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INTRODUCTION |
A general characteristic of inflammatory responses is the
migration of leukocytes from the blood to sites of injury or infection. A number of chemoattractants have been shown to cause the directed migration of leukocytes in vitro and in vivo.
These include complement fragment C5a, formylated bacterial peptides
(fMLP), arachidonic acid metabolites (LTB4), and a group of
low-molecular weight pro-inflammatory cytokines known as chemokines
(1-3). The superfamily of chemokines is subdivided into different
subsets based on the presence and positioning of highly conserved
cysteine residues. The family, based on the configuration of the first
two N-terminal located cysteines, is divided into CC, CXC, and
CX3C subfamilies. All known neutrophil-targeted human
chemokines belonging to the CXC subfamily
(IL-8,1 GRO
, GRO
,
NAP-2, ENA78, and GCP-2) are defined by the presence of a glutamic
acid-leucine-arginine motif (ELR motif) in the portion of the molecule
that lies N-terminal to the first highly conserved cysteine, thus
representing the ELR-CXC chemokine subclass of CXC chemokines
(3-5).
IL-8 and other neutrophil-directed chemokines stimulate neutrophils via
specific seven-transmembrane guanine nucleotide-binding protein-coupled
receptors (GPCRs) (5, 7). The two human IL-8 receptors, CXCR1 and
CXCR2, have 77% overall sequence homology. The two receptor subtypes
differ notably in their N-terminal extracellular domains, as well as in
their C-terminal intracellular domains, and possess differences in
their ligand specificities. CXCR1 displays greater ligand specificity
by binding to IL-8 and GCP-2 with high affinity, whereas CXCR2 binds
with high affinity multiple CXC chemokines in addition to IL-8,
including ENA 78, NAP-2, GRO
, and GRO
(6-9). Binding of the
ligand to high affinity IL-8 receptors initiates a variety of cellular
responses, including calcium translocation, chemotaxis, alterations in
cytoskeletal architecture as well as cellular morphology,
degranulation, and respiratory burst activation (3, 10-12). ELR-CXC
chemokines are produced by a variety of cell types including monocytes,
T lymphocytes, fibroblasts, and endothelial cells (3, 5, 6).
It has been well documented that IL-8 receptors become rapidly
desensitized and internalized upon agonist stimulation (13, 14). The
molecular mechanism(s) and cellular factors required for translocation
of these agonist-occupied receptors from the membrane to cytosolic
compartments are not well characterized. However, the rapid
sequestration and re-expression of CXCR1 (14, 16) is similar to the
well described model of
2-adrenergic receptor
(
2-AR) regulation.
In the case of
2-AR, agonist binding induces a change in
the receptor conformation, which is necessary for the interaction of
the receptor with G protein-coupled receptor kinases (GRKs) (17, 19).
GRK-mediated phosphorylation of the
2-AR C terminus promotes binding of arrestin proteins (
-arrestins) which when bound,
elicit uncoupling of the receptor from its G protein (18-20). Recent
data suggests that the synergistic action of cellular GRKs and
-arrestins determines the kinetics of
2-AR
internalization (21). Moreover, it was demonstrated that
-arrestins
serve as adaptor proteins, specifically targeting agonist-occupied
receptors to clathrin-coated vesicles (CCVs) (19, 20, 22). A critical step in receptor-mediated endocytosis of
2-AR is the
translocation of CCVs saturated with agonist-occupied receptor to the
cytosol, which is a dynamin-regulated event (23). Desensitized
2-ARs, internalized via CCVs, are thought to be
resensitized in the acidified endosomal environment and recycled back
to the cell surface to re-establish normal receptor signaling (24).
In the present work we examined the role of GRK2,
-arrestins, and
dynamin in regulating CXCR1 internalization. For this purpose a
CXCR1-green fluorescent protein (GFP) construct (CXCR1-GFP) was
transiently expressed in human embryonic kidney 293 (HEK 293) and rat
basophilic leukemia 2H3 (RBL-2H3) cell lines. We demonstrate that GRK2,
-arrestins, and dynamin are required for rapid agonist-induced internalization of CXCR1.
