required to elucidate the potential contribution of different
components of the endocytic pathway, as well as the ultimate fate of
internalized receptors.
Although multiple chemokine receptors appear to be capable of
mediating HIV entry, a recent report suggests that CXCR4 and CCR5 are
obligate for viral entry despite the presence of other coreceptors that
are capable of mediating HIV entry (79). This suggests that CXCR4 and
CCR5 are the most important targets for antiviral therapy. In contrast
to aminooxypentane-RANTES (40), there is no report of a small molecule
receptor agonist or antagonist of CXCR4 that induces receptor
down-modulation. The bicyclam compounds (23, 25, 26) and Alx-40C (24)
have both been shown to inhibit SDF-1-CXCR4 interaction. However,
they do not appear to promote receptor internalization themselves but
are capable of selectively inhibiting SDF-1-mediated CXCR4
internalization.3 Individuals
homozygous for a 32-base pair deletion in CCR5 that results in the lack
of functional or surface-expressed CCR5 are largely protected from HIV
infection but suffer no obvious detrimental effects (80, 81). In
contrast, studies on mice in which the CXCR4 or SDF-1 genes have
been knocked out reveal an important role for this chemokine-chemokine
receptor pair in B-cell lymphopoiesis, myelopoiesis, migration of
cerebellar neurons, vascular development, and cardiogenesis (82, 83).
Thus, strategies aimed at inhibiting the natural function of SDF-1
or CXCR4 will need to consider their apparently vital role in immune
system development. However, compounds that could induce clearance of
cell surface CXCR4 in addition to blocking virus interaction would be
very attractive. The ability to block surface CXCR4 expression may also
reduce the possibility that signaling as a result of interaction of
gp120, chemokines, or other molecules present during HIV infection with chemokine receptors could activate replication of latent viruses or
otherwise increase cellular transcriptional activity (27, 29, 31, 84).
Moreover, this might also prevent infection by X4 viruses that emerge
later in HIV infection. A better understanding of CXCR4 trafficking and
signaling will be required to understand how these processes may affect
HIV entry and replication and to evaluate the function and
effectiveness of compounds designed to influence HIV-coreceptor interaction.
,
TOP
ABSTRACT
INTRODUCTION
Both authors contributed equally to this work.
