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INTRODUCTION |
Chemokines are a large family of chemotactic cytokines that
mediate leukocyte activation and recruitment to sites of inflammation (1). They are small polypeptides of 8-10 kDa, which can be subdivided
into two major gene families depending on the relative position of four
conserved cysteines, the first two of which are adjacent in CC
chemokines or separated by one intervening residue in CXC chemokines.
Both CC and CXC chemokines bind to heptahelical G protein-coupled
receptors which transduce signals through members of the Gi
or Gq family of G proteins (2, 3). The ligand-binding repertoires of different chemokine receptors significantly overlap, as
do the sets of receptors expressed by different leukocytes and other
target cells. Further adding to the versatility of the chemokine
system, multiple and distinct signaling pathways exist for individual
receptors, which are differentially regulated depending on the ligand
and cell type involved (2, 4).
Like many other G protein-coupled receptors, chemokine receptors
demonstrate diminishing responsiveness upon repeated or prolonged exposure to agonist, a phenomenon generally referred to as receptor desensitization. Other regulatory processes are receptor sequestration and degradation, which summarily contribute to dampening the cellular response. Receptor phosphorylation by G protein-coupled receptor kinases (GRKs)1 has been
shown to be crucial in the rapid agonist-induced desensitization of
many G protein-coupled receptor systems (5). GRK-mediated receptor
phosphorylation promotes the binding of regulatory arrestin proteins,
which results in further uncoupling of receptor-G protein interactions.
Agonist-induced desensitization is clearly important in the control of
the cytotoxic potential of leukocytes in the response to
chemoattractant agonists. Furthermore, this regulatory mechanism may be
critical for chemokine receptors whose primary function is to
continuously sense small changes in the gradients of chemoattractants
and thereby direct cellular migration.
In the present study, we investigated early signaling events, which are
initiated after ligand binding to CC chemokine receptor CCR5 and
contribute to receptor desensitization. Under physiological conditions,
CCR5 interacts with RANTES, MIP-1
, MIP-1
, or MCP-2 (6, 7). In
addition, amino-terminal modifications of RANTES (Met-RANTES and
AOP-RANTES) have been described, creating receptor ligands with
antagonistic properties (8, 9). We show that these various CCR5 ligands
differ in their abilities to stimulate the receptor-G protein-PLC
system in RBL-2H3 cells and that the intrinsic activities of these
compounds directly correlate with their abilities to induce CCR5
phosphorylation and desensitization through a GRK-mediated mechanism.
Furthermore, we identify amino acid residues within the CCR5 carboxyl
terminus that are phosphorylated upon cellular stimulation with CC chemokines.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture media and cell culture supplies
were from Biochrom; RBL-2H3 and COS-7 cells were from the American Type
Culture Collection; protein G-Sepharose fast flow and ECL Western
blotting reagents were from Amersham Pharmacia Biotech;
bisindolylmaleimide, staurosporine, and pertussis toxin were from
Calbiochem; anti-hemagglutinin monoclonal antibody 12CA5 was from
Babco; horseradish peroxidase-conjugated streptavidin was from Jackson
Immunoresearch; restriction enzymes and T4 ligase were from Promega;
Taq DNA polymerase was from Life Technologies, Inc.; MCP-2,
MIP-1
, MIP-1
, and RANTES were from PeproTech; Met-RANTES (8) and
aminooxypentane (AOP)-RANTES (9) were produced as described;
32Pi and [
-32P]ATP were from
ICN; all other reagents, unless otherwise indicated, were purchased
from Sigma. The monoclonal anti-CCR5 antibodies MC-4 and
MC-52 were generated
following the same protocol, as described previously (10).
Plasmid Constructs--
The human CCR5 (6) was modified using
oligonucleotide-directed mutagenesis to include an influenza
virus hemagglutinin signal sequence (11) followed by the
FLAG epitope. The 5' mutagenic primer was:
5'-CGCGGGTCTAGAACC(ATGAAGACCATCATCGCCCTGAGCTACATCTTCTGCCTGGTGTTCGCC)[GACTACAAGGACGATGATGACGCC]GATTATCAAGTGTCAAGTCCA-3'. The signal sequence is surrounded by parentheses, the FLAG tag sequence
is surrounded by brackets, and the start codon and 21 bases
corresponding to amino acids 2-8 of CCR5 are underlined. The 3'
oligonucleotide primer was:
5'-CGCGGGTCTAGATCACAAGCCCACAGATATTTC-3', with
nucleotides 1039-1059 of the native sequence underlined. The resulting
PCR product was digested with XbaI and ligated into the
mammalian expression vector pEF-BOS (12). The fidelity of cDNA
amplification was verified with dideoxy DNA sequencing. The cDNA
construct for bovine GRK3 (13) has been described previously. The
chimeric G protein Gqo5 expression plasmid (14) was a
generous gift by Dr. Bruce Conklin of the J. David Gladstone Institutes of San Francisco, CA.
Site-directed Mutagenesis of CCR5--
Polymerase chain reaction
was employed to generate CCR5 mutants with serine to alanine mutations
at positions Ser-336, Ser-337, Ser-342, and Ser-349 by using the
following degenerate 3' primer: CGCGGGTCTAGATCACAAGCCCACAGMTATTTCCTGCTCCCCAGAGGMTCGGGTGTAAACTGMGGMTGCTCGCTCGGGAGCCTCTTG. Mutagenic base exchanges that resulted in the random substitution of serine residues by alanine are highlighted in boldface type; nucleotides 985-1059 of the native sequence are underlined. The mutated DNA fragments obtained were digested with XbaI and
ligated into the expression vector pEF-BOS. Recombinant clones were
isolated and sequenced. A total of 14 distinct sequences were
identified, which encoded for CCR5 variants with various combinations
of serine to alanine substitutions.