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EXPERIMENTAL PROCEDURES |
Materials--
HEK 293 and RBL-2H3 cell lines were obtained from
American Type Culture Collection (Manassas, VA). Dulbecco's modified
essential medium and Eagle's modified essential medium (EMEM) were
purchased from Biowhittaker (Walkerswille, MD). Chemiluminescent
substrates and horseradish peroxidase-coupled donkey anti-rabbit
antibody were purchased from Amersham (Amersham International). The
plasmid containing a variant of a green fluorescence protein (pEGFP-N1) and a green fluorescence protein directed polyclonal antibody were
purchased from CLONTECH (Palo Alto, CA). Human
recombinant interleukin-8 (IL-8) was obtained from R&D Systems
(Minneapolis, MN) and 125I-IL-8 was obtained from Amersham.
Texas Red-transferrin conjugates were purchased from Molecular Probes
(Eugene, OR).
Construction of CXCR1-GFP--
RNA from human neutrophils was
isolated using TriPureTM Isolation Reagent (Roche Molecular
Biochemicals) and CXCR1 cDNA synthesized using
SuperscriptTM II reverse transcriptase (Life Technologies,
Inc.) according to the manufacturer's instructions. The coding
sequence of CXCR1 was amplified using forward
(AAGAGGACATGTCAAATATTACAGAT) and reverse (TTCATCGATGGTTTTCCGAGG)
primers carrying EcoRI restriction enzyme recognition
sequence. Polymerase chain reaction products were 5' and 3' terminal
digested with EcoRI and cloned into pEGFP-N1 cloning vector.
The final construct was sequenced through the region that was generated
by polymerase chain reaction to confirm sequence fidelity.
Cell Cultures and Transfections--
HEK 293 cells were grown in
Dulbecco's modified essential medium whereas RBL-2H3 cells were grown
in EMEM, both containing 10% fetal bovine serum and 1:100 dilution of
penicillin/streptomycin (BioWhittaker) at 37 °C in a humidified
atmosphere of 95% air and 5% CO2. HEK 293 cells were
seeded 12 h prior to transfection in 35-mm glass bottom plates
(MatTek Corp., Ashland, MA) at a density of 2 × 105
cells per dish and RBL-2H3 cells were seeded at a density of 1 × 105 cells per dish 3 h prior to transfection. HEK 293 cells were transiently transfected with 5 µg of CXCR1-GFP and 7.5 µg of pcMV5 rat
-arrestin 1/pcMV5 rat
-arrestin 2, and/or 7.5 µg of pcDNA1-Amp rat GRK2. For RBL-2H3 cells, 3 h prior to
transfection the cells were washed and incubated in serum-free EMEM and
then transiently transfected with 5 µg of CXCR1-GFP alone or along
with 5 µg of pcDNA1-Amp rat
-arrestin 1-V53D or 5 µg of pCB1
rat dynamin I-K44A. Cell lines were transfected with LipofectAMINE
(Life Technologies, Inc.) following the manufacturer's instructions.
Following transfection the cells were maintained in fresh complete
medium for 12 h to recover. For stable transfections, RBL-2H3
cells were grown to 80% confluence in 100-mm dishes (Falcon) and
transfected with 10 µg of CXCR1-GFP in 30 µl of LipofectAMINE.
Three days after transfection, cells were harvested, diluted, and
replated in media supplemented with 1 mg/ml of Geneticin (Life
Technologies, Inc.). The media was replaced every 4 days and stable
transformants were isolated approximately 3 weeks after transfection.
Clonal selection was confirmed by observation of cells under confocal microscope.
