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Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas/Universidad Autonoma de Madrid, Campus de Cantoblanco, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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i activation condition subsequent signaling events,
including internalization and receptor
recycling. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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An N-terminal-modified RANTES, termed aminooxypentane
(AOP)3-RANTES, has
recently been described (10). This analogue binds to the
CCR5 as does RANTES, but does not induce chemotaxis, thus acting as a
RANTES antagonist (10, 11). We have shown that both RANTES
and (AOP)-RANTES activate early signaling events following binding to
CCR5, including triggering of tyrosine phosphorylation and activation
of the Janus activated kinase (JAK)/STAT pathway. They also promote
dimerization and tyrosine phosphorylation of the CCR5
(12), a phenomenon that has been described in other
chemokine receptors following chemokine binding (13).
Whereas both ligands promote Ca2+ influx, only
RANTES induces sustained cell polarization and chemotaxis. (AOP)-RANTES
promotes the activation and association of Gi
to the CCR5 more efficiently, although both association and
dissociation occur more rapidly (12). This conditions the
availability of free, active ß
subunits, which are required to
induce specific ligand-induced responses such as chemotaxis
(14).
After RANTES or (AOP)-RANTES binding to CCR5, there is a striking difference in their ability to trigger receptor down-modulation (11). (AOP)-RANTES thus causes a rapid decrease of >90% of cell surface-expressed CCR5, an effect that RANTES promotes less efficiently. This is due to the fact that (AOP)-RANTES inhibits recycling of internalized CCR5 to the cell surface, whereas RANTES does not (11). This effect has practical consequences, as (AOP)-RANTES is a strong inhibitor of macrophage infection by HIV-1 under conditions in which RANTES is barely effective (11).
Little is known of the regulation mechanisms in the cellular response to chemokines, or of the role of desensitization in lymphocyte migration. The trafficking of lymphocyte populations is a complex process controlled by a vast array of molecules. In this process, cells must be able to sense continuously small changes in chemoattractant gradients. Migration through a chemotactic gradient probably employs an on-off mechanism in which chemokine receptor desensitization, internalization, and recycling may be important steps. For a large number of related G protein-coupled receptors (GPCR), including chemokine receptors, rapid desensitization appears to involve agonist-promoted receptor phosphorylation by GPCR kinases (GRK) (15, 16). We demonstrated that desensitization of CCR2 in monocytes is mediated by GRK2, which translocates to the membrane following monocyte chemoattractant protein-1 (MCP-1) stimulation and phosphorylates the Ser/Thr residues in the carboxyl tail of CCR2, increasing its affinity for ß-arrestin (17). This macromolecular complex prevents any further coupling between the receptor and G proteins; uncoupled receptors are subsequently removed from the plasma membrane through internalization.
In this report, we extend this model by demonstrating that RANTES- and (AOP)-RANTES-promoted CCR5 desensitization involves GRK2 and ß-arrestin. We also show that chemokine receptor internalization takes place through the formation of clathrin vesicles, involving dynamin activity, and provide evidence that the differences in Gi activation between RANTES and (AOP)-RANTES condition subsequent signaling events, including internalization and receptor recycling.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human embryonic kidney (HEK) 293 cells (TIB202) were obtained
from the American Type Culture Collection (Manassas, VA), and
CCR5-transfected HEK 293 cells were donated by Dr. J. Gutierrez
(Department of Immunology and Oncology, Centro Nacional de
Biotecnología, Madrid, Spain). Abs used include
anti-phosphoserine and anti-threonine mAb (Biomol, Plymouth
Meeting, PA), anti-GRK2 Ab AB9 and anti-ß-arrestin-1 Ab Ab186
were donated by Dr. F. Mayor, Jr. (Centro de Biologia Molecular,
Madrid, Spain) (17), anti-JAK2 polyclonal Ab (Upstate
Biotechnology, Lake Placid, NY), anti-clathrin and anti-dynamin
II mAb (Transduction Laboratories, Lexington, KY), anti-MHC class I
(W6/32; American Type Culture Collection), and
anti-ß2-microglobulin (PharMingen, San
Diego, CA). The anti-hGH receptor (hGHR-05) used as isotype-matched
control Ab in FACS analysis was developed in our laboratory
(18). Anti-CCR5 mAb CCR5-02 and CCR5-03 were generated in
our laboratory (12). Recombinant human RANTES and stromal
cell-derived factor (SDF)-1 were from Peprotech (London, U.K.), and
(AOP)-RANTES was kindly donated by Drs. R. Offord, A. E. I.