Cell Culture, Plasmid Transfection, and Isolation of Leukocyte
Fractions--
RBL-2H3 cells 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), and 100 µg/ml streptomycin). RBL cells (1 × 107 cells in
250 µl of phosphate-buffered saline) were transfected by
electroporation (Bio-Rad Gene Pulser) at 260 V and a capacitance of 960 microfarads in the presence of ph
APr-1-neo plasmid (15) containing
the Geniticin resistance marker and mammalian expression vector pEF-BOS
(12) containing cDNA for FLAG-CCR5. Fresh medium containing 600 µg/ml Geniticin was added 24 h after transfection. Surviving
foci were isolated, expanded, and evaluated for expression of FLAG-CCR5
at the cell surface by flow cytometry using anti-FLAG M2 monoclonal
antibody (16). CHO-CCR1 cells, which stably express a
hemagglutinin-tagged version of CCR1 (17), were generously provided by
Dr. Ronald W. Barrett (Affymax Research Institute) and were maintained
in Dulbecco's modified Eagle's medium-F12 medium containing 10%
fetal bovine serum. COS-7 cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal bovine serum. Cells were
transfected with different DNAs by the DEAE-dextran method, and
phosphorylation experiments were performed 64 h after
transfection. The polymorphonuclear leukocyte and mononuclear cell
fractions were freshly isolated from EDTA blood of healthy donors by
density gradient centrifugation on Ficoll-Hypaque (Nycomed Pharma).
Granulocytes (>80% CD16+ cells) were isolated from the
cell pellets following the removal of erythrocytes by incubation with
Gey's lysis buffer. Monocytes (>80% CD14+ cells) were
purified after adherence (1 h/37 °C) of mononuclear cells to plastic
tissue culture plates in the presence of 10% heat-inactivated human AB
serum. Non-adherent lymphocytes consisted of 60-80% CD3+,
5-10% CD19+, and 10-20% CD56+ cells. Cell
lysates were obtained by scraping cells into detergent buffer.
Phosphorylation Experiments--
The agonist-induced
phosphorylation of CCR5 in intact cells was determined as described
previously (16). Briefly, RBL-CCR5 cells or COS-7 cells transiently
transfected with CCR5-pEF-BOS were metabolically labeled with
32Pi. After treatment with different stimuli as
indicated, cells were washed and solubilized in detergent buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Triton X-100, 0.05% SDS, with
phosphatase and protease inhibitors as described; Ref. 16). Receptors
were immunoprecipitated by incubating precleared cellular lysates with
12 µg of anti-FLAG M2 monoclonal IgG1 and protein G-Sepharose. Immune
complexes were dissociated and resolved by SDS-PAGE using 10% gels.
Radioactively labeled receptors were visualized by autoradiography and
analyzed with a Molecular Dynamics PhosphorImagerTM.
Receptor phosphorylation data were corrected for different receptor expression levels in transfected cells by multiplying the relative receptor expression as assessed by flow cytometry using
receptor-specific antibodies (percentage of positive cells multiplied
by the mean channel of fluorescence of positive cells) with the protein
content of each immunoprecipitation tube. The experimental protocol for determining receptor phosphorylation in permeabilized cells has been
described (18). In brief, RBL-CCR5 cells were permeabilized by
incubation for 5 min with 0.5 unit of streptolysin O in KG buffer (139 mM potassium glutamate, 5 mM glucose, 20 mM potassium salt of Pipes, 2.5 mM
MgCl2, 1 mM EDTA, pH 7.4). This procedure resulted in the permeabilization of >95% of the cells as assessed by
trypan blue staining. Cells were incubated with antibodies (500 µg/ml) for 10 min in KG buffer containing 10 µM ATP and
50 µM GTP in the presence or absence of staurosporine
(500 nM). [
-32P]ATP (100 µCi/ml) was
added, and cells were stimulated for 5 min with the indicated stimuli.
Cells were lysed with detergent buffer, and receptors were
immunoprecipitated using the same protocol as described for whole cell
phosphorylation experiments.
Phosphoamino Acid Analysis--
32P-Labeled
receptors were electrophoretically transferred to polyvinylidene
difluoride membranes (Immobilon PVDF), excised and hydrolyzed in 6 N HCl for 1 h at 110 °C. The hydrolysates were
lyophilized and resolubilized in pH 1.9 buffer (formic acid/acetic acid/H2O, 50/156/1794, (v/v/v)) containing phosphoamino
acid standards. The samples were spotted on thin-layer cellulose plates
and phosphoamino acids were separated by electrophoresis (900 V/1.5 h)
at pH 1.9, followed by a second electrophoresis (900 V/45 min) at pH
3.5 (pyridine/acetic acid/H2O, 10/100/1890, (v/v/v)) in the
orthogonal direction. After ninhydrin staining, thin layer plates were
exposed to PhosphorImager screens.
Calcium Fluorimetry and Internalization Assay--
RBL-CCR5 or
CHO-CCR1 cells were washed with buffer A (148 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM HEPES, 1 mM glucose) and incubated (30 min/37 °C) at 107 cells/ml in the same buffer containing
0.1% bovine serum albumin and 8 µM Fluo3-AM. After
washing the cells with buffer A, cells were resuspended at 5 × 106 cells/ml of buffer A. Chemokine-induced intracellular
calcium mobilization was determined by spectrofluorometry using a
Perkin Elmer MPF-44B fluorescence spectrophotometer with an excitation wavelength of 506 nm and an emission wavelength of 526 nm.
Intracellular calcium levels were calculated using the equation
[Ca2+]i = Kd × (F
Fmin)/(Fmax
F) (19), where Kd is the dissociation
constant of Fluo3-AM, Fmin is the
autofluorescence of cells that were incubated in the absence of calcium
chelator and Fmax is the maximal fluorescence
determined with detergent-lysed cells.
The agonist-induced internalization of CCR5 in RBL-CCR5 cells was
determined, in principal, as described previously (10). In brief,
5 × 105 RBL-CCR5 cells were incubated (30 min/37 °C) in 100 µl of medium containing various concentrations
of chemokines. Thereafter, cells were cooled to 4 °C and
surface-expressed CCR5 were detected by flow cytometry using anti-CCR5
mAb MC-4 and a fluorescein isothiocyanate-labeled rabbit anti-mouse
F(ab')2 fragment (Dako). The relative CCR5 surface expression was calculated as 100 × (mean channel of fluorescence [stimulated]
mean channel of fluorescence [negative
control])/(mean channel of fluorescence [medium]
mean channel of
fluorescence [negative control]) [%].
GRK-specific mAb and ELISA Procedures--
Synthetic peptides
that correspond to amino acids Val-658 to Leu-689 of bovine GRK2 and
Leu-658 to Leu-688 of bovine GRK3, respectively, were synthesized on an
Applied Biosystems 430A peptide synthesizer using FastMoc chemistry,
and the products were purified by high pressure liquid chromatography.
