From the the § Department of Veterans Affairs,
Nashville, Tennessee 37212-2637 and the Department of
Cancer Biology, Vanderbilt University School of Medicine,
Nashville, Tennessee 37212-2175
Received for publication, October 11, 2000, and in revised form, January 29, 2001
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ABSTRACT |
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Protein phosphatase 2A (PP2A) is postulated to be
involved in the dephosphorylation of G protein-coupled receptors. In
the present study, we demonstrate that the carboxyl terminus of CXCR2 physically interacts with the PP2A core enzyme, a dimer formed by PP2Ac
and PR65, but not with the PP2Ac monomer, suggesting direct interaction
of the receptor with PR65. The integrity of a sequence motif in the C
terminus of CXCR2, KFRHGL, which is conserved in all CC and CXC
chemokine receptors, is required for the receptor binding to the PP2A
core enzyme. CXCR2 co-immunoprecipitates with the PP2A core enzyme in
HEK293 cells and in human neutrophils. Overexpression of dominant
negative dynamin 1 (dynamin 1 K44A) in CXCR2-expressing cells blocks
the receptor association with the PP2A core enzyme, and an
internalization-deficient mutant form of CXCR2 (I323A,L324A)
also exhibits impaired association with the PP2A core enzyme,
suggesting that the receptor internalization is required for the
receptor binding to PP2A. A phosphorylation-deficient mutant of CXCR2
(331T), which has previously been shown to undergo internalization in
HEK293 cells, binds to an almost equal amount of the PP2A core enzyme
in comparison with the wild-type CXCR2, suggesting that the interaction
of the receptor with PP2A is phosphorylation-independent. The
dephosphorylation of CXCR2 is reversed by treatment of the cells with
okadaic acid. Moreover, pretreatment of the cells with okadaic acid
increases basal phosphorylation of CXCR2 and attenuates CXCR2-mediated
calcium mobilization and chemotaxis. Taken together, these data
indicate that PP2A is involved in the dephosphorylation of CXCR2. We
postulate that this interaction results from direct binding of the
regulatory subunit A (PR65) of PP2A to the carboxyl terminus of CXCR2
after receptor sequestration and internalization.
Chemokines comprise a family of about 50 low molecular weight
proteins that mediate inflammatory responses, chemotaxis, immune cell
development, and leukocyte homing. These have been classified into C,
CC, CXC, and CX3C chemokines, based on the presence and the position of
conserved cysteine amino acid residues (1-3). The biological functions
of chemokines are mediated through interaction with their cognate
receptors, which are members of the G protein-coupled receptor
(GPCR)1 superfamily. Like
other members of the GPCR superfamily, the functional status of many
chemokine receptors is determined largely by the phosphorylation state
(4-6). Agonist treatment enhances phosphorylation of the receptors by
protein kinases, presumably G protein-coupled receptor kinases
and protein kinase C, which results in desensitization of the receptors
(4, 7, 8). This phenomenon is common to many hormonal and
neurotransmitter signaling systems (9), but the underlying mechanisms
are still only partially understood, especially in the case of
chemokine receptors. Based on work on several chemokine receptors, the
phosphorylated receptor is then internalized via clathrin-coated pits
into early endosomes (6, 10-14) and subsequently dephosphorylated by
intracellular protein phosphatases (10). The dephosphorylated receptors
might be either recycled through sorting endosomes back to the plasma membrane or transported to the lysosomes for degradation. The recycling
and degradation rate might vary among different chemokine receptors.
For example, after down-modulation by interleukin-8 (CXCL8), the
expression of CXCR1 fully recovers within 1.5 h, while the
recovery rate of CXCR2 expression is very slow and never reaches 40%
of the control level during a 3-h culture period (15).
Several investigations have demonstrated that neutrophil chemotactic
responses require chemokine receptor internalization and recycling
(16). Recycling of chemokine receptors might be very important to
maintain the directional migration of cells toward a chemokine
concentration gradient. Moreover, dephosphorylation of the receptors
appears to play a key role in the recycling and resensitization of
chemokine receptors. Therefore, regulation of the receptor
dephosphorylation by protein phosphatases represents an important
mechanism for modulating the function of chemokine receptors.
