From the Department of Immunology, Georg-August-University, 37075 Göttingen, Germany
Received for publication, September 25, 2002, and in revised form, October 25, 2002
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ABSTRACT |
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Human CC chemokine receptor 5 (CCR5), a member of
the superfamily of G protein-coupled receptors, regulates the
activation and directed migration of leukocytes and serves as the main
coreceptor for the entry of R5 tropic strains of human immunodeficiency
viruses. We have previously shown that RANTES/CCL5 binding to CCR5
induces GPCR kinase (GRK)- and protein kinase C (PKC)-mediated
phosphorylation of four distinct C-terminal serine residues. To study
these phosphorylation events in vivo, we have generated
monoclonal antibodies, which specifically react only with either
phosphorylated or nonphosphorylated CCR5. These
phosphosite-specific antibodies reveal that following ligand
stimulation of the receptor serine 337 is exclusively phosphorylated by
a PKC-mediated mechanism, while GRKs phosphorylate serine 349. GRK-mediated receptor phosphorylation proceeds in a regular
time-dependent manner (t1/2 ~2 min)
with an apparent EC50 of 5 nM. In contrast, PKC
phosphorylates serine 337 at 50-fold lower concentrations and in a very
rapid, albeit transient manner. Protein phosphatases that are active at
neutral pH and are inhibited by okadaic acid rapidly dephosphorylate
phosphoserine 337, but less efficiently phosphoserine 349, in intact
cells and in an in vitro assay. Immunofluorescence
microscopy demonstrates that phosphorylated receptors accumulate in a
perinuclear compartment, which resembles recycling endosomes.
This study is the first to analyze in detail the spatial and
temporal dynamics of GRK- versus PKC-mediated
phosphorylation of a G protein-coupled receptor and its subsequent
dephosphorylation on the level of individual phosphorylation sites.
G protein-coupled receptors
(GPCR)1 comprise the largest
known family of signal-transducing proteins and respond to a large variety of external stimuli (1, 2). The receptors relay the information
encoded by the ligand through the activation of heterotrimeric guanine
nucleotide-binding proteins and intracellular effector molecules. Many
GPCR undergo a process of rapid desensitization, which involves
ligand-induced phosphorylation of serine and threonine residues located
in the third intracellular loop or C-terminal domain by two different
families of protein kinases. (i) GPCR kinases (GRKs) specifically
phosphorylate only the agonist-occupied GPCR and thus mediate
agonist-specific or homologous receptor phosphorylation (3, 4). (ii) In
contrast, the second messenger-activated kinases, such as cyclic
AMP-dependent protein kinase and protein kinase C (PKC),
potentially phosphorylate both the ligand-bound GPCR and multiple other
receptors in a heterologous manner. Receptor phosphorylation enhances
the affinity of the agonist-occupied receptor for interaction with
arrestin which interdicts signal transduction between the receptor and
G proteins by steric mechanisms. The nonvisual arrestins,
Although reversible receptor phosphorylation is a well recognized
mechanism that plays important roles in multiple aspects of GPCR
signaling, with few exceptions the exact sites of second messenger
kinase- and GRK-mediated receptor phosphorylation have not been
identified. Most insights into receptor phosphorylation derive from
in vitro assays with purified proteins in reconstituted systems or from mutagenesis studies with elimination of the presumed consensus sites for receptor phosphorylation under experimental conditions of protein overexpression. Results obtained by these various
methods can sometimes prove misleading, as illustrated by studies with
the In the present study we have taken a new approach to the analysis of
receptor phosphorylation in intact cells. By using phosphosite-specific monoclonal antibodies we determined with high temporal and spatial resolution the ligand-induced phosphorylation and dephosphorylation of
CC chemokine receptor CCR5 at two separate phosphorylation sites. This
receptor is well suited as a model protein for this kind of analysis
since its function as cofactor for the entry of R5 tropic strains of
human immunodeficiency viruses (HIV) has stimulated detailed
investigations into the mechanisms that regulate CCR5 signaling and
cell surface expression in recent years. In particular, this receptor
was shown to be a potential substrate for all major GRK isoforms (9,
10), and inhibitor studies revealed that PKC as well as GRK2 and/or
GRK3 are responsible for the chemokine-induced receptor phosphorylation
in rat basophilic leukemia (RBL) cells, which stably express CCR5 (10).
Two-dimensional phosphoamino acid analysis in combination with
site-directed mutagenesis identified four serine residues that are
located in the CCR5 C terminus as the only potential phosphorylation
sites on this receptor (10). Intact C-terminal phosphorylation sites
were found to be necessary for It was previously unknown whether phosphorylation of
different serine residues can be attributed to particular kinases or whether they compete for the same phosphoacceptor sites. The current report demonstrates that agonist-induced CCR5 phosphorylation at two
distinct sites proceeds with different kinetics and characteristic intracellular distribution in a dynamic manner that involves different protein kinases and phosphatases.
Materials--
Tissue culture reagents were from Biochrom;
RBL-2H3, HEK-293, and X63-Ag8.653 cells were from the American Type
Culture Collection; LipofectAMINE was from Invitrogen; geniticin,
detergents, potato acid phosphatase, activators and inhibitors of
protein kinase C, proteases, and phosphatase inhibitors were from
Calbiochem; synthetic phospho-/peptides were from Jerini; enhanced
chemiluminescence (ECL) Western blotting reagents and protein
G-Sepharose were from Amersham Biosciences; the CCR5 antagonist TAK-779
was kindly provided by Takeda; anti-phosphotyrosine antibody PY20 was
from Becton Dickinson Transduction Laboratories; horseradish-conjugated
and FITC-conjugated anti-mouse antibodies were from Dako; streptavidin peroxidase was from Jackson ImmunoResearch; all other reagents were
from Sigma-Aldrich.