Proudfoot, and T. Wells (Serono, Geneva, Switzerland).
Flow cytometry analysis
Cells were centrifuged (250 x g, 10 min, room
temperature) and plated in V-bottom 96-well plates (2.5 x
105 cells/well). Cells untreated or treated with
RANTES (10 nM), SDF-1 (10 nM), or (AOP)-RANTES (10 nM) for 30 min at
4°C were incubated with 50 µl/well biotin-labeled mAb (CCR5-03 or
hGHR-05 as an isotype-matched control, 5 µg/ml, 60 min, 4°C). Cells
were washed twice in PBS with 2% BSA and 2% FCS and centrifuged
(250 x g, 5 min, 4°C). FITC-labeled streptavidin
(Southern Biotechnologies Associates, Birmingham, AL) was added,
incubated (30 min, 4°C), and plates washed twice. Cell-bound
fluorescence was determined in a Profile XL flow cytometer at 525 nm
(Coulter Electronics, Miami, FL).
Calcium determination
Changes in intracellular calcium concentration were monitored
using the fluorescent probe Fluo-3AM (Calbiochem, La Jolla, CA). Cells
(0.5 x 106 cells/ml) were treated with 10
µg/ml of CCR5-03 mAb or 10 µg/ml of an isotype control mAb
(hGHR-05) during 30 min at 37°C. After washing, cells (2.5 x
106 cells/ml) were resuspended in RPMI 1640
containing 10% FCS and 10 mM HEPES and incubated with 10 µl of
Fluo-3AM/106 cells (300 mM in DMSO, 15 min,
37°C) (19). After incubation, cells were washed,
resuspended in complete medium containing 2 mM
CaCl2, and maintained at 4°C until just before
RANTES or (AOP)-RANTES addition to minimize membrane trafficking and to
eliminate spontaneous Ca2+ entry. Calcium
mobilization in response to 5 nM RANTES or (AOP)-RANTES was determined
at 37°C in an EPICS XL flow cytometer (Coulter) at 525 nm and
includes background level stabilization and determination of the probe
loading level for each sample. Only samples with a similar load, as
assessed by ionophore-induced Ca2+ mobilization,
were used (5 µg/ml ionomycin; Sigma, St. Louis, MO). For calcium
mobilization in cells pretreated with RANTES, SDF-1, or
(AOP)-RANTES, cells were loaded with Fluo-3AM, treated with 10 nM of
the different chemokines (15 min, 37°C), and, after washing, calcium
flux was determined as above.
Receptor internalization
For receptor internalization analysis, CCR5-transfected HEK 293 cells were serum-starved (0.5 x 106 cells/ml, 60 min, 37°C) in DMEM medium with 0.1% BSA. After washing, cells were incubated for the times indicated with 10 nM RANTES or (AOP)-RANTES at 37°C or 4°C. Cells were washed, and CCR5 expression was determined by flow cytometry analysis using an anti-CCR5 mAb (CCR5-03) compared with an isotype-matched mAb (hGHR-05). Results are expressed as percentage of staining observed in untreated CCR5-transfected HEK 293 cells.