Four major classes of serine/threonine-specific protein phosphatases
(PPs) have been described. These include PP1, PP2A, PP2B (calcineurin),
and PP2C. PP2B and PP2C are calcium-dependent, whereas PP1
and PP2A are not. PP1 and PP2A are widely expressed in the cytoplasm of
mammalian cells and have been reported to be involved in signal
transduction, proliferation, and metabolic events (17). Studies on the
The holoenzyme of PP2A contains a dimer (core enzyme) composed of a
36-kDa catalytic subunit (PP2Ac) and a 65-kDa regulatory subunit A
(PR65). In addition, there are several associated variable regulatory
subunits (B) that bind to PR65 and confer substrate specificity to the
dephosphorylating activity (17). Although In the process of investigating proteins that interact with chemokine
receptors using the yeast two-hybrid system and in vitro binding assay, we discovered that the CXC chemokine receptor, CXCR2,
binds to the PR65/PP2Ac dimer but not to the PP2Ac monomer, suggesting
direct interaction of the receptor with PR65. The binding requires the
integrity of a sequence motif that is conserved in all CC and CXC
chemokine receptors and in many other GPCRs. Moreover, CXCR2 associates
with the PP2A core enzyme in a phosphorylation-independent and
internalization-dependent manner.
Plasmid Construction--
Wild-type and truncated mutant (331T)
of CXCR2 were constructed previously (4). For the construction of
PAS2/CXCR2 tail for the two hybrid screen, the CXCR2 carboxyl-terminal
domain was cut from PRc/CMV-CXCR2 with NcoI and
HindIII, blunted with T4-DNA polymerase, and inserted into
the PAS2 bait vector that had been digested with NcoI and
blunted with T4-DNA polymerase. The correct orientation and in frame
fusion of the insert were determined by DNA sequencing. Constructs for
glutathione S-transferase (GST) fusion proteins of the
C-terminal tails of wild-type or mutant CXCR2 were generated using
PCR-amplified fragments. A BamHI site was included in the 5'
primer, and a HindIII site was included in the 3' primer.
pGEX-KG and the PCR-generated fragments were cut with BamHI
and HindIII, ligated, and used to transform
Escherichia coli DH5 Cell Culture and Transfection--
Human embryonic kidney 293 (HEK293) cells were grown in Dulbecco's modified Eagle's
medium (DMEM), containing 10% fetal bovine serum and a 1:100 dilution
of penicillin/streptomycin (BioWhittaker), at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. Cells were transfected
with wild-type or mutant CXCR2 using LipofectAMINE plus reagent (Life
Technologies, Inc.). Stably transfected cells were selected with 560 µg/ml Geneticin (G418) and evaluated for receptor expression using
125I-labeled melanoma growth-stimulatory
activity/growth-related protein (125I-CXCL1) binding
(PerkinElmer Life Sciences, catalog no. NEX-321).
Yeast Two-hybrid Assay--
Yeast two-hybrid techniques were
performed as described (23, 24). For screening cDNA libraries, the
bait plasmid PAS2/CXCR2 tail was transformed into yeast strain Y190
(CLONTECH) using a lithium acetate protocol
(CLONTECH manual). After confirming expression of
the bait protein, a human B lymphocyte library in the vector PACT2 was
transformed into the strain harboring the bait plasmid. The
transformants expressing both the bait and the prey proteins were
selected on medium lacking leucine, tryptophan, and histidine (SD/ Filter Lift Assay--
A dry nitrocellulose filter was placed on
the yeast colonies. The filter was then carefully lifted, transferred
(colonies facing up) into liquid nitrogen, completely submerged for
10 s, and then allowed to thaw at room temperature. The filter was
then placed on a Whatman filter paper soaked in Z buffer (60 mM Na2HPO4, 40 mM
NaH2PO4, 10 mM KCl, 1 mM Mg2SO4, pH 7.0) containing 50 mg/ml 5-bromo-4-chloro-3-indolyl-galactopyranoside, with colonies face up until blue color appeared.
In Vitro Binding Assay--
Bacteria encoding GST or GST fusion
proteins were cultured overnight at 37 °C, and then
isopropyl-1-thio- Co-immunoprecipitation and Western Blot--
Human neutrophils
were isolated from fresh heparinized peripheral blood from single human
donors as described previously (25). HEK293 cells stably expressing
CXCR2 were serum-starved overnight in DMEM containing 0.5% fetal
bovine serum before the experiment. The cells were treated with
or without agonists, and then the cells were washed three times with
ice-cold PBS and lysed in 1 ml of RIPA buffer. The cell debris was
removed by centrifugation for 4 min at 13,000 rpm in an Eppendorf
microcentrifuge. The supernatant was precleared for 1 h to reduce
nonspecific binding by the addition of 40 µl of protein A/G-agarose
(Pierce). After removal of the protein A/G-agarose by centrifugation in
an Eppendorf microcentrifuge at 3000 rpm for 1 min, the cleared
supernatant was collected, and 10 µl of affinity-purified anti-CXCR2
antibody (prepared in our laboratory) was added for overnight
precipitation at 4 °C. 40 µl of protein A/G was then added and
incubated at 4 °C for 2 h. The protein A/G-antibody-antigen
complex was then collected by washing three times with ice-cold RIPA
buffer. The final pellet was resuspended in 50 µl of SDS sample
buffer containing 5% In Vitro Phosphorylation and Dephosphorylation--
CXCR2 was
immunoprecipitated with a rabbit anti-CXCR2 antibody and protein A/G
beads as described above. The immunoprecipitates were then washed four
times with kinase assay buffer containing 50 mM Tris-HCl,
pH 7.4, 10 mM MgCl2, 500 µM
CaCl2. The immunoprecipitates were incubated with 100 µg/ml phosphatidylserine, 2.5 µCi of [ In Vivo Phosphorylation and Dephosphorylation--
Receptor
phosphorylation assay was performed as described previously (10). In
brief, the transfected cells were replated on six-well plates 1 day
after the transfection. On the following day, after incubating in
serum-and phosphate-free medium for 1 h, cells were labeled by
incubating in [32P]orthophosphate (100 µCi/ml)
(PerkinElmer Life Sciences) in the same medium at 37 °C for 2 h. Cells were then stimulated with or without agonists.