Peptides were coupled through an additional cysteine residue at the
peptide amino terminus to bovine serum albumin using the
heterobifunctional cross-linking reagent succinimidyl
4-(N-maleimidomethyl)cyclohexane-1-carboxylate as described
(20). Monoclonal antibodies were generated according to standard
techniques following the immunization of BALB/c mice with
peptide-bovine serum albumin conjugates. Hybridoma supernatants were
screened by solid phase ELISA techniques or immunoblotting employing
purified recombinant GRKs or cellular lysates from cells that
overexpressed individual GRKs as the antigens. While several mAbs were
obtained that specifically react with either GRK2 alone or both GRK2
and GRK3, no antibody was identified that solely recognizes GRK3. ELISA
procedures were established that allowed the sensitive quantitation of
GRK2 or GRK3 in cellular lysates. For the quantitation of GRK2, the mAb
C5/1 (18), which recognizes a conserved sequence (Ala-480 to Phe-488)
present in both GRK2 and GRK3, was adsorbed into wells of microtiter
plates (5 µg/ml) in 50 mM carbonate, pH 10.6. Purified
recombinant GRK2 as the protein standard or cellular lysates were
diluted in phosphate-buffered saline-0.05% Tween and were allowed to
bind for 2 h. GRK2 that had bound to the first mAb was detected by
adding, in sequence, the biotinylated GRK2-specific mAb E23/8 (1 µg/ml; 1 h), a 4000-fold dilution of streptavidin-peroxidase (1 h) and 2.2-azino-di-(3-ethylbenzthiazoline sulfonate) as substrate. The
quantitation of GRK3 by ELISA followed the same protocol except for the
following modifications. Recombinant GRK3 as the protein standard or
GRK3 present in cellular lysates was detected by adding the
biotinylated GRK2/3-specific mAb G7/5 (1 µg/ml) in the presence of a
100-fold excess of mAb E23/8 (which blocks G7/5 binding to GRK2) to
wells containing mAb C5/1 as the first antibody. Detection limits of
these assays are 15 ng/ml GRK2 or GRK3. Purified recombinant GRK2 and
GRK3 were kindly provided by Dr. Robert J. Lefkowitz (Duke University,
Durham, NC).
Co-immunoprecipitation and Immunoblotting--
Agonist-induced
association of GRKs with CCR5 was analyzed essentially as described
previously (21). Briefly, RBL-CCR5 cells were treated with the
indicated stimuli for 3 min in serum-free medium and were then
incubated for 30 min with Hank's balanced salt solution containing 10 mM HEPES, pH 7.8, 10% dimethyl sulfoxide, and 2.5 mM of the cell-permeant homobifunctional cross-linking agent dithiobis(succinimidylproprionate). After cellular lysis in
detergent buffer, receptor-containing complexes were immunoprecipitated with FLAG M2 antibody and were dissociated under reducing conditions. Equivalent amounts of receptor protein were separated on 10%
SDS-polyacrylamide gels and electrotransferred to nitrocellulose
membranes. Following the incubation with biotinylated GRK2/3-specific
mAb C5/1 (2 µg/ml) and a 4000-fold dilution of streptavidin
peroxidase, chemiluminescence of GRK2/3 was detected using an enhanced
chemiluminescence kit.
Data Analysis--
Experimental procedures were performed three
times, unless otherwise indicated. Results were analyzed for
statistical significance by an unpaired Student's t test,
and one-sided p values were calculated using
ExcelTM software (Microsoft).
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RESULTS |
Chemokine-induced Receptor Activation, Desensitization, and
Internalization--
To investigate the signaling properties of CCR5
in response to different chemokines, RBL-2H3 cells were stably
transfected with a cDNA construct encoding human CCR5. Fig.
1 shows that RANTES, AOP-RANTES,
Met-RANTES, MIP-1
, and MIP-1
induced calcium mobilization in
RBL-CCR5 cells, albeit with different efficiencies. Dose-response curves indicated that the maximal MIP-1
or MIP-1
induced calcium mobilization amounted to only 30% of the signal seen in cells that
were stimulated with AOP-RANTES or RANTES. Met-RANTES was the least
effective of all CCR5 ligands tested. Half-maximal effects were
observed at concentrations between 1.5 and 6 nM for the
different chemokines, with AOP-RANTES being the most potent agonist. To assess the effect of pertussis toxin on agonist-induced calcium mobilization, RBL-CCR5 cells were pretreated with 100 ng/ml pertussis toxin for 20 h. This treatment completely abrogated CCR5 signaling in response to chemokines (data not shown).

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Fig. 1.
Chemokine-induced calcium mobilization in
RBL-CCR5 and CHO-CCR1 cells. Fluo-3/AM-loaded cells were
challenged with the indicated concentrations of RANTES ( ),
AOP-RANTES ( ), Met-RANTES ( ), MIP-1 ( ), and MIP-1 ( ).
Data are values (mean ± S.E.) from at least two
experiments.
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Other groups, using different cells and assay systems, reported rank
orders of signaling efficacies of CC chemokines in the CCR5 system that
partially deviate from our findings (6, 7, 10, 22). This may point to
cell type-specific effects or to the differential activation of the
various intracellular effectors by these chemokines. Alternatively,
varying biological activities of the different preparations of
recombinant chemokines may account for these discrepancies. We
therefore compared chemokine effects on CCR5 with those on the closely
related chemokine receptor CCR1. Here, RANTES and MIP-1
were
essentially equally effective (Fig. 1). Interestingly, AOP-RANTES
failed to activate intracellular calcium mobilization in
CCR1-expressing cells.
RANTES-induced calcium mobilization was effectively desensitized in
RBL-CCR5 cells by prior treatment with RANTES or with AOP-RANTES (Fig.
2). In contrast, pretreatment with
MIP-1
, MIP-1
, or Met-RANTES reduced the subsequent RANTES-induced
cellular stimulation by only 15-20%. Thus, the abilities of the
different receptor ligands to activate calcium mobilization via CCR5
paralleled their abilities to induce receptor desensitization. This
generally good fit between the efficacies of chemokines in receptor
activation and desensitization was also observed in the CCR1 system
(Fig. 2). Activation of PKC by treatment with PMA inhibited the
subsequent RANTES-induced calcium mobilization via both CCR1 and
CCR5.

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Fig. 2.
Differential effects of CC chemokines on the
desensitization of CCR1 and CCR5. Fluo-3AM-loaded CHO-CCR1 or
RBL-CCR5 cells were pretreated with or without chemokines (15 nM) or PMA (2 µM). Cells were washed and
restimulated 7 min later with 15 nM RANTES. Thrombin (1 unit/ml), which causes calcium mobilization in RBL cells, was used to
verify normal responsiveness of RBL-CCR5 cells after each stimulus.