Cell Culture and Transfection--
Rat basophilic leukemia
cells, which stably express wild-type CCR5 (RBL-CCR5 (10)) or CCR5
mutants with alanine exchange of three serine residues at amino acid
positions 336, 337, and 342 (RBL-CCR5-AAAS, Ref. 12) or of all four
C-terminal serine phosphorylation sites (RBL-CCR5(P-), Ref. 11) were
maintained in 80:20-10 medium (80 parts RPMI 1640, 20 parts medium 199, supplemented with 10% heat-inactivated fetal bovine serum, penicillin
(100 units/ml), 100 µg/ml streptomycin, and 600 µg/ml geniticin) in a 5% CO2 incubator at 37 °C. Human embryonic kidney
(HEK-293) cells were grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. Cells were seeded at a
density of 3 × 105 cells per 600-mm dish and
transfected using LipofectAMINE with pEF-BOS expression vectors (1.5 µg/dish), which encode various CCR5 Ser/Ala mutants (10).
Transfection efficiencies as determined by flow cytometry with an
anti-CCR5 mAb (Q10/19) ranged between 43 and 68%.
Generation of Phosphosite-specific Antibodies--
A
phosphopeptide
(CEAPERA(pS)(pS)VYTR(pS)TGEQI(pS)VGL)
corresponding to the 22 C-terminal amino acid residues of human CCR5
with phosphoserine (highlighted in boldface type) incorporated at the
four known phosphorylation sites as well as a nonphosphorylated version
of the same peptide were synthesized by standard solid phase methods.
The peptides were purified to >70% purity by reversed phase HPLC and
displayed the correct mass spectrum. The peptides were conjugated via
the N-terminal cysteine residues to bovine serum albumin using
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and
BALB/c mice were immunized at four monthly intervals with peptide-BSA conjugates (75 µg per animal and injection). Monoclonal antibodies were generated according to standard techniques by the fusion of spleen
cells with X63-Ag8.653 myeloma cells and were identified initially by
differential ELISA reactivities toward phosphorylated and
nonphosphorylated receptor peptides. Polyclonal phosphopeptide-specific antisera directed against a putative tyrosine phosphorylation site
within a highly conserved Asp-Arg-Tyr sequence at the end of the third
transmembrane domain of CCR5 (15) were generated by immunizing New
Zealand White rabbits with the following synthetic phosphopeptide which
corresponds to amino acids Ile-119 to Val-131 coupled to bovine serum
albumin: C( Immunoblotting--
RBL-2H3 cells which express wild-type or
phosphorylation-deficient CCR5 (7 × 105 cells per
60-mm dish) were stimulated with varying concentrations of RANTES for
10 min at 37 °C, washed once with PBS, and solubilized in detergent
buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 0.05% SDS with phosphatase and
protease inhibitors as described, Ref. 16) on ice. Experiments that
focused on the detection of tyrosine phosphorylation were performed in the presence of 10 µM sodium orthovanadate. Receptors
were immunoprecipitated by the incubation (2 h/4 °C) of cellular
lysates with 20 µg of anti-CCR5 R22/7 and protein G-Sepharose.
Receptors were eluted by incubation at 37 °C for 30 min in SDS
sample buffer containing 2% SDS and 5% 2-mercaptoethanol and
subjected to 10% SDS-polyacrylamide gel electrophoresis. Immunoblots
were performed using monoclonal anti-pS337 V14/2 (5 µg/ml), anti-pTyr
PY20 (0.1 µg/ml), or polyclonal anti-pY127 (10 µg/ml) antibodies in
Tris-buffered saline containing 0.1% Tween 20/5% nonfat dry milk.
Enhanced chemiluminescence detection of antigens was achieved with
horseradish peroxidase-conjugated secondary antibodies. Afterward
membranes were stripped and reprobed for total cellular receptors with
anti-CCR5 mAb R22/7 (10 µg/ml).
Enzyme Immunoassay--
The agonist-induced phosphorylation of
CCR5 was determined in RBL-CCR5 cells grown to confluency in 24-well
dishes, which were stimulated for various incubation times with the
indicated concentrations of RANTES or PMA in 80:20-10 medium. The
cells were transferred to ice and scraped in 200 µl of lysis buffer containing protease and phosphatase inhibitors. Cell debris was removed
by centrifugation at 3,000 × g for 10 min and
supernatants (100 µl) were applied either directly or after 2-fold
dilution in lysis buffer into wells of microtiter plates, which were
adsorbed with anti-CCR5 mAb T21/8 (5 µg/ml in 50 mM
carbonate, pH 10.6) as the capture antibody. The same cellular lysates
were probed, in parallel, with several biotinylated mAb (anti-pS349
E11/19; anti-pS337 V14/2; anti-S337 R-C10; 1 µg/ml in PBS, 0.05%
Tween 20), which react with distinct phosphorylation states and sites on the CCR5 C terminus. After a 2-h incubation period
(non-)phosphorylated CCR5 were detected by adding a 4000-fold dilution
of streptavidin peroxidase (Jackson ImmunoResearch) in PBS/Tween for
1 h and 2.2-azino-di-(3-ethyl-benzthiazoline sulfonate) as
substrate. Incubation periods were terminated by 3 wash cycles with
PBS/Tween, respectively. The assays were calibrated with a standard
protein, which was obtained by the conjugation of bovine serum albumin
with synthetic N-terminal and non-/phosphorylated C-terminal CCR5
peptides at 1:5:5 molar ratios using SMCC as a cross-linking reagent.
Results were expressed in arbitrary units (1 AU equals 1 ng of
BSA-peptide per ml).