Dephosphorylation was performed by allowing cells to recover in fresh
serum-free media at 37 °C for 1 h. The cells were then
lysed in RIPA buffer. CXCR2 was immunoprecipitated as described above
with a specific antibody. The immunoprecipitates were electrophoresed
through a 10% SDS-polyacrylamide gel and transferred to a
nitrocellulose membrane (Bio-Rad). The phosphorylated receptors were
then detected by autoradiography. The amount of receptor
immunoprecipitated was determined by blotting the membrane with a
monoclonal antibody against CXCR2 to evaluate relative receptor phosphorylation.
Chemotaxis Assay--
A 96-well chemotaxis chamber (Neuroprobe
Inc.) was used for chemotaxis assays, and the lower compartment of the
chamber was loaded with 400-µl aliquots of 1 mg/ml ovalbumin/DMEM
(chemotaxis buffer) or CXCL8 diluted in the chemotaxis buffer (1-200
ng/ml). Polycarbonate membranes (10-µm pore size) were coated on both sides with 20 µg/µl human collagen type IV, incubated for 2 h at 37 °C, and then stored at 4 °C overnight. To prepare cells for
chemotaxis assay, they were removed by trypsinization, washed with
Hanks' solution, and incubated in 10% fetal bovine serum/DMEM for
2 h at 37 °C to allow time for restoration of receptors. The cells were washed with chemotaxis buffer and then loaded into the upper
chamber in the chemotaxis buffer. The chamber was incubated for 4 h at 37 °C in humidified air with 5% CO2, and then the
membrane was removed, washed, fixed, and stained with a Diff-Quik kit. Cell chemotaxis was quantified by counting the number of migrating cells present in 10 microscope fields (× 20 objective).
Calcium Fluorimetry--
HEK293 cells stably expressing CXCR2
were grown until confluent. Cells were released by shaking, collected
by centrifugation at 300 × g for 6 min, and washed
with Hanks' buffer containing 5 mM HEPES. Cells were
resuspended at 2 × 106 cells/ml and incubated with
2.5 µM Fluo-3 (Molecular Probes, Inc., Eugene, OR) for 30 min at 37 °C. After incubation, the cells were washed once with
Hanks' buffer containing 5 mM HEPES and 2 mM
Ca2+. The cells were finally adjusted to 2 × 106 cells/ml. Ca2+ mobilization experiments
were performed as described previously (26).
In an attempt to isolate chemokine receptor-associated proteins,
we used the yeast two-hybrid system to identify proteins that interact
with the carboxyl terminus of the chemokine receptor, CXCR2 (Fig.
1A). Screening of a human B
lymphocyte library fused to the GAL4 transactivation domain (generous
gift from Dr. Stephen J. Elledge) yielded several potential candidate
genes that were both His+ and LacZ+. The prey
cDNAs were recovered from yeast and transformed into bacteria. The
cDNAs were then sequenced using primers complementary to 5' or 3'
ends of the inserts. Among them, one (clone 91) encoding PR65 was
chosen for further study based on its moderately strong LacZ+. The specificity of the interaction in yeast was
tested by retransforming PACT2/clone 91 along with the original bait
PAS2/CXCR2 tail or PAS2 alone back into yeast strain Y190. The
interaction between the receptor C terminus and PR65 specifically
allowed growth on SD medium lacking leucine, tryptophan, and
histidine (SD/
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptor (
2-AR) suggest that
PP2A is involved in the dephosphorylation of the receptor. Pitcher
et al. (18) have identified a plasma and vesicular
membrane-associated phosphatase that dephosphorylates the
2-AR phosphorylated by G protein-coupled receptor
kinases. This phosphatase, referred to as G protein-coupled receptor
phosphatase, is a subclass of PP2A. The
2-AR interacts
with a G protein-coupled receptor phosphatase in an acidic pH condition
in endosomal vesicles (19). Increasing the endosomal pH not only blocks
the association of the receptor with the phosphatase but also prevents
the receptor dephosphorylation. In addition, PP2A has been indicated to
be the chief enzyme acting on the cholecystokinin receptor from
pancreatic acinar cells (20), rhodopsin (21), and C5a receptor in HL60
cells (22).