Percent desensitization was calculated as a function of the
RANTES-induced calcium mobilization in naive and prestimulated cells:
100 × (naive-prestimulated)/(naive). Data are mean ± S.E.
from three experiments.
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Because of the importance of agonist-induced receptor internalization
for receptor desensitization and resensitization in many G
protein-coupled receptor systems, we also analyzed the effects of
chemokines on CCR5 endocytosis in RBL-CCR5 cells. As shown in Fig.
3, AOP-RANTES was more effective than
RANTES and Met-RANTES at receptor endocytosis. While 10 nM
AOP-RANTES induced internalization of >80% of CCR5, treatment of
RBL-CCR5 cells with equivalent concentrations of RANTES or Met-RANTES
resulted in the down-regulation of surface-expressed CCR5 of only 60%
or <20%, respectively. This result confirms previous findings with
chemokine-induced CCR5 endocytosis in CHO-CCR5 cells (10).

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Fig. 3.
Differential effects of CC chemokines on CCR5
internalization. RBL-CCR5 cells were incubated (30 min/37 °C)
with the indicated concentrations of RANTES ( ), AOP-RANTES ( ), or
Met-RANTES ( ). Surface CCR5 were detected with monoclonal anti-CCR5
antibody MC-4 and analyzed by flow cytometry. The figure is
representative of two experiments with identical results.
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Receptor Phosphorylation by Chemokines and PMA--
To determine
whether receptor phosphorylation correlated with the observed
chemokine-induced receptor desensitization and internalization we
immunoprecipitated CCR5 from RBL cells which were metabolically labeled
with 32Pi and subsequently stimulated with
various agonists. In a dose-dependent fashion, RANTES
promoted within 5 min the phosphorylation of a 40-kDa band, which was
absent in untransfected 32P-labeled control cells. At
receptor-saturating concentrations, RANTES increased the
phosphorylation of CCR5 in RBL cells 4.5-fold above basal levels (Fig.
4). After stimulation of these cells with
MIP-1
or MIP-1
, maximal 32P incorporation into this
receptor amounted to only 18 ± 4% or 12 ± 2% of the
signal observed after RANTES stimulation. Yet, these signals were
significantly (p < 0.01) different from basal values
in the absence of stimulus. While Met-RANTES was the least effective of
all CCR5 ligands tested, the maximal AOP-RANTES induced receptor
phosphorylation was 2.8 ± 0.3-fold higher than in cells that had
been stimulated with RANTES. Pretreatment of RBL-CCR5 cells with
pertussis toxin reduced the maximal AOP-RANTES-induced receptor
phosphorylation by 55% (data not shown). Again, MIP-1
and RANTES
induced CCR1 phosphorylation equally well, while stimulation with
AOP-RANTES and Met-RANTES did not result in significant CCR1 phosphorylation.

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Fig. 4.
Differential effects of CC chemokines on the
phosphorylation of CCR1 and CCR5. 32P-Labeled CHO-CCR1
or RBL-CCR5 cells were stimulated with chemokines (100 nM)
or PMA (2 µM) for 5 min at 37 °C. Immunoprecipitated
receptors were subjected to 10% SDS-PAGE under reducing conditions.
A, the autoradiogram from one representative experiment is
shown. B, the data in the figure are expressed as a
percentage of 32P incorporation measured in cells treated
with 100 nM RANTES and represent mean ± S.E. from
three experiments.
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Since our calcium mobilization experiments had indicated that in RBL
cells CCR5 is coupled to phospholipase C, we sought to determine the
extent to which PKC might participate in the agonist-induced CCR5
phosphorylation. The phorbol ester PMA significantly increased CCR5
phosphorylation above basal values (Fig.
5). However, pretreatment of RBL-CCR5
cells with the PKC inhibitor bisindolylmaleimide at concentrations that
abolished PMA-induced phosphorylation inhibited RANTES- or
AOP-RANTES-induced CCR5 phosphorylation by only 16 ± 1% or
22 ± 10%, respectively. While this modest effect of the PKC
inhibitor on chemokine-induced receptor phosphorylation was a
significant (p < 0.05) and reproducible finding,
it could be due to direct, PKC-mediated CCR5 phosphorylation
or, alternatively, reflect the stimulatory effect of PKC on GRK2
activity (23). We conclude that PKC can phosphorylate the receptor, but
is not the main kinase that is responsible for the rapid
agonist-induced CCR5 phosphorylation.

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Fig. 5.
Agonist-induced phosphorylation of CCR5:
effects of PKC inhibition. 32P-Labeled RBL-CCR5 cells
were incubated with or without bisindolylmaleimide (BIM; 2 µM) for 10 min. Cells were then treated with either 250 nM PMA or with 100 nM AOP-RANTES for 10 min.
Receptors were immunoprecipitated, resolved by SDS-PAGE, and subjected
to autoradiography (A) and quantitative PhosphorImager
analysis (B). Radioactive counts in the receptor bands were
normalized to those in cells stimulated with 100 nM RANTES
in the absence of PKC inhibitor. Data represent mean ± S.E. of
three experiments; **, p < 0.001; *, p < 0.05 compared with samples without bisindolylmaleimide.
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This conclusion is supported by a kinetic analysis of the
RANTES-induced CCR5 phosphorylation in COS-7 cells. In these cells CCR5
does not couple to PLC unless cells are co-transfected with G
proteins that restore signaling through PLC (7). We therefore compared
the RANTES-induced receptor phosphorylation in COS-7 cells that
expressed CCR5 either alone or together with Gqo5. In
Gqo5, the carboxyl-terminal five amino acids of
G
o, which mediate receptor binding, replace those of
G
q. As shown in Fig. 6,
CCR5 was rapidly phosphorylated even in the absence of
Gqo5. Upon co-transfection of COS-7 cells with expression
plasmids for CCR5 and Gqo5, agonist-promoted receptor
phosphorylation was further enhanced up to 2-fold. This effect was, in
part, sensitive to bisindolylmaleimide (data not shown). These results
indicate that rapid chemokine-induced CCR5 phosphorylation derives
principally from second messenger-independent kinases. Upon prolonged
exposure to receptor ligand, however, PKC may significantly contribute to the agonist-induced receptor phosphorylation.

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Fig. 6.