In Vitro CCR5 Dephosphorylation Assay--
RBL-CCR5 cells were
incubated with 30 nM RANTES (10 min) and 200 nM
PMA (5 min) to induce maximal receptor phosphorylation. Cells were
scraped in lysis buffer and filled directly into wells of ELISA plates,
which were precoated with anti-CCR5 mAb T21/8. Receptors bound to the
solid phase were incubated for up to 1 h at 37 °C with potato
acid phosphatase (0.6 units/ml in 40 mM PIPES, 1 mM dithiothreitol, pH 6.0 with protease inhibitors). Alternatively, RBL-2H3 cells were scraped in 50 mM
Tris-HCl, pH 7.0, 0.1 mM EDTA, 50 mM
2-mercaptoethanol with protease inhibitors and homogenized by
sonication (four 10-s bursts at 100 watts). Nuclei were removed by
centrifugation (10 min; 300 × g) and the supernatant
was incubated for 1 h at 37 °C with receptors bound to the
ELISA plate in the presence or absence of 200 nM okadaic acid.
Immunofluorescence Microscopy--
RBL-CCR5 cells were grown
overnight on glass coverslips in 24-well plates. Cells were washed once
in warm 80:20-10 medium and then treated with 25 nM RANTES
in medium for varying time intervals at 37 °C. Thereafter, the
coverslips were placed on ice, washed with cold medium, and fixed with
an ice-cold solution of 3% paraformaldehyde, pH 7.4 in PBS for 20 min.
Free aldehyde groups were quenched with 50 mM
NH4Cl in PBS for 30 min. After permeabilization with cold
PBS containing 0.05% saponin and 0.2% gelatin for 15 min, cells were
washed with the same buffer and stained with anti-CCR5 (T21/8),
anti-pS337 (V14/2), or anti-pS349 (E11/19) antibodies (5 µg/ml in
PBS/saponin) for 1 h on ice. After washing with
PBS/saponin/gelatin, secondary antibody staining was carried out with a
FITC-conjugated goat anti-mouse IgG (1:100 dilution) for 1 h.
After further washes in PBS, coverslips were mounted in Mowiol
containing 0.1% p-phenylenediamine. The samples were
analyzed by confocal laser-scanning microscopy utilizing a Leica TCS
SP2 system, images were assembled in Corel Draw.
Characterization of Phosphospecific Antibodies Directed against
CCR5--
Synthetic (phospho-)peptides that encompass the four
C-terminal serine phosphorylation sites of CCR5 were used to generate monoclonal antibodies which differentially bind either to
phosphorylated or nonphosphorylated versions of the immunogenic
peptides. Two hybridomas (E11/19, IgG1/
It has been proposed that chemokine binding to their receptors exposes
the tyrosine residue within the highly conserved Asp-Arg-Tyr motif in
the second intracellular receptor loop which is then rapidly
phosphorylated by Janus kinases (JAKs) (15, 17). We tested this
hypothesis by generating phosphopeptide-specific antibodies, which
specifically recognize phospho-Tyr-127 and probed the ligand-activated CCR5 with this and anti-phosphotyrosine antibodies, in parallel, by
immunoblotting and enzyme-linked immunosorbent assay. We did not
observe phosphorylation of either this specific or any other tyrosine
residue of this receptor after incubation of RBL-CCR5 cells with
saturating concentrations of RANTES (30 nM) within a time
frame of 30 s up to 20 min (Fig.
2).
To exactly map the epitopes that are recognized by the various
phospho-CCR5-specific mAb we determined ELISA reactivities of RBL
cellular lysates containing maximally phosphorylated CCR5 Ser/Ala
mutants (Fig. 3). Substitution of serine
337 by alanine either alone or in combination with any other mutation
resulted in the complete loss of V14/2 binding to the receptor, whereas the mutation of serine 349 eliminated E11/19 reactivity. This indicates
that V14/2 and E11/19 recognize phosphoserines at positions 337 and
349, respectively, and independent of the phosphorylation state of any
of the other three serine residues. Using the same cellular lysates in
the absence of receptor activation we found that the mAb R-C10
exclusively reacts with nonphosphorylated serine 337 (not shown).
Epitope mapping with monophosphorylated CCR5 peptides independently
confirmed these results.
Phosphorylation of Ser-337 and Ser-349 by Different Protein
Kinases--
Exposure to CC-chemokines produces a rapid increase in
CCR5 phosphorylation, which is mediated by protein kinases belonging to
the PKC and GRK families. We tested the hypothesis that both protein
kinases phosphorylate distinct CCR5 C-terminal serine residues. To this
end ELISA procedures were established that are based on a capture
antibody with specificity for a CCR5 N-terminal epitope and different
(non)phospho-CCR5-specific detecting antibodies. These assays enabled
us to determine, in parallel, the phosphorylation status of serine
residues at positions 337 and 349 after stimulation of CCR5-expressing
cells with various agonists. Stimulation with PMA caused maximal
phosphorylation at serine 337 and pretreatment with bisindolylmaleimide
I, a selective and potent inhibitor of classical and novel PKC
isoforms, completely prevented phorbol ester- and ligand-induced
receptor phosphorylation (Fig. 4). In contrast, in the same experiment neither the PMA-induced activation nor
the inhibition of PKC by bisindolylmaleimide in cells that were exposed
to receptor-saturating concentrations (30 nM) of RANTES had
any noticeable effect on the phosphorylation state of serine 349. Several other protein kinase inhibitors, including the protein kinase A
inhibitor H-89 (5 µM), the calmodulin kinase inhibitor
KN-93 (10 µM), the phosphatidylinositol 3-kinase
inhibitor wortmannin (200 nM), or the broad spectrum
protein kinase inhibitor staurosporine (500 nM) also did
not inhibit the chemokine-stimulated phosphorylation of Ser-349.
Pretreatment of cells with pertussis toxin for 16 h at a
concentration (100 ng/ml) known to disrupt G protein-mediated signaling
in RBL-CCR5 cells (11) abrogated RANTES-induced phosphorylation at
serine 337 but, again, had no effect on phosphorylation of serine
349.