Radioligand Sequestration Assay--
RBL-2H3 cells were
subcultured overnight in 6-well plates (1 × 106
cells/well) in complete EMEM. Cells were washed twice with serum-free EMEM containing 1% bovine serum albumin and 25 mM HEPES
(pH 7.2) and preincubated in the same media 1 h prior to
125I-IL-8 treatment. Nontransfected RBL-2H3 and RBL-2H3
cells stably expressing CXCR1-GFP were stimulated with 50 nM 125I-IL-8 (3000 Ci/mmol) at 4 and 37 °C
for 45 min. The reaction was stopped with 1 ml of ice-cold PBS (pH 7.4)
supplemented with 1% bovine serum albumin, the cells were washed
with the same buffer three times and lysed in the lysis buffer (0.5%
Nonidet P-40 and 0.5% Triton X-100 in PBS) on ice for 30 min. After
incubation, the total volume of the well was transferred onto 10%
sucrose, PBS cushion and pelleted at 10,000 rpm for 20 min. Equal
volumes of the supernatant (100 µl) were aliquoted and the amount of
125I-IL-8 in supernatants was measured using a
-counter
(LKB/Wallac, Turku, Finland). Incorporation of nonspecific
radioactivity was determined in supernatants of nontransfected RBL-2H3 cells.
Secretion of
-Hexosaminidase--
Cells were seeded as
described for the "Radioligand Sequestration Assay," washed, and
preincubated in serum-free EMEM for 30 min. Nontransfected and RBL-2H3
cells stably expressing CXCR1-GFP were then stimulated with 50 nM IL-8 for 60 min at 37 °C. The reaction was terminated
by placing 6-well plates on ice for 15 min. The amount of the secreted
-hexosaminidase was determined by incubating 50 µl of the
overlaying medium with 50 µl of 1 mM p-(nitrophenyl)-N-acetyl-
-D-glucosamide
in 0.1 M sodium citrate buffer (pH 4.5) at 37 °C for
1 h. At the end of the incubation 500 µl of a 0.1 M
Na2CO3, NaHCO3 buffer (pH 4.5) was
added and the absorbance was measured at 400 nm.
Confocal Microscopy of Single Cell Time Courses and
Colocalization Studies--
Confocal microscopy was performed on a
Bio-Rad MRC-600 confocal microscope under × 60 oil immersion
objective, using a fluorescein isothiocyanate filter with the emission
wavelength of 488 nm. Transiently transfected HEK 293 and RBL-2H3 cells
were maintained in fresh complete media. For time course studies, the
cells were treated with increasing concentrations of IL-8 (10, 25, 40, 50, and 75 nM) and events following agonist stimulation
were observed in 5-min time intervals up to 90 min post-agonist
stimulation. To determine the effect of de novo protein
synthesis that occurs during the time of observation, RBL-2H3 cells
transiently expressing CXCR1-GFP were first pretreated with
cyclohexamide (10 ng/ml) for 45 min at 37 °C and then stimulated
with 50 nM IL-8. Events following agonist treatment were
observed under confocal microscope.
For colocalization studies, RBL-2H3 cells transiently expressing
CXCR1-GFP were stimulated with 50 nM IL-8 and labeled with Texas Red-transferrin conjugates (15 ng/ml) for 45 min at 37 °C. The
reaction was terminated by washing the cells twice with ice-cold PBS
(pH 7.4). The cells were fixed in 3.6% paraformaldehyde solution and
confocal microscopy was performed as described above.
Subcellular Cell Fractionation--
HEK 293 cells transiently
expressing CXCR1-GFP, CXCR1-GFP, and GRK2, and CXCR1-GFP, GRK2, and
-arrestin 1 were stimulated with 50 nM IL-8 for 45 min
at 37 °C. The cells were washed twice with ice-cold PBS (pH 7.4),
removed from plates by gentle washing, and pelleted at 100 rpm for 10 min. The cell pellet was resuspended in 3 ml of buffer A (10 mM Tris-HCl, pH 7.4, 2 mM EDTA), incubated on
ice for 30 min and homogenized using a Dounce homogenizer. Nuclei were
removed by centrifugation at 200 rpm for 10 min. The supernatant was
loaded on a stepwise sucrose cushion (35 and 5% sucrose in PBS) and
centrifuged at 35,000 rpm for 90 min at 4 °C. The supernatant was
removed and the 35% sucrose interface fraction containing endosomes
(the light vesicular fraction) was collected, diluted in buffer A, and
re-centrifuged at 35,000 rpm for 45 min at 4 °C. The pellets were
resuspended in 100 µl of buffer A containing 2 × SDS sample
buffer and 100 µg of each protein sample was loaded onto
SDS-polyacrylamide gel electrophoresis.