RANTES-induced phosphorylation of CCR5:
effects of co-transfected Gqo5. COS-7 cells were
transfected with plasmids encoding FLAG-CCR5 along with expression
plasmids encoding either no protein (Control) or the
chimeric G protein Gqo5 (+Gqo5).
32P-Labeled cells were challenged with 100 nM
RANTES for the indicated time periods. Immunoprecipitation of labeled
receptors was followed by 10% SDS-PAGE and quantitative PhosphorImager
analysis. Data are mean ± S.E. from three experiments; *,
p < 0.05 compared with control cells.
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GRK Expression Levels--
The rapid time course of RANTES-induced
CCR5 phosphorylation by a second messenger-independent protein kinase
is suggestive of a GRK-mediated mechanism. If CCR5 phosphorylation and
desensitization under physiological conditions is to be attributed to
the action of one or several GRK(s), GRKs need to be expressed in the
same cells that normally express CCR5. We therefore determined
expression levels of the widely expressed kinases GRK2, GRK3, GRK5, and
GRK6 in human peripheral blood leukocytes and the various cell lines that were used in this study. First, we performed immunoblot analysis of concentrated cellular lysates using GRK-specific antibodies (data
not shown). While GRK5 and GRK6 were readily detected in cells that
overexpressed these kinases, they were absent (or below the detection
limit of our assay) in leukocytes and cell lines relevant to this
study. In contrast, GRK2/3-specific antibodies immunoprecipitated
80-kDa proteins from cellular lysates which comigrated with recombinant
GRK2 or GRK3. To more accurately determine cellular expression levels
of these two closely related receptor kinases, we established ELISA
procedures for the quantitation of GRK concentrations in cellular
lysates which are based on GRK-specific monoclonal antibodies. Human
leukocyte subpopulations contain approximately 1 × 105 molecules GRK2 per cell and 2 × 105
molecules GRK3 per cell (Table I). GRK2
is the predominant receptor kinase in RBL cells, where it is expressed
at high levels. In contrast, GRK levels in COS-7 cells were at the
lower detection limit (10 ng/mg of cellular protein) of the assays.
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Table I
Expression levels of GRK2 and GRK3 in human leukocytes and in cell
lines
Cellular concentrations of GRK2 and GRK3 were determined by ELISA as
described under "Experimental Procedures." Total protein in
cellular lysates was determined with the Bio-Rad DC protein assay kit.
Values are represented as mean ± S.D. ND, not determined.
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Phosphorylation of CCR5 by GRK2 and GRK3 and Translocation of GRK2
to the Receptor--
If GRK2 or GRK3 are involved in RANTES-induced
receptor phosphorylation, overexpression of these kinases can be
expected to enhance CCR5 phosphorylation upon agonist stimulation. As
shown in Fig. 7, overexpression of GRK3
together with CCR5 in COS-7 cells significantly (p < 0.05) enhanced receptor phosphorylation following cellular stimulation
with chemokines. Among the different CCR5 ligands, overexpression of
GRK3 had the least effect on the AOP-RANTES-induced receptor
phosphorylation. This result suggests that, with AOP-RANTES, most of
the CCR5 phosphorylation sites available to GRKs are phosphorylated by
endogenous kinases. In contrast, receptor phosphorylation by
Met-RANTES, MIP-1
, or MIP-1
, which hardly induced any CCR5
phosphorylation in the absence of overexpressed GRKs, was enhanced up
to 12-fold in the presence of high intracellular GRK3 levels. We also
tested whether individual GRKs differ in their abilities to enhance
CCR5 phosphorylation in response to agonist. Overexpression of GRK2,
GRK3, GRK5, or GRK6 augmented RANTES-induced receptor phosphorylation
3-5-fold over that determined in control cells (data not shown).
Significant differences between individual GRKs with regard to receptor
phosphorylation were not observed.

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Fig. 7.
Chemokine-induced phosphorylation of CCR5:
augmentation by overexpressed GRK3. COS-7 cells were transfected
with plasmids encoding CCR5 along with expression plasmids encoding
either no protein (Control) or GRK3 (+GRK3).
32P-Labeled cells were stimulated with 100 nM
of the indicated chemokines for 5 min. Receptor immunoprecipitation and
analysis proceeded as described for Fig. 3. A, shown is an
autoradiogram representative of three experiments. B, data
(mean ± S.E.) from quantitative PhosphorImager analysis were
normalized to values obtained from RANTES-stimulated cells in the
absence of overexpressed GRK3.
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To more directly address the question of which endogenously expressed
protein kinases mediate CCR5 phosphorylation in RBL cells, we used
monoclonal antibodies as intracellular inhibitors of endogenous GRKs in
permeabilized cells. As shown in Fig. 8, the RANTES-stimulated receptor phosphorylation in permeabilized RBL-CCR5 cells was reduced by 60 ± 14% by antibodies (mAb C5/1) that block GRK2 and GRK3 while GRK4-6-specific antibodies (mAb A16/17)
had no effect. A GRK2-specific mAb (E23/8), which is directed against
an GRK2 carboxyl-terminal epitope and does not inhibit GRK2 activity,
also did not affect CCR5 phosphorylation in permeabilized cells. In the
presence of staurosporine, which suppressed basal levels of receptor
phosphorylation, the inhibitory effect of anti-GRK2/3 antibodies was
even more pronounced (92 ± 11% inhibition compared with control
cells).

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Fig. 8.
Inhibition of RANTES-induced CCR5
phosphorylation in permeabilized RBL-CCR5 cells by GRK-specific
antibodies. Cells were permeabilized by streptolysin O (0.5 unit/ml) and incubated with anti-glutathione S-transferase
mAb O3/5 (control), anti-GRK2/3 mAb C5/1, anti-GRK2 mAb E23/8, or
anti-GRK4-6 mAb A16/7 (0.5 mg/ml) in buffer containing
[ -32P]ATP with or without staurosporine. After
stimulation with RANTES (100 nM/5 min), cells were lysed
and receptors were immunoprecipitated. A, the autoradiogram
from one representative experiment is shown. B, data are
mean ± S.E. from three experiments. *, p < 0.005 compared with RANTES-stimulated cells.
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Translocation of cytosolic kinases to the membrane is an essential step
in the agonist-induced receptor phosphorylation by GRK2 or GRK3. Given
the relatively high expression levels of GRK2 and GRK3 in RBL cells, we
asked whether these kinases can be found associated with CCR5 after
chemokine stimulation. We therefore co-immunoprecipitated endogenous
GRKs together with CCR5 from stimulated RBL-CCR5 cells. The result from
one representative Western blotting experiment is depicted in Fig.