Kinetic Analysis of CCR5 Phosphorylation at Different Agonist
Concentrations--
Previous studies that employed receptor mutants
(18) or various kinase inhibitors (19, 20) suggested that second
messenger-dependent kinases and GRKs differently contribute
to GPCR phosphorylation upon exposure to different agonist
concentrations and at different time intervals. Phosphosite-specific
mAb now allowed us to monitor the kinetics and dose-dependence of the
RANTES-induced phosphorylation of non-mutated CCR5 by different protein
kinases under close to physiological conditions. When RBL-CCR5 cells
were stimulated with increasing concentrations of agonist for various
time intervals half-maximal phosphorylation of Ser-349 was observed at
5 nM RANTES and ~4-fold higher concentrations were
required to achieve maximal phosphorylation (Fig.
5B). The concentration
dependence of RANTES-induced phosphorylation paralleled ligand binding
to the receptor (11) and thus conformed to the characteristics of a
GRK-mediated mechanism. In contrast, phosphorylation of Ser-337 by the
second messenger-activated kinase (PKC) was detected at 50-fold lower
concentrations when cells were exposed to agonist for 1 min (Fig.
5A). This indicates significant signal amplification
downstream of the receptor under these experimental conditions.
Unexpectedly, the dose dependence of this effect shifted to higher
concentrations when cells were exposed to RANTES for longer incubation
times. A detailed kinetic analysis revealed that stimulation with low
RANTES concentrations results in the phosphorylation of Ser-337 in a
very rapid, albeit transient manner (Fig. 5C). Maximal CCR5
phosphorylation at this site was obtained by stimulation for 40 s,
and thereafter the receptor was rapidly dephosphorylated. In the
presence of receptor-saturating concentrations of ligand
dephosphorylation of phospho-Ser-337 was significantly retarded.
When lysates of RBL-CCR5 cells that had been stimulated with 0.5 nM RANTES for various times were probed with a mAb (R-C10), which specifically recognizes only nonphosphorylated Ser-337 the results represented a mirror image of the values obtained with anti-phospho-Ser-337 mAb V14/2 (Fig.
6A). This experiment shows that stimulation of only a small fraction of the cellular complement of
CCR5 with low concentrations of RANTES is capable of causing the
transient phosphorylation of ~50% of all receptors in a heterologous manner. A strikingly different time course of Ser-337 phosphorylation was observed after incubation of cells with 50 nM PMA.
Phosphorylation was irreversible within the time frame of this
experiment and did not peak until after 5 min (Fig. 6B).
Ligand-induced CCR5 Phosphorylation Proceeds in a Non-hierarchical
Manner--
Phosphorylation of substrates by serine/threonine protein
kinases often requires phosphorylation of a nearby priming site by
another protein kinase (21, 22) and this mechanism was also proposed to
underlie the sequential phosphorylation of phosphoacceptor sites in
certain G protein-coupled receptors (23-25). We therefore asked
whether in the case of the ligand-stimulated CCR5 the rapid PKC-mediated phosphorylation of Ser-337 is necessary for the subsequent phosphorylation of Ser-349 by GRKs. However, in a CCR5 mutant with
simultaneous replacement of serine residues at positions 336, 337, and
342 by alanine the GRK-mediated phosphorylation of the remaining serine
349 was not affected (Fig. 7). We
conclude from this experiment that GRK- and PKC-mediated
phosphorylation of CCR5 proceed independently and in a
non-hierarchical manner.
Site-specific Regulation of Receptor Dephosphorylation--
Since
different kinases appear to independently phosphorylate distinct sites
on CCR5 with characteristic time courses we investigated the
possibility that this also applies to the mechanisms involved in
receptor dephosphorylation. We therefore determined the kinetics of
Ser-337 and Ser-349 dephosphorylation in intact cells after a short
pulse (2 min) of stimulation with a saturating concentration (30 nM) of RANTES. Thereafter, agonist was quenched by adding a
large excess (2 µM) of the CCR5 antagonist TAK-779 (26).
As illustrated in Fig. 8, Ser-337 is
rapidly and completely dephosphorylated within 3 min
(t1/2 ~90 s) after withdrawal of the agonist. In
contrast, dephosphorylation of Ser-349 proceeded much slower
(t1/2 ~12 min). This indicated that either
phospho-Ser-337 is a much better substrate for the same receptor
phosphatase, which also dephosphorylates phospho-Ser-349, or that
different protein phosphatases are involved in receptor
dephosphorylation at the two sites.
To address this question, we established an in vitro CCR5
dephosphorylation assay that allowed determining site-specific receptor dephosphorylation after the exposure of fully phosphorylated CCR5 to
phosphatases from various sources. Under these experimental conditions
purified acid phosphatase (0.6 units/ml; 1 h at 37 °C)
completely dephosphorylated both phosphoserine residues. Incubation with RBL-2H3 cell lysates at pH 7.0 eliminated more than 50% of phosphate associated with Ser-337 (Fig.
9A) and ~30% of phosphate associated with Ser-349 (Fig. 9B). Preincubation of cell
lysates with 200 nM okadaic acid, a potent inhibitor of
protein phosphatases 1 (PP1) and 2a (PP2A), significantly
(p < 0.05) inhibited dephosphorylation of the
phosphoserine located at amino acid position 337 and had little effect
on the dephosphorylation of phospho-Ser-349.
Intracellular Localization of Phosphorylated Receptors--
To
identify the subcellular compartments in which CCR5 is either
phosphorylated or dephosphorylated by the various protein kinases and
phosphatases we determined the intracellular localization of
phosphorylated receptors in RBL-CCR5 cells by immunofluorescence. In
untreated cells the majority of receptors were present at the cell
surface in saponin-permeabilized cells (Fig.