Immunoprecipitation, SDS-PAGE, and Western blot analysis
RANTES- or (AOP)-RANTES-stimulated cells (20 x 106) were lysed in a detergent buffer (20 mM triethanolamine, pH 8.0, 300 mM NaCl, 2 mM EDTA, 20% glycerol, 1% digitonin, with 10 mM sodium orthovanadate, 10 µg/ml leupeptin, and 10 µg/ml aprotinin) for 30 min at 4°C with continuous rocking, then centrifuged (15,000 x g, 15 min). Immunoprecipitations were performed essentially as described earlier (19). Protein extracts precleared by incubation with 20 µg of anti-mouse IgM-agarose (Sigma) were centrifuged (15,000 x g, 1 min), immunoprecipitated with the CCR5-03 mAb (5 µg/sample, 120 min, 4°C), and followed by anti-mouse IgM-agarose. Immunoprecipitates or protein extracts were separated in SDS-PAGE and transferred to nitrocellulose membranes. Western blot analysis was performed as described (18), using 2% BSA in TBS as a blocking agent for the anti-phosphoserine/threonine analysis. When stripping was required, membranes were incubated (60 min, 60°C) with 62.5 mM Tris-HCl, pH 7.8, containing 2% SDS and 0.5% 2-ME. After washing with 0.1% Tween-20 in TBS for 2 h, membranes were reblocked, reprobed with the appropriate Ab, and developed as above. In all cases, protein loading was controlled using a protein detection kit (Pierce, Rockford, IL) and, when necessary, by reprobing the membrane with the immunoprecipitating Ab.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokine receptors undergo internalization after ligand
activation. RANTES and (AOP)-RANTES promote CCR5 internalization in
both freshly isolated primary cells and in transfected CHO cells
(11). We analyzed this process in CCR5-transfected HEK 293
cells. Cells were starved for 60 min before stimulation for different
periods with RANTES, (AOP)-RANTES, or SDF-1 as control. After
washing to eliminate free ligand, cell-surface CCR5 was measured by
FACS analysis using the specific CCR5-03 Ab. This mAb does not
interfere with RANTES binding to CCR5 at either 4°C or 37°C, as
assessed by FACS (Fig. 1
A) and
Ca2+ mobilization assays (Fig. 1B).
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, A and B).
However, (AOP)-RANTES was more effective than RANTES, showing 20%
down-regulation at 5 min and 55% at 15 min, whereas only 5% and 15%
down-regulation was promoted by RANTES at the same times (Fig. 2, A and B). When experiments were performed at
4°C, a condition that inhibits receptor internalization
(20), disappearance of surface CCR5 was minimal following
either stimulus (Fig. 2A). This indicates that the decrease
induced in cell-surface CCR5 was due to receptor internalization and
not to ligand-mediated inhibition of CCR5-03 binding. This receptor
down-modulation has functional consequences; cells pretreated for 15
min with (AOP)-RANTES display minimal calcium mobilization in response
to RANTES, whereas cells pretreated with RANTES still mobilize calcium
in response to RANTES, although this response is reduced compared with
the response when cells are pretreated with SDF-1 (Fig. 2C).
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We previously demonstrated that the rapid uncoupling of the CCR2
chemokine receptor is mediated by MCP-1-promoted translocation of GRK2
to CCR2 and subsequent receptor phosphorylation (17).
Analysis of the CCR5 carboxyl-terminal sequence reveals serine and
threonine residues that may be potential GRK phosphorylation sites, as
are also found in the CCR2. Therefore, we explored whether RANTES and
(AOP)-RANTES treatment of CCR5-transfected HEK 293 cells promotes CCR5
phosphorylation in Ser/Thr residues. Cells treated for different times
with either ligand were lysed and extracts immunoprecipitated with the
CCR5-specific Ab (CCR5-03). Western blot analysis of immunoprecipitates
with a mixture of anti-phosphoserine/threonine Abs showed a
phosphorylated protein in the position corresponding to that of CCR5.
The time course of phosphorylation differed depending on the ligand
used to stimulate the cells. Whereas CCR5 phosphorylation after RANTES
stimulation peaks/reaches maximum at 15 min, similar to the times
described for MCP-1-activated CCR2 (17), (AOP)-RANTES
appears to be more efficient; phosphorylated CCR5 Ser/Thr residues were
apparent as soon as 5 min after stimulation (Fig. 3), confirming the ability of
(AOP)-RANTES to activate GRK2 more rapidly. Protein loading equivalence
was confirmed by reprobing the membrane with specific anti-CCR5 mAb
(CCR5-02).