2-AR
co-immunoprecipitates with PP2Ac in response to agonist stimulation
(19), it has not been determined whether there is a direct binding
event and, if so, to which subunit of PP2A the receptor directly binds.
For chemokine receptors, the association between receptor and protein
phosphatase(s) has not been characterized.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
.
Leu/
Trp/
His). Colonies capable of growing on the
SD/
Leu/
Trp/
His medium were then tested for
-galactosidase activity (LacZ+) using the filter lift
assay. Clones that were consistently phenotypically His+
and LacZ+ were further characterized. Approximately
2.6 × 106 transformants were screened, and several of
them were His+ and LacZ+. A single clone was
chosen for further pursuit based on its strong His+/LacZ+ phenotype.
-D-galactopyranoside was added, and
incubation was continued for another 3 h to induce protein
expression. The bacteria were lysed in RIPA buffer (25 mM
Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 10 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg each of
leupeptin and aprotinin) and then sonicated on ice for 10 s. The
supernatant of the bacterial lysate was incubated with
glutathione-Sepharose at 4 °C for 30 min. After washing three times
with RIPA buffer, the beads were resuspended in RIPA buffer. Purified
PP2A subunits (a generous gift from Dr. Brian Wadzinski) or HEK293 cell
lysates were incubated with the GST or GST fusion proteins bound to
glutathione-Sepharose for 2 h at 4 °C with rotation. Beads were
pelleted by centrifugation (12,000 rpm) for 2 min and washed four times
with RIPA buffer. Bound proteins were released by boiling in SDS-PAGE
sample buffer for 5 min and detected by SDS-PAGE and Western blot.
-mercaptoethanol and heated to 50 °C for 10 min. 20 µl of this preparation was electrophoresed on a 10%
SDS-polyacrylamide gel, and the proteins on the gel were transferred to
nitrocellulose membranes (Bio-Rad) as previously described (10).
Co-precipitated PR65 or PP2Ac was detected using a goat polyclonal
antibody (catalog no. SC6112; Santa Cruz Biotechnology, Inc., Santa
Cruz, CA) and a mouse monoclonal antibody (catalog no. P47720;
Transduction Laboratories), respectively.
-32P]ATP,
and 50 microunits of purified protein kinase C (Pierce) at 30 °C for
30 min in kinase assay buffer in a final volume of 20 µl. 1 unit of
protein kinase C activity is defined as the amount of enzyme required
to transfer 1 µmol of phosphate from ATP to histone H1 per min at
30 °C. The immunoprecipitates were then washed with phosphatase
assay buffer containing 5 mM Tris-HCl, pH 7.0, 0.1 mM EDTA, 0.1% 2-mercaptoethanol, and 1 mg/ml bovine serum
albumin. Dephosphorylation was carried out by incubation of the
immunoprecipitates with different concentrations of purified PP2A core
enzyme in the phosphatase assay buffer in a volume of 30 µl at
30 °C for 30 min. The reaction was terminated by adding Laemmli
sample buffer and heating at 50 °C for 10 min. Samples were
subjected to 10% SDS-PAGE, and phosphorylated CXCR2 was detected by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Leu/
Trp/
His) (Fig.
2A, left
panel). Neither the bait protein, PAS2/CXCR2 tail, nor the
prey was able to activate transcription of the reporter genes in the
presence of only empty prey or bait vectors, respectively (Fig.
2A, left panel). Using the
-galactosidase assay, we found that only the yeast co-transformed
with PACT2/clone 91 and the PAS2/CXCR2 tail displayed LacZ+
(Fig. 2A, right panel).
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Fig. 1.
The carboxyl-terminal sequence of CC and CXC
chemokine receptors and mutagenesis of the CXCR2 carboxyl tail. A,
schematic of the carboxyl terminus of CXCR2 and mutations. The
indicated amino acid residues were changed to alanine
(underlined). B, alignment of the
carboxyl-terminal amino acid sequence of CC and CXC chemokine
receptors. Conserved residues potentially involved in the binding to
PP2A are boxed.
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Fig. 2.