10) when we used an anti-CCR5 mAb
(T21/8) with specificity for an N-terminal epitope to detect CCR5. No
phosphorylated receptors were detected in the absence of ligand. Within
30 s after exposure to a saturating concentration of RANTES (25 nM) punctate areas of fluorescence formed at or close to
the membrane, which corresponded to CCR5 that was phosphorylated by PKC
at serine 337. After 10 min of incubation at 37 °C the fluorescence
was predominantly in small diffusely distributed cytoplasmic vesicles
which contained receptors phosphorylated at both serine 337 and serine
349. By 30 min most of the receptors had concentrated in a region of
the cell adjacent to the nucleus. A faint fluorescent signal, which
corresponded to an extracellular CCR5 epitope was visible at the cell
surface after 30 min or longer stimulation times, but these receptors were never stained by phosphosite-specific mAb.
Receptor phosphorylation by two major classes of protein kinases,
the second messenger-dependent kinases and GRKs, is of
critical importance in the homologous and heterologous desensitization of many G protein-coupled receptors and promotes their internalization through clathrin-coated vesicles by a
In the current study we use phosphosite-specific antibodies to analyze
in intact cells the ligand-induced phosphorylation of the CC chemokine
receptor CCR5 by two different protein kinases in a cellular
environment which resembles natural conditions. We show that in RBL
cells, which express physiological levels of the protein kinase, PKC
exclusively phosphorylates Ser-337, while GRKs phosphorylate Ser-349.
The evidence for PKC-mediated phosphorylation of Ser-337 is derived
from experiments with selective PKC activators and inhibitors. Reagents
that selectively modulate GRK activity in whole cells are currently not
available. Our conclusion that GRKs are responsible for Ser-349
phosphorylation is therefore indirect and primarily based on the lack
of effect of a large number of different protein kinase inhibitors,
including staurosporine. Ligand-induced phosphorylation at this site
was also not affected by pertussis toxin, which indicates that the
kinase that phosphorylates Ser-349 is independent of G protein
activation and second messengers. The concentration dependence of
RANTES-induced phosphorylation of Ser-349 paralleled ligand binding to
the receptor, which further supports our conclusion that GRKs
phosphorylate this particular site (10). Previously, we concluded from
radiolabeling experiments on the phosphorylation of CCR5 serine to
alanine triple mutants in GRK-overexpressing cells that GRKs
participate in the ligand-induced phosphorylation of all four
C-terminal serine residues (31). These divergent results emphasize that
phosphorylation experiments with receptor mutants in the presence of
unphysiological concentrations of protein kinases reveal, at best,
potential phosphorylation sites and the identity of protein kinases,
which are likely to be involved in substrate phosphorylation. The
unequivocal identification of phosphorylation sites and corresponding
kinases requires that experiments are performed with non-mutated
proteins at physiological intracellular concentrations of protein kinases.
The observation that mutation of tyrosine 139 within the conserved DRY
sequence motif in the second intracellular loop of the CCR2 chemokine
receptor to phenylalanine eliminated functional activity of the
receptor has lead to the hypothesis that this critical tyrosine residue
is phosphorylated by JAKs following ligand binding to this or other
related chemokine receptors (17). By using phosphosite-specific or
anti-phosphotyrosine antibodies and in accord with earlier experiments
with two-dimensional phosphoamino acid analysis (10) we found no
evidence that this mechanism may operate in RBL-CCR5 cells.
In the present study we observed that GRK-mediated receptor
phosphorylation after RANTES stimulation was significantly slower compared with phosphorylation caused by PKC. Half-maximal
phosphorylation of Ser-349 was achieved after 1.5 to 2 min. The
kinetics of GPCR phosphorylation by GRKs versus second
messenger-dependent kinases has been investigated only in
relatively few receptor systems. In permeabilized A431 cells the
GRK-mediated phosphorylation and desensitization of
An unexpected finding of the present work related to the kinetics of
PKC-mediated CCR5 phosphorylation. Upon RANTES stimulation Ser-337 was
rapidly phosphorylated by PKC and half-maximal phosphorylation was
observed after 10 s. In the presence of low concentrations of
ligand Ser-337 phosphorylation was a transient event and
dephosphorylation proceeded with a calculated t1/2 of 1 min. In contrast, treatment of cells with PMA induced a sustained phosphorylation of Ser-337, which was maximal after 5 min. Several PKC
isoenzymes undergo reversible membrane translocation in response to
GPCR activation. Our data show that the kinetics of ligand- or phorbol
ester-induced CCR5 phosphorylation by these kinases closely follows the
time course of membrane recruitment of PKC PKC-mediated receptor phosphorylation after stimulation with low
concentrations of RANTES is tightly controlled by protein phosphatases,
which rapidly dephosphorylate phospho-Ser-337. The kinetics and
dose-dependence of Ser-337 de-/phosphorylation suggest that these
phosphatases are different from the GPCR phosphatase (GRP), a latent
endosome-associated form of PP2A, which was previously identified as
the major phosphatase that dephosphorylates the GRK-phosphorylated
The ligand-induced endocytosis and recycling of CCR5 was previously
described in detail using immunofluorescence and immune electron
microscopy (40, 41). These studies revealed that after RANTES
stimulation the intracellular distribution of CCR5 largely overlaps
with that of the transferrin receptor, a marker of early and recycling
endosomes. Immunofluorescence with phosphosite-specific antibodies
allowed us to assign receptor phosphorylation and dephosphorylation at
the GRK and PKC sites as defined by biochemical assays to these intracellular compartments. Shortly (30 s) after treatment with a high
concentration of RANTES we observed strong staining with the antibody,
which recognizes PKC-phosphorylated Ser-337 at the plasma membrane and
in small punctate vesicles close to the cell surface. Vesicular
staining in the periphery of the cell was observed after 10 min with
both antibodies, which recognize GRK- or PKC-phosphorylated CCR5. At
later time points phosphorylated receptors accumulated in perinuclear
clusters, which are probably equivalent to recycling endosomes as
previously described in CHO cells (41). We showed by flow cytometry
that prolonged stimulation of RBL-CCR5 cells with RANTES reduces
receptor expression at the cell surface by no more than 60% (10).