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As we previously demonstrated that GRK2 is involved in Ser/Thr
phosphorylation of CCR2 in both Mono Mac 1 and CCR2-transfected HEK 293
cells (17), we tested whether this is also the case for
CCR5. As expected, a fraction of cytoplasmic GRK2 associates with the
ligand-stimulated receptor in the plasma membrane. When extracts from
RANTES- and (AOP)-RANTES-stimulated cells are immunoprecipitated with
the CCR5-03 mAb and tested for the presence of GRK2 in Western blot,
GRK2 association to CCR5 is observed in a ligand-dependent manner (Fig. 4). The association is highest at 15 min
after RANTES induction, but (AOP)-RANTES is more efficient, because
GRK2 association is evident at 5 min after activation. Assay
specificity was demonstrated by immunoprecipitating the same cell
extracts with an unrelated control mAb
(anti-ß2-microglobulin) (Fig. 4). The GRK2
AB9 Ab shows slight cross-reactivity with GRK3, but this band migrates
at a slightly different molecular mass, as previously described
(17). Changes in GRK2 association are not a consequence of
variation in the amount of CCR5 immunoprecipitated, because Western
blot analysis of the same membrane after reprobing with the CCR5-02 mAb
confirms equivalent CCR5 protein loading (Fig. 4). These data
demonstrate that 1) GRK2 forms a complex with CCR5 as consequence of
ligand stimulation and 2) that the (AOP)-RANTES derivative is more
efficient than RANTES in triggering this association.
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). In both cases, equal protein loading
in the gel was assessed by reprobing the membrane with the CCR5-02 mAb.
Control experiments with the unrelated
anti-ß2-microglobulin mAb confirm the
specificity of the ß-arrestin-CCR5 interaction (Fig. 5).
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RANTES and (AOP)-RANTES induce rapid internalization of CCR5
(11), although as shown above, RANTES is less efficient
than (AOP)-RANTES in promoting CCR5 down-regulation (Fig. 2A
, B). To
examine whether the internalization process occurs via clathrin
vesicles, RANTES- or (AOP)-RANTES-stimulated cell extracts were
immunoprecipitated with CCR5-03 mAb and tested for clathrin in Western
blot. Clathrin associates to CCR5 following stimulation by either
ligand; this association is most evident at 30 min after RANTES
induction, whereas in (AOP)-RANTES-stimulated cells, clathrin
associates to the receptor by 5 min, with a maximum at 15 min (Fig. 6). Assay specificity was demonstrated by
immunoprecipitating the same extracts with the unrelated
anti-ß2-microglobulin mAb used as control;
equivalent protein loading was confirmed by stripping and reprobing the
membranes with anti-CCR5 mAb (Fig. 6).
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Endocytosis of many GPCR requires the GTPase activity of dynamin
(21). Receptor stimulation leads to recruitment of
cytosolic dynamin to coated pits, where it induces constriction of the
pits and fission of vesicles (22, 23). We also analyzed
whether dynamin associates to CCR5 after RANTES and (AOP)-RANTES
activation. CCR5-transfected HEK 293 cells, untreated or treated with
RANTES or (AOP)-RANTES, were lysed, and cell extracts
immunoprecipitated with CCR5-03 mAb were tested for the presence of
dynamin in Western blot. Dynamin associates to CCR5 following
stimulation by either ligand. This association is evident earlier for
RANTES than for (AOP)-RANTES, but the latter ligand triggers a more
prolonged association. Whereas RANTES promotes maximum association at 5
min, decreasing thereafter, maximum association with (AOP)-RANTES
occurs at 30 min and is maintained throughout the activation studies
(Fig. 7). Thus, we conclude that
(AOP)-RANTES triggers more rapid and sustained dynamin association to
CCR5. Assay specificity was demonstrated by immunoprecipitating cell
extracts from treated cells with the unrelated
anti-ß2-microglobulin mAb used as control;
equivalent protein loading was confirmed by reprobing the membrane with
anti-CCR5 mAb (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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i association to and dissociation
from the CCR5. This gives rise to differences in the pool of free,
active ß subunits, subsequently affecting long-term ligand-induced
responses such as chemotaxis, internalization, and recycling.