Protein Determination--
Protein levels in the whole cell
lysates of HEK 293 and RBL-2H3 cells were determined using Bio-Rad
protein assay (Richmond, CA) with bovine serum albumin as a standard.
Western Blotting--
Expression levels of
-arrestin 1,
-arrestin 2, and GRK 2 in HEK 293 and RBL-2H3 cells were examined
using specific polyclonal antisera as described previously (20).
Equivalent amounts (100 µg) of total cell protein were separated on a
10% polyacrylamide gel and transferred onto nitrocellulose membrane
(Bio-Rad). The endogenous amounts of
-arrestin 1,
-arrestin 2, and GRK 2 were determined using anti-
-arrestin 2 and anti-GRK2
rabbit polyclonal sera at a dilution of 1:2500 and horseradish
peroxidase-conjugated anti-rabbit secondary antibodies using the ECL
system (Amersham) according to manufacturer's instructions. The amount
of total
-arrestin 1,
-arrestin 2, and GRK 2 in RBL-2H3 cells
were determined relative to their respective endogenous expression in
HEK 293 cells. Amounts of CXCR1-GFP in the light vesicular
subcellular fraction were determined using GFP-directed polyclonal
antibody at dilution 1:1000 (CLONTECH).
Statistical Analysis of the Sequestration Data--
The relative
membrane and cytosol luminosity was measured using SigmaScan Pro
software. Data was statistically analyzed and plotted using Microsoft
Excel software. Results are the average ± S.D. from three
separate identical experiments.
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RESULTS |
Agonist-promoted Internalization of CXCR1-GFP in HEK 293 and
RBL-2H3 Cells--
Cells transfected with CXCR1-GFP were positive for
fusion protein expression within 24-36 h post-transfection as
evidenced by robust membrane fluorescence in 15% of the RBL-2H3 and
70% of the HEK 293 cells visualized by confocal microscopy. In some transfected cells under nonstimulated conditions the CXCR1-GFP conjugate accumulated in the Golgi apparatus. Dose-response experiments indicated that maximal internalization of CXCR1-GFP occurred in a range
of 40-75 nM IL-8. Over a 45-min time period,
agonist-occupied CXCR1-GFP conjugates carried in specific
membrane-associated vesicles, gradually translocated from the plasma
membrane to the cytosol (Fig.
1A, i).
Sequestration data acquired in RBL-2H3 cells showed a mean decrease of
61% in membrane luminosity and a 4.2-fold increase in cytosol
fluorescence intensity in response to agonist stimulation over the
45-min time period (Fig. 1B). Although there was a
significant decrease of membrane luminosity in response to IL-8
treatment in RBL-2H3 cells transiently expressing the CXCR1-GFP
construct not all of the expressed receptor was internalized 45 min
post-stimulation. Pretreatment of RBL-2H3 cells transiently expressing
the CXCR1-GFP fusion construct with cyclohexamide resulted in rapid
CXCR1-GFP sequestration with no membrane fluorescence after 45 min
post-stimulation, indicating that residual membrane fluorescence was
due to de novo synthesis of CXCR1-GFP conjugates (data not
shown). Unstimulated cells showed very little redistribution of
CXCR1-GFP over the same time frame (Fig. 1A, ii).