Accordingly, CCR5 immunofluorescence at the cell surface was reduced,
but still detectable even after agonist stimulation for more than 30 min. Since these receptors were not detected by phospho-CCR5-specific
antibodies they appear to be dephosphorylated by phosphatases within
the perinuclear recycling compartment. It has been hypothesized that
dephosphorylation of a GRK-phosphorylated C-terminal retention domain
within this perinuclear compartment precedes and even may be required
for the return of intracellular receptors to the plasma membrane (42, 43). Our data in the CCR5 system do not prove, but are compatible with
this concept. In summary, we have shown that phosphosite-specific antibodies provide unique and sensitive means for studying GPCR phosphorylation and dephosphorylation in a native cellular environment.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-1 and
-arrestin-2, also promote clathrin-mediated
endocytosis of phosphorylated receptors and have been implicated in
cross-talk with other signaling pathways. Once internalized, GPCR are
targeted to recycling or degradative pathways (5). Some GPCR are
dephosphorylated by membrane-associated G protein-coupled receptor
phosphatases and recycled rapidly to the plasma membrane where they can
again respond to agonists while other receptors appear to be retained
within the cell.
2-adrenergic receptor (6-8). The observed discrepancies in the outcome of these studies are probably explained by
unspecific effects of receptor mutagenesis and by the poor substrate
specificity of receptor kinases in reconstituted systems. Furthermore,
very little is known about the different kinetics and intracellular
localization of agonist-induced GPCR phosphorylation, which is mediated
by second messenger-dependent kinases versus GRKs at normal levels of protein expression.
-arrestin binding as well as
efficient receptor desensitization and internalization, but not for
CCR5-mediated chemotaxis (11, 12). HIV-1 entry does not require
receptor phosphorylation (13), yet a fully phosphorylation-deficient CCR5 mutant is largely resistant to the inhibitory effect of CC chemokines on in vitro HIV infection (14).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-aminocaproic
acid)IILLTIDR(pY)LAVV (phosphotyrosine highlighted in
boldface type). IgG was purified using protein A chromatography, and
antibodies reactive with the corresponding nonphosphorylated receptor
sequence were removed by adsorption to a nonphosphopeptide affinity
column. Two murine mAb (R22/7 and T21/8; both IgG1/
) which bind with
high affinity to a CCR5 N-terminal epitope were generated following the
immunization of mice with intact RBL-CCR5 cells (10).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
; V14/2, IgG1/
) were
identified, which selectively react only with ligand-activated CCR5 in
a phosphorylation-dependent manner as shown by several
independent immunological techniques. Another mAb (R-C10, IgG1/
) was
derived from a separate fusion after immunization with a
nonphosphorylated C-terminal receptor peptide which exclusively
recognizes non-activated CCR5. The immunoblot shown in Fig.
1 demonstrates that the pCCR5-specific
mAb V14/2 reacts with CCR5, which migrates as a broad 40-kDa protein in SDS-PAGE analysis typical of a glycosylated G protein-coupled receptor,
but only in its ligand-activated, i.e. phosphorylated form.
Under the experimental conditions used in this study we did not observe
higher molecular weight forms of CCR5. Despite RANTES activation this
mAb did not recognize a phosphorylation-deficient CCR5 mutant, which
was generated by alanine replacement of all four C-terminal serine
phosphorylation sites. Similar results were obtained with the mAb
E11/19, whereas the mAb R-C10 displayed reverse reactivity and lost its
ability to react with CCR5 upon ligand activation in a
dose-dependent manner.
View larger version (57K):
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Fig. 1.
Specificity of the anti-pCCR5 mAb V14/2
through immunoblot analysis. Lysates from non-transfected RBL-2H3
cells (RBL) or from RBL-2H3 cells expressing either wild
type CCR5 (RBL-CCR5) or phosphorylation-deficient CCR5 with
alanine replacement of all four C-terminal serine residues
(RBL-CCR5(P-)) were exposed to the indicated concentrations
of RANTES for 10 min. Receptors were immunoprecipitated and analyzed by
SDS-PAGE and serial immunoblotting with anti-phosphoCCR5 mAb V14/2
(top panel) and anti-CCR5 mAb R22/7 (bottom
panel).
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Fig. 2.
Lack of RANTES-induced Tyr-127
phosphorylation in RBL-CCR5 cells. RBL-CCR5 cells were stimulated
(0-20 min) with 30 nM RANTES and lysed, and
immunoprecipitated with anti-CCR5 mAb R22/7. Immunoblots were probed,
in parallel, with anti-pTyr PY20 (A), polyclonal rabbit
anti-phospho-Tyr-127 (B), anti-phospho-Ser-349 mAb E11/19
(C), or anti-CCR5 mAb R22/7 (D). Positive
controls (lanes C) were 10 µg of EGF-stimulated A431 cell
lysate in A, and 500 ng of BSA-pTyr-127 phosphopeptide
conjugate in B.
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[in a new window]
Fig. 3.
Epitope mapping of pCCR5-specific mAb.
HEK-293 cells were transfected with expression plasmids encoding CCR5
mutants with replacement of the four C-terminal serine residues at
positions 336, 337, 342, and 349 by alanine in various combinations
(WT denotes wild-type; AAAA represents the mutant
with alanine exchange of all four serine residues) and stimulated with
50 nM RANTES and 2 µM PMA for 10 min to
induce maximal receptor phosphorylation. Identical amounts of cells
lysates were probed for immunoreactivity by using ELISA procedures that
are based on two different phospho-CCR5-specific mAb (black
bars, E11/19; gray bars, V14/2) in parallel. Data
(means ± S.D.) were normalized to values obtained with cells
expressing wild-type CCR5.