9. Only small amounts of GRK2/3 were
found to be associated with CCR5 in cells that had been treated with
medium or with MIP-1
. In contrast, stimulation of RBL-CCR5 cells
with AOP-RANTES or RANTES led to a significant increase in
receptor-associated GRK2/3. Thus, at least for these three agonists,
ligand-induced association of GRKs with CCR5 appears to correlate with
receptor phosphorylation and desensitization.

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Fig. 9.
Chemokine-induced association of GRKs with
CCR5. RBL-CCR5 cells (3 × 107 cells/145-mm dish)
were exposed to 100 nM of the indicated stimuli for 5 min
at 37°. After cross-linking of cellular proteins with
dithiobis(succinimidylproprionate) cells were lysed in detergent buffer
and receptors were immunoprecipitated. GRK2-CCR5 complexes were
dissociated by incubation with electrophoresis buffer containing 20 mM DTT, and GRKs were detected by immunoblotting with
anti-GRK2/3 mAb C5/1. Reprobing these blots with biotinylated anti-FLAG
M2 antibody confirmed that equivalent amounts of receptor were loaded
in each lane. 20 ng of purified recombinant bovine GRK2 was loaded
separately onto the gel and served as a protein standard. The figure is
representative of three experiments with similar results.
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Phosphoamino Acid Analysis and Identification of Phosphorylation
Sites--
The carboxyl-terminal domain of CCR5 contains several
serine and threonine residues that represent potential phosphate
acceptor sites for GRK-mediated receptor phosphorylation. Phosphoamino acid analysis revealed that CCR5 phosphorylation through both GRK- and
PKC-mediated mechanisms occurs exclusively on serine residues (Fig.
10).

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Fig. 10.
Two-dimensional phosphoamino acid analysis
of CCR5. COS-7 cells were transfected with expression plasmids
encoding CCR5. 32P-Labeled cells were exposed for 5 min to
100 nM RANTES or for 12 min to 250 nM PMA.
Phosphoamino acid analysis of the immunoprecipitated receptors was
performed as described under "Experimental Procedures." Positions
of phosphoserine (S), phosphothreonine
(T), and phosphotyrosine (Y) were
assessed by ninhydrin staining of phosphoamino acid standards, which
were separated together with the radioactive sample. The figure is
representative of three experiments with similar results.
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We used a mutational approach to define the precise location of those
serines that are phosphorylated upon cellular stimulation with RANTES
or AOP-RANTES. First, a CCR5 truncation mutant was generated by
introducing a stop codon at position CCR5-334. This truncated version
of CCR5 was not phosphorylated upon agonist stimulation (not shown),
indicating that Ser-325 is not a GRK phosphorylation site. The
remaining four serine residues at positions 336, 337, 342, and 349 were
substituted with alanine either individually or in various
combinations. As shown in Fig. 11,
alanine-mutagenesis of any single or any two serines did not
significantly affect the RANTES-induced CCR5 phosphorylation as
compared with wild type receptor. In the triple mutants RANTES-induced
receptor phosphorylation was reduced 50-70% relative to CCR5 wild
type, and alanine substitution of all four serines completely abrogated
agonist-stimulated receptor phosphorylation. In contrast,
AOP-RANTES-stimulated receptor phosphorylation was reduced by 15-45%
in CCR5 single mutants, by 45-70% in double mutants, and by 85-90%
in triple mutants. Overall, all four serines equally contributed to
ligand-induced receptor phosphorylation.

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Fig. 11.
Effects of Ser Ala mutagenesis on
agonist-induced CCR5 phosphorylation. COS-7 cells were transfected
with expression plasmids encoding CCR5 wild type or CCR5 mutants which
contained Ser Ala mutations of four carboxyl-terminal serine
residues (Ser-336, Ser-337, Ser-342, Ser-349) in various combinations
(e.g. A - - - denotes Ala mutation of Ser-336). Receptor
phosphorylation was monitored after cellular stimulation with 100 nM RANTES or 50 nM AOP-RANTES. Agonist-induced
receptor phosphorylation was calculated by subtracting the values of
basal phosphorylation in the absence of stimulus for each receptor
variant and was normalized to that obtained in CCR5 wild type cells.
Shown are data (mean ± S.E.) from experiments performed in
triplicate.
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Furthermore, we observed a stepwise shift in the electrophoretic
mobility of phosphorylated receptor variants, depending on the number
of Ser to Ala mutations (Fig. 12,
left). The immunoblot (Fig. 12, right) revealed
the presence of additional receptor bands in lanes
B-D with the same electrophoretic mobility as found in non-stimulated cells. These bands probably correspond to intracellular receptors, which form a significant portion of the total cellular receptor complement in transiently transfected cells and which are,
consequently, neither exposed to ligand nor phosphorylated by receptor
kinases. The observed stepwise shift in the electrophoretic mobility of
phosphorylated CCR5 suggests that AOP-RANTES stimulates 32P
incorporation at three to four acceptor sites per receptor. In
contrast, RANTES apparently induces receptor phosphorylation by GRKs at
a lower stoichiometry with no preference for any specific residue(s)
among the four serines.

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Fig. 12.
AOP-RANTES-induced phosphorylation and shift
in electrophoretic mobility of CCR5(Ser Ala) mutants. COS-7
cells were transfected with expression plasmids encoding CCR5 wild type
(B), or CCR5 mutants containing Ser Ala mutations at
positions 336 (C), 336 and 337 (D), 336, 337, and
342 (E), or 336, 337, 342, and 349 (F);
A are empty vector-transfected control cells.
32P-Labeled cells were stimulated with 50 nM
AOP-RANTES for 5 min. Left, autoradiogram from one
representative experiment. Receptors were immunoprecipitated and
resolved by 10% SDS-PAGE. Right, immunoblot depicting the
shift in electrophoretic mobility of phosphorylated CCR5 mutants. After
electrophoretic separation (10% SDS-PAGE) of cellular lysates and
electrotransfer to nitrocellulose, receptors were visualized with
anti-CCR5 monoclonal antibody MC-5 (2 µg/ml), a 1000-fold dilution of
horseradish peroxidase-conjugated rabbit anti-mouse antibodies (Dako),
and an enhanced chemiluminescence kit. In the absence of stimulus, all
receptor variants migrated as narrow bands at the same position as the
phosphorylation-deficient CCR5 mutant shown in lane
F.