Stably transfected CXCR1-GFP cells internalized 125I-IL-8
at 37 °C whereas at 4 °C, CXCR1-GFP transfected cells failed to
internalize 125I-IL-8 (Fig. 1C), indicating that
internalization of CXCR1-GFP is an agonist and
temperature-dependent process, which is similar to IL-8
receptor internalization observed in neutrophils (14). To assess
whether CXCR1-GFP transduced functional responses, we performed
-hexosaminidase assays on IL-8 stimulated and unstimulated CXCR1-GFP
stably transfected cells.

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Fig. 1.
IL-8 induces internalization of CXCR1-GFP in
RBL-2H3 cells. A, RBL-2H3 cells transiently expressing
CXCR1-GFP fusion protein were treated with saturating concentrations of
IL-8 (50 nM) (i) or unstimulated
(ii). Events following agonist addition were observed using
a confocal microscope. A representative single cell time courses are
shown. B, the relative membrane and cytosolic luminosity of
RBL-2H3 cells overexpressing CXCR1-GFP was plotted versus
time prior to and post-agonist stimulation for n = 3 (± S.D.) experiments. , membrane; , cytoplasm. C,
detection of 125I-IL-8 binding was performed as described
under "Experimental Procedures." 125I-IL-8 accumulation
in control RBL-2H3 cells was compared with 125I-IL-8
accumulation in CXCR1-GFP expressing RBL-2H3 cells at 4 and 37 °C.
*, represents statistical significance (p < 0.05)
using one-way ANOVA as compared with control nontransfected RBL-2H3
cells. , RBL-2H3 cells nontransfected; , RBL-2H3 cells CXCR1-GFP
transfected (4 °C); , RBL-2H3 cells CXCR1-GFP transfected
(37 °C). D, the release of -hexosaminidase was
performed as described under "Experimental Procedures." Data are
represented as percentage of total -hexosaminidase in the cell. *,
represents statistical significance (p < 0.05) using
one-way ANOVA as compared with group 1 (RBL-2H3 cells nonstimulated).
Lanes indicate RBL cells: 1, nonstimulated; 2,
IL-8 stimulated; 3, MCP-1 stimulated; 4,
CXCR1-GFP transfected, nonstimulated; 5, CXCR1-GFP
transfected, IL-8 stimulated; 6, CXCR1-GFP transfected,
MCP-1 stimulated.
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IL-8 stimulation of CXCR1-GFP transfected cells resulted in a 13.4%
release of hexosaminidase compared with a 4.8% release from
untransfected RBL-2H3 cells stimulated with IL-8 (Fig. 1D). Stimulation with MCP-1, a CC chemokine that does not bind CXCR1, did
not induce hexosaminidase release. These results show that CXCR1-GFP
expressed in RBL-2H3 cells retains several features of the wild type
receptor expressed in neutrophils; the receptor can transduce signals
that result in granule release, undergo agonist-induced
internalization, and sequester IL-8.
In contrast to RBL-2H3 cells, HEK 293 cells transiently expressing the
fusion protein construct did not internalize CXCR1-GFP when stimulated
with IL-8 (Fig. 2A,
i). Since previous studies (30) have demonstrated that HEK
293 cells require increased expression of
-arrestins and GRKs for
internalization of some GPCRs we explored whether co-expression of
these two classes of molecules with CXCR1-GFP could restore
agonist-induced receptor internalization in HEK 293 cells.

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Fig. 2.
Visualization and statistical determination
of CXCR1-GFP internalization in HEK 293 cells. A, HEK
293 cells are shown transiently expressing CXCR1-GFP alone
(i), CXCR1-GFP and GRK2 (ii), CXCR1-GFP, and
-arrestin 1 (iii), and CXCR1-GFP in combination with
-arrestin 1 (iv), -arrestin 2 (v) and GRK2.
Cells were stimulated with IL-8 (50 nM) and observed using
a confocal microscope. B, the relative membrane and
cytosolic luminosity for HEK 293 cells expressing CXCR1-GFP,
-arrestin 2 and GRK2 was plotted versus time prior to and
post-agonist treatment for n = 3 (± S.D.) experiments.