Interaction of CXCR2 carboxyl terminus with
PP2A. A, yeast strain Y190 containing the PAS2/CXCR2
tail, the PACT2/PR65, or both plasmids were replica-plated on SD medium
without tryptophan, leucine, and histidine. The cotransformation of p53
and SV40 provided by the manufacturer was performed as a positive
control (left). A -galactosidase assay was carried out
using a YPD plate replicate (right) as described under
"Experimental Procedures." B, the indicated GST or
GST-CXCR2 carboxyl tail fusion protein was incubated with the purified
PP2Ac monomer (C monomer) or PR65/PP2Ac dimer (AC dimer) and then
absorbed onto glutathione-Sepharose beads. After washing, the beads
were resuspended in loading buffer. Proteins were separated using 10%
SDS-PAGE and transferred to a nitrocellulose membrane. Coprecipitated
PR65 and PP2Ac were analyzed by immunoblotting with anti-PR65 and
anti-PP2Ac. The membrane was stripped and reblotted with a mouse
monoclonal anti-GST antibody to confirm the protein expression and
equal loading.
To confirm the specific biochemical interaction between the C terminus of CXCR2 and PR65, we used an in vitro binding assay to test for direct interaction. A GST fusion protein containing the C terminus of CXCR2 was made (Fig. 1A) and tested for binding to the purified PP2Ac monomer or PP2Ac/PR65 dimer. We observed that the GST-CXCR2 tail fusion protein specifically bound the purified PP2Ac/PR65 dimer but not the PP2Ac monomer (Fig. 2B), suggesting that the C terminus of CXCR2 only binds to PR65. Several GST fusion proteins show a consistent pattern of lower molecular weight bands in the purified sample. These are believed to be either unstable degradation products or incompletely translated products.
We next identified the region of CXCR2 involved in the PP2A binding by
using GST fusion proteins encoding various fragments of the C terminus
of CXCR2 (Fig. 1A). We found that the
GST-CXCR2-(311-330) bound to an equal amount of the PP2A core
enzyme as compared with GST-CXCR2 tail, whereas the
GST-CXCR2-(331-355) did not bind to the PP2A core enzyme, implying
that the minimal CXCR2 binding region resides in residues 311-330 of
the C-terminal domain of CXCR2 (Fig.
3A). Interestingly, this
domain is immediately upstream of the phosphorylation sites, suggesting
that PP2A does not bind to the C-terminal phosphorylation sites of the
receptor.
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To identify which residues are required for the binding of the PP2A core enzyme, in vitro binding assays using GST fusion proteins with group or single mutations in the C terminus of CXCR2 were performed (Fig. 1A). We found that the GST-CXCR2 carboxyl-terminal mutants GQK313-315A, FRH316-318A, and GLL319-321A bound less PP2A core enzyme than the wild-type GST-CXCR2 carboxyl terminus fusion protein (Fig. 3B). A GST pull-down assay using single mutants revealed that residues Lys315, Arg317, His318, and Leu320 were important for the receptor binding to the PP2A core enzyme (Fig. 3C). These data indicate that the integrity of the sequence motif, KFRHGL, is required for the interaction of CXCR2 with PP2A. This motif is conserved in all CC and CXC chemokine receptors (Fig. 1B) and in some other GPCRs (not shown).
We next examined whether a functional complex consisting of CXCR2 and
the PR65/PP2Ac dimer could be detected in HEK293 cells overexpressing
the receptors using an immunoprecipitation assay. Immunoprecipitation
of CXCR2 from HEK293 cells revealed a weak basal association of the
receptors with the PP2A core enzyme, and CXCL1 (100 ng/ml) treatment
time-dependently increased the association between the
immunoreactive PP2A core enzyme and the receptor, which peaked at 10 min (Fig. 4, A and
B). We also tested the interaction of CXCR2 with PP2A in
human neutrophils. Treatment of the cells with CXCL8 (100 ng/ml) for 10 min significantly increased the association of CXCR2 with the PP2A core
enzyme (Fig. 4C).
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Our previous study has shown that inhibition of CXCR2 internalization
blocks the receptor dephosphorylation (10), suggesting that receptor
internalization is required for the association of the receptors with
PP2A. The internalization of CXCR2 and other chemokine receptors can be
blocked by mutation of the carboxyl-terminal dileucine motifs (26) or
co-transfection of dominant negative dynamin 1 (dynamin 1 K44A) (10,
27). As shown in Fig. 5, A and
B, mutation of Ile323-Leu324 greatly
impaired the receptor binding to the PP2A core enzyme. Overexpression
of dynamin 1 K44A in HEK293 cells stably transfected with CXCR2 also
significantly decreased the interaction of the receptors with the PP2A
core enzyme (Fig. 5, C and D). These data indicate that the receptor internalization is required for the agonist-dependent association of the receptors with
PP2A.