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To directly investigate whether each of the four serines at positions
336, 337, 342, and 349 are GRK substrates, we co-expressed GRK3
together with CCR5 triple mutants in COS-7 cells (Fig.
13). Overexpression of GRK3 resulted in
a significant increase in the RANTES-induced phosphorylation not only
of CCR5 wild type, but also of every of these four receptor variants.
Since GRK3 overexpression had no effect on the CCR5 mutant that had all
four serines mutated to alanine, these residues represent the only
sites on CCR5 available to GRK3 for agonist-induced
phosphorylation.

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Fig. 13.
Effect of GRK3 overexpression on the
phosphorylation of CCR5 (Ser Ala) mutants. COS-7 cells were
transfected with expression plasmids encoding CCR5 wild type or CCR5
(Ser Ala) mutants alone (open bars) or together with DNA
encoding GRK3 (filled bars).
32P-Labeled cells were exposed to 100 nM
RANTES for 5 min; receptors were immunoprecipitated and resolved by
SDS-PAGE. RANTES-induced receptor phosphorylation was quantitated by
PhosphorImager analysis and was calculated by subtracting basal levels
in the absence of stimulus. Results are mean ± S.E. of three
experiments. *, p < 0.05 compared with cells with a
normal GRK complement.
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DISCUSSION |
Several lines of evidence from the current study point to a
central role for GRK2 and/or GRK3 in the agonist-induced
phosphorylation of CCR5. First, CCR5 transiently expressed in COS-7
cells was still rapidly phosphorylated upon stimulation with RANTES
despite the fact that COS-7 cells apparently lack signaling elements
that would allow efficient coupling of CCR5 to PLC (7, 24). This suggests that homologous CCR5 phosphorylation, at least after short
term exposure to agonist, is largely independent of second messenger-dependent protein kinases. Our finding of a
significant reduction of receptor phosphorylation in pertussis
toxin-treated RBL-CCR5 cells does not preclude a major role for GRKs in
receptor phosphorylation. ADP-ribosylation of G
i
prevents receptor-induced dissociation of heterotrimeric G proteins and
thereby interferes with 
-mediated targeting to membrane-bound
receptors of GRK2 and GRK3 (25). Second, the time course of
RANTES-induced CCR5 phosphorylation conforms with the rapid kinetics of
GRK-mediated receptor phosphorylation observed in previous studies
(16;26). Third, overexpression of GRKs in COS-7 cells was shown to
significantly enhance chemokine-induced CCR5 phosphorylation. While
this finding shows that agonist-occupied CCR5 is a potential substrate
for GRKs, it does not directly address the question of which
endogenously expressed kinases actually mediate receptor
phosphorylation in RBL cells or in leukocytes. We therefore adapted a
previously described method for the intracellular inhibition of
receptor kinases by anti-GRK antibodies in permeabilized cells (18) to the CCR5 system in RBL cells. These experiments revealed that GRK2
and/or GRK3 are the major kinases that mediate RANTES-induced CCR5
phosphorylation in RBL cells. Consistent with a major role for GRK2 and
GRK3 in CCR5 phosphorylation under physiological conditions, we
determined high intracellular levels of these kinases in human
leukocytes and in RBL cells. In this study we provide, to our knowledge
for the first time, quantitative estimations of intracellular GRK
concentrations in biological materials. These data not only support a
role for GRKs in the regulation of chemotactic leukocyte receptors,
they also shed light on the stoichiometry of signaling components that
participate in the regulation of G protein-coupled receptors in native
cells. With 1-2 × 105 molecules of GRK2 or GRK3 per
cell, these kinases are expressed in leukocytes in numbers that are
roughly equivalent to expression levels of many G protein-coupled
receptors, but much lower than concentrations of G
subunits (27).
The relative abundance of signaling elements that transduce signals
compared with levels of receptor kinases may ensure that ligand binding
will still lead to cellular activation until receptor inactivation by
phosphorylation and arrestin binding gradually takes over.
Although GRK2 and/or GRK3 are the protein kinases that are mainly
responsible for the rapid agonist-induced phosphorylation of CCR5 in
RBL cells and possibly in human peripheral blood leukocytes, this
receptor is also a substrate for PKC, the kinase that is activated by
signaling pathways downstream of CCR5. Inhibitor studies using
bisindolylmaleimide or anti-GRK antibodies revealed that PKC only
modestly contributes to rapid RANTES-induced receptor phosphorylation
in RBL-CCR5 cells. In COS-7 cells, restoration of the receptor-PLC-PKC
system by co-transfection with Gqo5 resulted in a
significant increase in CCR5 phosphorylation only upon prolonged (>10
min) stimulation with agonist. The kinetics of GRK- versus PKC-mediated CCR5 phosphorylation resembles the time course of GRK- and
second messenger-dependent kinase-mediated phosphorylation and desensitization in other receptor systems (16, 26). By analogy with
these earlier studies, functional differentiation of PKC-mediated from
GRK-mediated phosphorylation of CCR5 seems to relate to their differing
time courses of action. Additional significance for our finding of
PKC-mediated CCR5 phosphorylation derives from the fact that any agent
that activates PKC will induce heterologous phosphorylation of CCR5.
Serine or threonine residues of the CCR5 carboxyl terminus most likely
represent phosphorylation sites for GRKs. By using a combination of
phosphoamino acid analysis and alanine-scanning mutagenesis, we
identified residues Ser-336, Ser-337, Ser-342, and Ser-349 as the
phosphoacceptor sites on CCR5 that are phosphorylated upon agonist
stimulation of this receptor. This result has several implications.
First, the finding that these four serines are (the only) substrates
for GRK3 on CCR5 lends further support to the notion that GRK3 or a
GRK3-related GRK is the endogenous kinase that is responsible for
agonist-induced CCR5 phosphorylation.
Second, since the extent of agonist-stimulated receptor phosphorylation
was determined by the number of serines present in the CCR5 (Ser
Ala) mutants, but not by the position of the individual serines, all
four serines seem to be phosphorylated independently and in a
non-preferential manner. Similar results were obtained for the
2A-adrenergic receptor (28) and the
1B-adrenergic receptor (29), where GRK-mediated
phosphorylation was shown to proceed non-sequentially. In contrast, a
hierarchy of phosphorylation sites seems to exist for rhodopsin (30),
the C5a receptor (31), and the N-formyl peptide receptor
(32). In these receptors phosphorylation of a primary phosphoacceptor
site is necessary to allow kinases to proceed further on secondary
sites. Subtle differences in the conformations of receptor
phosphorylation sites may determine whether GRKs phosphorylate
receptors in a sequential or non-sequential manner.