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[in a new window]
Fig. 4.
Effect of pertussis toxin treatment and
modulation of PKC activity on CCR5 phosphorylation. Site-specific
phosphorylation of CCR5 at Ser-337 and Ser-349 was examined in the
absence or presence of 200 nM PMA (5 min) or 30 nM RANTES (10 min) in RBL-CCR5 cells, which had been
pretreated with medium, bisindolylmaleimide (2 µM for 30 min), or pertussis toxin (100 ng/ml for 16 h). Samples were
processed and analyzed, in parallel, by the two ELISA procedures as
described under "Experimental Procedures." The assays were
calibrated with a BSA-phosphopeptide standard protein and results
expressed in arbitrary units (AU). Means ± S.D. from a single
experiment performed in duplicate representative of three independent
experiments are shown.
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Fig. 5.
Time- and dose-dependent
phosphorylation of CCR5 Ser-337 and Ser-349. RBL-CCR5 cells were
exposed to the indicated concentrations of RANTES for 1 min ( ), 5 min (
), or 15 min(
) in A and B, or were
stimulated for up to 30 min with 0.5 nM (
), 5 nM (
), or 50 nM (
) RANTES in C
and D. Cell lysates from the same experiments were analyzed
for CCR5 phosphorylation at Ser-337 (A and C) or
Ser-349 (B and D) by ELISA procedures that are
based on phosphosite-specific mAb. Mean values from one representative
experiment performed in duplicates are shown.
View larger version (21K):
[in a new window]
Fig. 6.
Reciprocal binding of phospho- and
nonphospho-Ser-337 specific mAb to RANTES- or PMA-treated CCR5.
RBL-CCR5 cells were treated with 0.5 nM RANTES
(A) or 50 nM PMA (B) for different
times. ELISA reactivities of the same cell lysates using detecting mAb
with specificity for phospho-Ser-337 (V14/2; ) or nonphospho-Ser-337
(R-C10;
) are shown. Each data point represents the mean of
duplicate determinations from one representative out of three separate
experiments.
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[in a new window]
Fig. 7.
RANTES-induced phosphorylation of a mutant
CCR5 lacking all phosphorylation sites except for Ser-349.
Time-dependent phosphorylation of Ser-337 ( ) or Ser-349
(
) in lysates from RANTES (5 nM)-stimulated RBL cells,
which express a CCR5-AAAS mutant lacking all C-terminal phosphorylation
sites except for Ser-349 was determined by ELISA.
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[in a new window]
Fig. 8.
Kinetics of CCR5 Ser-337 and Ser-349
dephosphorylation in intact cells. RBL-CCR5 cells were stimulated
at 37 °C with 30 nM RANTES in 80:20-10 medium. One set
of treated cells was incubated with RANTES for up to 3 h, whereas
the other was briefly washed after 2 min and incubated with prewarmed
medium that contained 2 µM TAK-779. At the indicated time
points, cells which were incubated in the presence ( ) or absence
(
) of the CCR5 antagonist were scraped into lysis buffer and
phosphorylation of Ser-337 (A) or Ser-349 (B) was
quantitated by parallel ELISA procedures.
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[in a new window]
Fig. 9.
In vitro dephosphorylation of CCR5
by acid phosphatase and phosphatases from RBL cellular lysates.
Lysates of RBL-CCR5 cells, which had been treated with 30 nM RANTES (15 min) and 200 nM PMA (5 min) to
induce maximal CCR5 phosphorylation were directly filled into anti-CCR5
mAb T21/8 precoated wells of ELISA plates. After washing with the
respective phosphatase buffers, CCR5 bound to the solid phase were
incubated for up to 1 h at 37 °C with potato acid phosphatase
( ), lysates of RBL-2H3 cells (
), or RBL-2H3 cell lysates in the
presence of 200 nM okadaic acid (
). Phosphorylated
receptors were detected by adding, in sequence, biotinylated
phosphosite-specific anti-pS337 mAb V14/2 (A) or anti-pS349
E11/19 (B) and streptavidin horseradish peroxidase.
View larger version (107K):
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Fig. 10.
Subcellular localization of phosphorylated
CCR5 in RBL-CCR5 cells. RBL cells expressing CCR5 were treated
with 25 nM RANTES for 30 s, 10 min, or 30 min at
37 °C or left untreated. After fixation and permeabilization, the
cells were stained for total CCR5 with mAb T21/8, for phospho-Ser-337
with mAb V14/2 or for phospho-Ser-349 with mAb E11/19 and viewed by
confocal microscopy. The immunofluorescent signal was fully saturable
with an excess (1 µM) of N-terminal (T21/8) or
phosphorylated C-terminal (V14/2; E11/19) receptor peptide during
incubation with the primary antibodies (not shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-arrestin-dependent mechanism (3, 5). In addition to
terminating the cellular response, receptor phosphorylation was also
shown to initiate
-arrestin-mediated signaling pathways and to
switch coupling of the
2-adrenergic receptor away from
Gs in favor of enhanced coupling to Gi (27, 28). Despite its significance for many aspects of GPCR signaling, little is known about the dynamic regulation of receptor
phosphorylation and dephosphorylation at distinct sites by different
protein kinases and phosphatases. Previously, regulation of receptor
phosphorylation by second messenger-dependent and
-independent kinases could be described only through removal of
presumptive consensus sites of receptor phosphorylation or under
experimental conditions of overexpressed kinases. Accurate mapping of
in vivo phosphorylation sites of a GPCR under physiological
conditions has been achieved only for rhodopsin and, more recently, the
bradykinin B2 receptor (29, 30). In the case of rhodopsin two
C-terminal serine residues were differentially phosphorylated by
rhodopsin kinase (GRK1) after a bright flash or under constant
illumination. Receptor dephosphorylation at these two sites proceeded
with markedly different kinetics and appeared to be spatially
controlled within rod outer segments (29).