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To test whether agonist-induced phosphorylation is required for the
association of the receptors with the PP2A core enzyme, the
carboxyl-terminal truncated mutant of CXCR2 (331T) was used to
co-immunoprecipitate with the PP2A core enzyme. Because 331T, which no
longer undergoes agonist-induced phosphorylation, still transduces
downstream signaling and undergoes agonist-induced internalization in
HEK293 cells (26), it can be used as a model to investigate the
potential role of phosphorylation in the association of the receptor
with PP2A. Interestingly, compared with wild-type CXCR2, 331T
coimmunoprecipitated an almost equal amount of the PP2A core enzyme in
response to agonist treatment (Fig. 6,
A and B). These data support the hypothesis that
agonist-induced phosphorylation of the receptor is not required for the
receptor binding to PP2A.
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To assess the potential involvement of PP2A in the dephosphorylation of
CXCR2, we used okadaic acid (OA), a potent cell-permeant inhibitor of
PP1 and PP2A (28, 29). As shown in Fig.
7A, exposure of HEK293 cells
expressing CXCR2 to CXCL8 resulted in a robust phosphorylation of the
receptors (Fig. 7A, lane 1). The
phosphorylation was reversed after withdrawal of the agonist followed
by continued incubation for 1 h at 37 °C (Fig. 7A,
lane 2). Treatment of the cells with
concentrations of OA ranging from 0.1 nM to 1 µM inhibited the dephosphorylation of the receptors in a
concentration-dependent fashion (Fig. 7A,
lanes 3-7). A maximal effect was obtained for concentrations of OA equal to or higher than 100 nM.
Quantification of the phosphorylation of CXCR2 indicated that the
IC50 for OA to inhibit the dephosphorylation of CXCR2 was
about 10 nM (Fig. 7B). In addition, OA was also
found to increase the basal level of phosphorylation in the absence of
CXCL8 stimulation (Fig. 7C, compare lane
2 with lane 1). Thus, the results
indicate that a protein phosphatase(s) sensitive to OA, presumably PP2A
and/or PP1, is involved in regulating the phosphorylation state of
CXCR2. Although the inhibition of PP2A has been reported to occur
in vitro at subnanomolar concentrations of OA, whereas that
of PP1 requires 100-fold higher concentration (30), the pharmacological sensitivity of the inhibitor is likely to be quite different in intact
cells. The relatively high concentration of OA (equal to or higher than
100 nM) that is required for a maximal effect might reflect
the abundance of PP2A present in our experimental conditions, i.e. at high cell density, rather than a preference for PP1,
since treatment of the cells with the predominant PP1 inhibitor,
tautomycin (500 nM), did not affect the receptor
dephosphorylation (data not shown).
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To confirm that CXCR2 is dephosphorylated by PP2A, an in vitro phosphorylation and dephosphorylation experiment was performed. HEK293 cells stably expressing CXCR2 were immunoprecipitated with an anti-CXCR2 antibody. The immunoprecipitates were phosphorylated by purified protein kinase C, which has been reported to be capable of phosphorylating CXCR2 (4). The phosphorylated receptors were then incubated with purified PP2A core enzyme. As expected, PP2A dephosphorylated the receptors in a concentration-dependent manner (Fig. 7D).
To assess the functional role of PP2A in the signaling of CXCR2, OA was
used to pretreat HEK293 cells stably expressing CXCR2, and
CXCL8-induced calcium mobilization and chemotaxis were observed. Pretreatment of the cells with OA (100 nM) for 1 h
significantly attenuated CXCR2-mediated calcium mibilization (Fig.
8A) and chemotaxis (Fig.
8B).
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DISCUSSION |
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One of the most important functions of chemokine receptors is to mediate chemotaxis of neutrophils and lymphocytes. The receptor desensitization and resensitization processes are postulated to provide an on-off mechanism for the receptor-mediated chemotaxis (10). Compared with the well established mechanisms for the receptor desensitization (5, 31-34), the mechanisms underlying the receptor resensitization are poorly understood. Dephosphorylation appears to play a key role in the recycling and the subsequent resensitization of the receptors (10). However, little is known about the mechanisms underlying the receptor dephosphorylation. We present evidence in this study that PP2A is involved in the dephosphorylation of the chemokine receptor, CXCR2, by physically interacting with the receptors. It is surprising that CXCR2 only binds to the purified PP2A core enzyme, a dimer composed of the regulatory subunit A (PR65) and the catalytic subunit (PP2Ac), but not the PP2Ac monomer, suggesting that the receptor interacts exclusively with PR65. More interestingly, a binding domain in the C terminus of CXCR2, which is localized upstream of the potential phosphorylation sites and is conserved in all CC and CXC chemokine receptors as well as many other kinds of GPCRs, was identified. Several charged residues in this domain are required for the receptor binding to PP2A. We were unable to test the significance of this binding domain in an in vivo study, since mutation of any residue in this domain seriously impaired the receptor localization on the cell membrane (data not shown). In future experiments, it will be of interest to investigate the binding domain in the sequence of PR65 that is involved in the interaction between PP2A and CXCR2 or other types of GPCRs.