To understand the mechanism by which these two ligands trigger
different responses, we used anti-CCR5-specific mAb to analyze CCR5
desensitization and internalization following RANTES or (AOP)-RANTES
stimulation in CCR5-transfected HEK 293 cells. As has been shown for
freshly isolated primary cells and CCR5-transfected Chinese hamster
ovary cells (11), both ligands induce receptor
down-regulation in transfected HEK 293 cells, although (AOP)-RANTES is
more efficient in promoting faster disappearance of CCR5 from the cell
surface. These differences have functional implications, because
RANTES- and AOP-RANTES-induced loss of CCR5 surface expression
correlates with a reduced RANTES-induced calcium signal at this time
point. This observation concurs with previously described differences
in the ability of other CCR5 ligands (macrophage inflammatory protein
(MIP)-1, MIP-1ß, Met-RANTES) to promote calcium flux, receptor
phosphorylation, and receptor down-modulation (11, 26).
We next analyzed the mechanism implicated in CCR5 down-regulation and show that following stimulation both RANTES and (AOP)-RANTES trigger the formation of a macromolecular complex between CCR5, GRK2, the regulatory protein ß-arrestin, and clathrin. These results extend and confirm earlier data for the MCP-1-stimulated CCR2 (17) and assign a clear role to the complex associated to the chemokine receptor during the internalization process.
Supporting these results, recent reports also show that GRK2 and
arrestin colocalize with another GPCR, the ß-adrenergic receptor, in
internalization vesicles (27, 28), and GRK3 has been
associated to ligand-promoted internalization of CCR5 in transfected
rat basophilic leukemia-2H3 cells (26). Our data also lead
to the conclusion that the improved capacity of (AOP)-RANTES to promote
CCR5 internalization is a consequence of improved ability to trigger
complex formation, that is, GRK2 association, phosphorylation in CCR5
Ser/Thr residues, and association of ß-arrestin1 and clathrin. It has
recently been shown that the CCR5 C-terminal serine residues are
phosphorylated following agonist stimulation (26). In
addition, Gß acts as a docking complex, providing an interface for
the GPCR, which would facilitate GPCR interaction with diverse
signaling pathways (29). This is the case for GRK coupling
to other receptors (29), in which ß interacts with
the third intracellular loop of the M2-M3 muscarinic receptors. The
association of ß with the activated GPCR allows formation of a
ternary complex with GRK2, which is required for effective receptor
Ser/Thr phosphorylation, necessary in turn for ß-arrestin
recruitment. Our previous observations showed that (AOP)-RANTES is more
efficient than RANTES in promoting both Gi
association to and dissociation from CCR5 (12). We now
link this to the differential efficiency of these molecules to activate
the Gi pathway, thus making active ß
subunits available, which recruit activated GRK2. Given that
Gi association to CCR5 is a consequence of
ligand-induced receptor activation, it appears obvious that receptor
activation, probably as a result of conformational changes, may differ
following RANTES or (AOP)-RANTES binding. This concurs with the report
of affinity differences between RANTES and (AOP)-RANTES in their
interaction with CCR5, reflected by differences in their binding
properties (10), because RANTES shows biphasic binding to
the receptor whereas (AOP)-RANTES has a single phase competition
curve.