2-adrenergic receptors was reported to transpire with a
t1/2 of less than 20 s, whereas phosphorylation
and desensitization by the cyclic AMP-dependent protein
kinase (PKA) had a t1/2 of at least 2 min (19). The
desensitization and phosphorylation of odorant receptors from detached
olfactory cilia preparations proceeded even more rapidly, within 0.2-1
s, and both GRK3 and second messenger-activated kinases appeared to
function in series in this particular receptor system (32). A
limitation of these early studies is that they were performed with
permeabilized cells and kinase inhibitors such as heparin, which lack
specificity. Nonetheless, these varying results indicate that the
kinetics of GPCR phosphorylation largely differ between receptor
systems, depending on such variables as different receptor substrates
and cellular expression levels of receptor kinases. Variability in the
mechanisms which regulate GPCR phosphorylation is also illustrated by
the finding in the present study that GRK- and PKC-mediated CCR5
phosphorylation proceed independently and in a non-hierarchical manner,
in contrast to other receptor systems (23-25).
after GPCR activation, as
reported in other receptor systems which are coupled to the activation
of phospholipase C (33, 34). Whereas PMA induced the persistent
redistribution of the kinase from the cytosol to the membrane after
2-5 min, G
q-coupled receptor signaling was reported to
lead to only one rapid and brief peak of PKC membrane translocation
(33). Within 1 min after translocation the kinase returned to the
cytoplasm by a mechanism that involves PKC autophosphorylation (35).
Agonist stimulation of intact cells that express the substance P
receptor induced a very rapid and transient translocation of a green
fluorescent protein (GFP)-PKC
construct, whereas GFP-GRK2 was
recruited to the plasma membrane shortly after PKC and persisted over a
longer time period (34).
2-adrenergic receptor (36). Whereas GRP dephosphorylates
receptors only in the acidic milieu of endosomal vesicles and, thus,
requires receptor endocytosis, the phosphatases that dephosphorylate
Ser-337 are active at neutral pH and efficiently dephosphorylate
receptors at the cell surface that have been phosphorylated by a
heterologous mechanism. At higher ligand concentrations a larger
fraction of receptors at the cell membrane are present in the
ligand-bound form, which then leads to GRK-mediated receptor phosphorylation,
-arrestin binding, and receptor endocytosis. We
have previously shown that
-arrestin binding to CCR5 strictly follows ligand occupancy and is maximal after stimulation of receptors with RANTES for 3-10 min (12). Our data are consistent with the
hypothesis that at high concentrations of ligand a stable complex
between
-arrestin and phosphorylated CCR5 is formed, which prevents
the phosphatase from dephosphorylating Ser-337. This notion is
supported by our observation that treatment of cells that express
maximally phosphorylated CCR5 with a receptor antagonist, which quickly
dissociates receptor-
-arrestin
complexes,2 leads to the
rapid dephosphorylation of Ser-337. An inhibitory effect of arrestin on
the dephosphorylation of rhodopsin, a GPCR that does not internalize,
by a retinal protein phosphatase 2A was previously suggested by
Palczewski et al. (37). In this study we show that
stimulation with a low concentration of agonist induces the
PKC-mediated reversible phosphorylation of a significant fraction of
all CCR5 receptors in a heterologous manner. We suggest that a protein
phosphatase which efficiently dephosphorylates PKC-phosphorylated GPCR
at the plasma membrane fulfils an important role in maintaining cell
membrane receptors in a nonphosphorylated state. In contrast to the
endosome-associated GPR this phosphatase does not require receptor
endocytosis. Whether protein phosphatases that either directly (38) or
indirectly through anchoring proteins (39) bind to receptors also
contribute to this mechanism remains to be investigated in future
studies. A different mechanism appears to be responsible for the
dephosphorylation of GRK-phosphorylated Ser-349. In chase experiments
with the CCR5 antagonist TAK-779 phosphoserine 349 was much slower
dephosphorylated compared with phosphoserine 337 and the phosphatase(s)
present in RBL cell lysates that dephosphorylated phosphoserine 337 in vitro at neutral pH less efficiently dephosphorylated
phospho-Ser-349.
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ACKNOWLEDGEMENTS |
---|
We thank Maren Wüstefeld, Saskia Esser, and Jennifer Ücer for excellent technical assistance and Mark Marsh for critical comments on the article. We are grateful to Amanda Proudfoot for kindly providing RANTES and Takeda for providing TAK-779.
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FOOTNOTES |
---|
* This work was supported by the Deutsche Forschungsgemeinschaft SFB 523 TPA10 and SFB 402 TPB7.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
Immunology, University of Göttingen, Kreuzbergring 57, 37075 Göttingen, Germany. Tel.: 49-551-395822;
Fax: 49-551-395843; E-mail: mopperm@gwdg.de.
Published, JBC Papers in Press, October 27, 2002, DOI 10.1074/jbc.M209844200
2 M. Wüstefeld, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are: GPCR, heterotrimeric GTP-binding protein-coupled receptor; AU, arbitrary unit; CCR5, CC chemokine receptor 5; ELISA, enzyme-linked immunosorbent assay; GRK, GPCR kinase; HEK-293, human embryonic kidney 293; HIV, human immunodeficiency virus; mAb, monoclonal antibody; pCCR5, phosphorylated CCR5; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; RANTES, released on activation normal T cell expressed and secreted (also known as CCL5); RBL-2H3 cells, rat basophilic leukemia cells; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; PBS, phosphate-buffered saline; FITC, fluorescein isothiocyanate; BSA, bovine serum albumin; HPLC, high performance liquid chromatography; PIPES, 1,4-piperazinediethanesulfonic acid.
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