An in vivo interaction of CXCR2 with PP2A was demonstrated
in this study. Agonist treatment induced an association of CXCR2 with
the PP2A core enzyme in a time-dependent manner. The
association peaked at about 10 min and lasted for at least 30 min,
whereas, within the same time frame, most portions of CXCR2 and other
GPCRs become sequestered into endosomes (26, 27, 31, 35, 36). We
previously demonstrated that inhibition of CXCR2 internalization impairs the receptor dephosphorylation (10). In addition, studies of
2-AR indicate that the receptor is dephosphorylated in
endosomal vesicles. It has been shown that
2-ARs in
vesicular fractions are in a less phosphorylated state than are
receptors in the plasma membrane (37). Suppressing the receptor
internalization by internalization-blocking reagents or mutation of the
internalization motifs impairs the receptor dephosphorylation and the
subsequent resensitization (38, 39). Moreover, an increase in endosomal
pH suppresses the receptor dephosphorylation and co-immunoprecipitation
with PP2Ac (19). Based on these data, we sought to investigate the potential requirement of the internalization of CXCR2 for its association with PP2A.
CXCR2 internalization is regulated differentially in a variety of cell
types. The carboxyl-terminal domain of CXCR2, which includes the
phosphorylation domain appears to be required for the receptor
internalization in 3ASubE cells but is not required for internalization
in HEK293 cells (4, 26). Although the underlying mechanisms are not yet
fully understood, it is postulated that different adaptor proteins
present in these cell lines might be responsible for the difference in
CXCR2 internalization (26). Previous studies have demonstrated that
CXCR2 binds to AP-2 and -arrestins, two adaptor proteins involved in
the internalization of CXCR2, through its C-terminal dileucine motifs
and the downstream phosphorylation sites, respectively (26). It has
been suggested that in HEK293 cells, the internalization of the
truncated mutant CXCR2 (331T), which loses all of the C-terminal
phosphorylation sites and fails to bind to
-arrestins, is mediated,
at least in part, by interaction with AP-2 (26). We postulate that the level of AP-2 in 3ASubE cells is not sufficient to mediate the internalization of 331T in the absence of
-arrestin association with
the carboxyl-terminal domain of the receptor. To investigate the
potential role of CXCR2 internalization in the interaction of the
receptor with PP2A, the receptor internalization was blocked by
overexpression of a dominant negative mutant (K44A) of dynamin 1, a
major component of clathrin-coated pits (10), or by mutation of the
dileucine motifs in the receptor C terminus (26). The results
demonstrate that blocking the receptor internalization impaired the
receptor binding to the PP2A core enzyme. These data strongly support
the hypothesis that the internalized CXCR2 associates with PP2A in the endosomes.
We purpose that PP2A binding to the receptors is to reverse the phosphorylated state of the receptors and that agonist-induced phosphorylation of the receptors facilitates the receptor association with PP2A. CXCR2 provides an ideal model to investigate the potential role of phosphorylation in the interaction between GPCRs and PP2A, because truncation of the C-terminal domain containing all of the potential phosphorylation sites totally abolishes agonist-induced phosphorylation of the receptor yet does not affect the receptor/G protein coupling and internalization in HEK293 cells (4, 26, 31). Interestingly, the present data clearly demonstrate that the agonist-induced phosphorylation of CXCR2 is not required for the receptor binding to the PP2A core enzyme, since the C-terminal truncated mutant (331T) of CXCR2 associated with an almost equal amount of PP2A, as compared with the wild-type CXCR2. These data, taken together with the evidence from the GST pull-down assay, indicating that truncation of the C-terminal domain containing the serine/threonine residues did not affect the receptor C terminus binding to the PP2A core enzyme, strongly suggest a modest effect of the receptor phosphorylation in the direct interaction with PP2A. These data support the hypothesis that it is the agonist-induced conformational change and not the phosphorylation of the receptors that facilitates the association of the receptors with PP2A.
The involvement of PP2A in the dephosphorylation of CXCR2 is demonstrated by in vivo and in vitro experiments. OA has been demonstrated to enter cells and inhibit PP2A at low concentrations without affecting PP1, another phosphatase that is only inhibited by higher concentrations of OA (28, 29). In the present study, OA inhibited the dephosphorylation of CXCR2 in a concentration-dependent manner with an IC50 value of about 10 nM. Penetration of OA through the cell membrane is time- and concentration-dependent and is affected by temperature and pH (40). Higher concentrations of OA are needed in some cell types to overcome the high concentration of PP2A in intact cells (41). This may account for the 10 times higher value of the IC50 of OA to inhibit the intracellular PP2A than the IC50 of OA to inhibit purified PP2A activity (42). A maximal inhibitory effect on CXCR2 dephosphorylation was obtained for concentrations of OA equal to or higher than 100 nM, and this concentration of OA has been reported to specifically inhibit intracellular PP2A without affecting PP1 activity in intact cells (43, 44). Moreover, the phosphorylated state of CXCR2 was reversed by purified PP2A core enzyme in a concentration-dependent manner. These data strongly suggest the critical role of PP2A in regulating the phosphorylation of CXCR2.