Third, this study contributes to the characterization of consensus
phosphorylation site motifs for GRKs. Phosphorylation studies using
synthetic peptides have indicated that GRK2 is an acidotropic kinase
that prefers acidic amino acids juxtaposed to serines or threonines
(33). Interestingly, of the four CCR5-carboxyl-terminal serines that
were identified as GRK phosphorylation sites in this study, only
Ser-349 is located in close proximity to an upstream glutamic acid
residue. Yet, experiments with overexpression of GRK3, which is closely
related to GRK2, indicated that this kinase phosphorylates all four
serines equally well. These findings support the view that the overall
structure of an activated receptor rather than a specific amino acid
sequence determines whether a serine or threonine residue will be
phosphorylated by GRKs.
Fourth, the four serine residues that constitute phosphorylation sites
on CCR5 for GRKs are highly conserved in several members of the CC
chemokine receptor family (6). Thus, the results of this study may also
be relevant to the identification of phosphorylation sites on closely
related members of this receptor family. Of note, alanine mutation of 7 out of the 10 carboxyl-terminal Ser/Thr residues in the CCR2b receptor,
including 4 amino acids homologous to the CCR5 phosphorylation site,
almost completely prevented the inhibitory effect of GRK3 on the
MCP-1-induced calcium response in Xenopus oocytes (34).
Rapid and dose-dependent receptor phosphorylation has been
linked to homologous and heterologous desensitization in several CC and
CXC chemokine receptor systems (22, 34-36). Experiments with
overexpression of GRK2 and/or GRK3 together with CCR2b in either the
Xenopus oocyte expression system (34) or in HEK-293 cells
(37) have directly implied a role for GRKs in the phosphorylation and
desensitization of this receptor. Using a similar approach, Aramori
et al. have recently demonstrated that CCR5 in HEK293 cells
are rapidly phosphorylated and desensitized upon cellular stimulation
with MIP-1
(22). However, this effect was only observed in cells
that overexpressed receptor kinases. The authors hypothesized that
MIP-1
-induced phosphorylation and desensitization of CCR5 may still
be physiologically relevant in cells that express high cellular levels
of GRKs, such as leukocytes. In this study we show that, in RBL cells
which express GRK2 and GRK3 at levels comparable to those determined in
human leukocytes, MIP-1
has only a marginal effect on CCR5
phosphorylation and desensitization. We conclude that for CCR5
regulation under physiological conditions intracellular levels of
receptor kinases are a much less important determinant than the nature
of the respective CCR5 ligand.
The finding that various ligands which bind to CCR5 with comparable
affinities (7) differ largely in their abilities to induce cellular
signaling as well as receptor desensitization and phosphorylation was
unexpected. The results of our study imply that these molecules may
function as full or partial agonists with a wide range of ligand
efficacies. Most interestingly, the amino-terminal modification of
RANTES with either an additional methionine (Met-RANTES) or with the
structurally closely related aminooxypentane group (AOP-RANTES)
drastically affected ligand efficacy in opposite directions. Our
findings support the concept that the amino-terminal region of RANTES
is an important triggering domain which is involved in chemokine
receptor activation (38, 39). In general, we observed a close
correlation between the efficacies of receptor ligands in CCR5
activation, phosphorylation and desensitization, with rank order
AOP-RANTES > RANTES > MIP-1
, MIP-1
> Met-RANTES. Our
results conform with a previous study in the
-adrenergic receptor
system using purified components, which showed that the abilities of
partial agonists to stimulate adenylate cyclase activity directly
correlated with their abilities to promote receptor phosphorylation by
GRKs (40).
By co-immunoprecipitation of CCR5 together with GRKs, we find that GRK2
forms a macromolecular complex with CCR5 after chemokine stimulation. A
similar approach was previously used to demonstrate receptor
specificities among different GRKs in the endothelin and angiotensin II
receptor systems (21). We show here that differences in ligand
efficacies are also reflected in the abilities of chemokines to induce
GRK association with the receptor. Mechanistically, this may be the
result of varying numbers of dissociated 
subunits that are
released from the
subunit after agonist-induced receptor activation. G protein 
subunits are essential in the
translocation of GRK2 and GRK3 to the membrane (25). Alternatively,
full agonists such as AOP-RANTES may induce conformational changes of
CCR5 that allow a more stable association of GRKs with the receptor.
The results of our study suggest a molecular mechanism that may explain
the differential effects of CC chemokines on CC chemokine receptor
internalization, observed previously (10, 17) and confirmed for the
RBL-CCR5 cell system in this study. Mack et al. reported
that natural CC chemokines and the amino-terminal modifications of
RANTES induced varied degrees of CCR5 down-regulation from cellular
surfaces (10), which followed the same rank order of ligand efficacy as
was determined in the present study. According to this concept binding
of the different ligands to CCR5 leads to conformational changes of the
receptor that activate G proteins as well as GRKs with varying
efficacies. Arrestin binding to the phosphorylated receptor precludes G
protein interaction with the receptor, leading to functional
desensitization. Furthermore, as with other receptors, arrestin-like
proteins may be instrumental in the removal of CCR5 from the cell
surface via clathrin-coated pits (41). The finding that overexpression
of
-arrestin 2 together with GRK2 results in a significant increase
in the MIP-1
-induced CCR5 sequestration in COS-7 cells (22) lends
support to this notion. Clearly, more work needs to be done to
establish the functional significance of GRK-mediated receptor
phosphorylation for the agonist-induced endocytosis of CCR5.
By comparison of chemokine effects on CCR1 and CCR5, we show that
different rank orders of ligand efficacies exist within these two
closely related receptor systems. This is most strikingly illustrated
by the different effects of AOP-RANTES on CCR1 and CCR5. While
AOP-RANTES has the highest potency on CCR5 among the various CC
chemokines used in this study, it did not induce CCR1 signaling or
phosphorylation. Therefore, the original observation that AOP-RANTES
acts as a chemokine antagonist of monocyte chemotaxis (9) may well be
due to the predominant expression of CCR1 over CCR5 in these cells.
This study demonstrates that CC chemokines largely differ in their
agonistic efficacies within the CCR5 and CCR1 systems. The differential
ability of chemokines to induce receptor activation, phosphorylation,
desensitization, and internalization provides yet another mechanism
that allows chemokines to fine-tune the inflammatory response.