All together, our data explain the increased efficiency of (AOP)-RANTES in promoting CCR5 down-regulation. However, the enhanced ability of (AOP)-RANTES to trigger GRK2 translocation and ß-arrestin-1/clathrin association to the CCR5 does not explain the capacity of (AOP)-RANTES to inhibit normal CCR5 recycling, which does not occur when receptors are activated by RANTES (11). Although endocytosis of chemokine receptors has not been studied in detail, there is evidence that these receptors can either recycle or enter a degradation pathway via the lysosomal compartment; in fact, CXCR2 is reported to undergo degradation after internalization (30). It is believed that receptor recycling from endocytic organelles requires ligand dissociation from the receptor and subsequent dephosphorylation. Here again, we show clear evidence that explains the enhanced efficiency of (AOP)-RANTES-induced internalization, because clathrin association to CCR5 persists longer after (AOP)-RANTES treatment. Nonetheless, this is insufficient to account for the absence of recycling after (AOP)-RANTES stimulation.
Synaptic vesicle recycling and endocytosis of many receptors, including
GPCR, require the GTPase activity of dynamin (31, 32); in
fact, coexpression of CXCR2 and a dominant negative mutant of dynamin
inhibits receptor internalization (33). After ligand
activation, and as a consequence of Src-mediated tyrosine
phosphorylation (21), dynamin is recruited to coated pits,
where it binds to the appendage domain of -adaptin, a component of
the clathrin-coated pits (34). In endocytosis, dynamin
catalyzes a GTP-dependent pinching-off of endocytic vesicles from the
plasma membrane (31). Dynamin is depleted from coated
vesicles relative to coated pits and there is a pool of available
cytosolic dynamin; it is thus assumed that GDP-bound dynamin is
disassembled and recycled via the cytosol for repeated rounds of
vesicle budding (32). This process also takes place in
RANTES- and (AOP)-RANTES-mediated CCR5 internalization. Nonetheless,
whereas RANTES promotes transient association between the dynamin and
CCR5 in the clathrin vesicles, (AOP)-RANTES promotes sustained
association between dynamin, clathrin, and the receptor. The outcome of
the differential dynamin association to CCR5 is that normal CCR5
recycling is altered when cells are stimulated with (AOP)-RANTES. This
suggests that dynamin association to clathrin is a critical step in
normal CCR5 recycling. Furthermore, the loss of receptor recycling
alters receptor expression and therefore the ability of cells to
respond to a continuous signal generated over a concentration gradient
(33). Thus, we conclude that distinct ligands are able to
trigger the differential association of signaling complexes that
further modulate biological responses, and we identify the molecular
complexes that control these processes.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carlos Martínez-A., Department of Immunology and Oncology, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Cientificas/Universidad Autonoma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain. E-mail address:
3 Abbreviations used in this paper: AOP, aminooxypentane; JAK, Janus activated kinase; GPCR, G protein-coupled receptor; GRK, GPCR kinase; MCP, monocyte chemoattractant protein; SDF, stromal cell-derived factor; MIP, macrophage inflammatory protein; HEK, human embryonic kidney.
Received for publication April 26, 1999. Accepted for publication June 29, 1999.
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K. Kraft, H. Olbrich, I. Majoul, M. Mack, A. Proudfoot, and M. Oppermann Characterization of Sequence Determinants within the Carboxyl-terminal Domain of Chemokine Receptor CCR5 That Regulate Signaling and Receptor Internalization J. Biol. Chem., September 7, 2001; 276(37): 34408 - 34418. [Abstract] [Full Text] [PDF]
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 A. J. Vila-Coro, M. Mellado, A. Martin de Ana, P. Lucas, G. del Real, C. Martinez-A., and J. M. Rodriguez-Frade HIV-1 infection through the CCR5 receptor is blocked by receptor dimerization PNAS, March 28, 2000; 97(7): 3388 - 3393. [Abstract] [Full Text] [PDF]
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50 kDa. A
CCR5-transfected HEK 293 cell lysate was included in the gel as a
control of ß-arrestin migration. As a negative control,
RANTES-stimulated cell lysates were immunoprecipitated with
anti-ß2-microglobulin mAb. CCR5 protein loading was
controlled by reprobing the membrane with CCR5-02 mAb. One
representative experiment of three performed is shown.