The functional role of phosphorylation/dephosphorylation in the signaling of CXCR2 and other GPCRs is still not fully understood. Phosphorylation of the receptors in response to agonist stimulation appears to be a key step in termination of the receptor/G protein coupling, which results in desensitization of the receptors (4, 7, 8). Receptor phosphorylation may also play a role in facilitating certain chemokine receptor internalization (4). It is increasingly clear that dephosphorylation is an important step in reestablishing a normal responsiveness after agonist removal. Studies of the C5a receptor have pointed out that internalized C5a receptors are recycled to the plasma membrane with a time course consistent with the kinetics of dephosphorylation, suggesting that dephosphorylation of the C5a receptor might be essential to receptor recycling and resensitization during chemotaxis (22). In view of the basal association of CXCR2 with the PP2A core enzyme in the absence of agonist stimulation and of the higher incorporation of phosphate in the unoccupied CXCR2 after OA treatment, it appears that the basal level of serine/theonine dephosphorylation has to be relatively active to maintain a low state of phosphorylation of unoccupied CXCR2, which is necessary for the normal responsiveness of the receptors. Thus, a fraction, if not all, of the unoccupied CXCR2 appears to undergo a constitutive phosphorylation-dephosphorylation cycle, presumably controlled by protein kinases such as G protein-coupled receptor kinases and/or protein kinase C and by protein phosphatases such as PP2A. Disrupting the phosphorylation-dephosphorylation cycle would affect the receptor signaling. Studies on 3ASubE cells and on RBL-2H3 cells have demonstrated that truncation of the cytoplasmic tail of CXCR2 (331T) prolonged its signaling relative to wild-type CXCR2, increased its resistance to internalization, and induced phospholipase D activation (4, 33). Although 331T undergoes internalization in HEK293 cells, the calcium mobilization is significantly prolonged in response to activation of the mutant receptor (26). On the other hand, pretreatment of the cells with OA results in an increase in the basal level of CXCR2 phosphorylation and significantly attenuates the CXCR2-mediated signaling such as calcium mobilization and chemotaxis. However, we cannot rule out the possibility that other intracellular molecules required for calcium mobilization and chemotaxis might also be affected by OA treatment.
In conclusion, the present study demonstrates for the first time that
the G protein-coupled chemokine receptor, CXCR2, interacts with the
PP2A core enzyme, probably through its C-terminal association with the
regulatory subunit A (PR65) of PP2A. A conserved sequence motif in the
carboxyl-terminal domain, upstream of the serine/threonine residues, is
the potential PP2A binding domain. Agonist-induced increase in the
interaction of the receptors with the PP2A core enzyme is
phosphorylation-independent but internalization-dependent. PP2A is involved in the dephosphorylation of CXCR2 and plays an important role in regulating the receptor signaling.
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ACKNOWLEDGEMENTS |
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We thank Stephen J. Elledge (Baylor College of Medicine) for the generous gift of the yeast two-hybrid screen kit and Brian E. Wadzinski (Vanderbilt University School of Medicine) for generously providing the purified PP2A proteins. We also thank Jinming Yang and Yinchun Yu (our laboratory) and Li Zhang (Department of Microbiology and Immunology, Vanderbilt University Medical Center) for technical help.
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FOOTNOTES |
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* This work was supported by a career scientist grant from the Department of Veterans Affairs (to A. R.), by NCI, National Institutes of Health, Grant CA 34590 (to A. R.), and Vanderbilt-Ingram Cancer Center Grant CA68485.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 Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-7777; Fax: 615-343-4539. Tel.: 615-343-7777; Fax: 615-343-4539; E-mail: ann.richmond@mcmail.vanderbilt.edu.
Published, JBC Papers in Press, February 26, 2001, DOI 10.1074/jbc.M009292200
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ABBREVIATIONS |
---|
The abbreviations used are:
GPCR, G
protein-coupled receptor;
2-AR,
2-adrenergic receptor;
PP, protein phosphatase, HEK293
cells, human embryonic kidney 293 cells;
GST, glutathione
S-transferase;
DMEM, Dulbecco's modified Eagle's
medium;
OA, okadaic acid;
PAGE, polyacrylamide gel
electrophoresis;
RIPA buffer, radioimmune precipitation buffer;
PCR, polymerase chain reaction.
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