, membrane; , cytoplasm.
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Co-expression of CXCR1-GFP with either GRK2 (Fig. 2A,
ii) or
-arrestin 1 alone (Fig. 2A,
iii) in HEK 293 cells failed to facilitate IL-8 induced
CXCR1-GFP internalization. However, expression of CXCR1-GFP with GRK2
and
-arrestins together resulted in IL-8-induced receptor
internalization (Fig. 2A, iv) showing a 3.5-fold
increase of cytosolic fluorescence and a 60% decrease in membrane
fluorescence (Fig. 2B). The increased cytosolic receptor
fluorescence was associated with increased receptor labeling of the
intracellular vesicles. Similar results were obtained with
-arrestin
2, another closely related member of the
-arrestin family, again
co-expressed with GRK2 and CXCR1-GFP (Fig. 2A,
v). These results suggest that both GRKs and
-arrestins
are required for CXCR1 internalization. Western analysis of GRK2,
-arrestin 1, and
-arrestin 2 from HEK 293 cells and RBL-2H3 cells
(Fig. 3, A and B),
shows a substantial difference in
-arrestin 1,
-arrestin 2, and
GRK2 expression between HEK 293 cells and RBL-2H3 cells. The higher
levels of GRK2 and
-arrestin 2 expression in RBL-2H3 cells likely
explain why CXCR1-GFP undergoes agonist-induced internalization in
RBL-2H3 cells without the requirement for co-expression with GRKs or
-arrestins.

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Fig. 3.
Expression of
-arrestins and GRK2 in RBL-2H3 and HEK 293 cells. A, the amount of endogenously expressed
-arrestin proteins in HEK 293 and RBL-2H3 cells was detected using a
specific anti- -arrestin 2 antibody in Western blot analysis.
Overexpressed -arrestin 1 in HEK 293 cells (lane 3) is
shown as a control. Overexpressed -arrestin 1 runs slower than
endogenously expressed -arrestin 2 in HEK 293 (lane 1)
and RBL-2H3 (lane 3) cells. B, shows endogenous
cellular amounts of GRK2 in HEK 293 (lane 2) and RBL-2H3
(lane 3) cells. Lane 1 indicates the size of
purified GRK2 (100 µg) that was run as a control.
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Inhibition of CXCR1-GFP Sequestration in RBL-2H3 Cells by
Overexpression of
-Arrestin 1-V53D and Dynamin I-K44A
Mutants--
To explore the role of
-arrestins in CXCR1-GFP
internalization in RBL-2H3 cells we co-expressed CXCR1-GFP along with
the
-arrestin 1-V53D dominant negative mutant in RBL-2H3 cells and stimulated with IL-8. In the presence of
-arrestin 1-V53D there was
no redistribution of membrane fluorescence that followed IL-8 stimulation (Fig. 4, A,
ii, and B). This was in sharp contrast to cells
expressing CXCR1-GFP alone (Fig. 4A, i) or cells
expressing CXCR1-GFP and wild type
-arrestin 1 (data not shown),
which showed marked receptor internalization following IL-8
stimulation. These observations complement the results obtained with
HEK 293 cells and clearly demonstrate a role of
-arrestins in
agonist-induced CXCR1 internalization.

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Fig. 4.
Effects of -arrestin
1-V53D and dynamin I-K44A mutants overexpression on the
agonist-promoted internalization of CXCR1-GFP conjugate in RBL-2H3
cells. A, RBL-2H3 cells transiently expressing
CXCR1-GFP alone (i), CXCR1-GFP and -arrestin 1-V53D
(ii), or dynamin I-K44A mutant (iii). Cells were
stimulated with IL-8 (50 nM) and observed using a confocal
microscope. B, the relative membrane and cytosolic
luminosity of RBL-2H3 cells overexpressing CXCR1-GFP and -arrestin
1-V53D mutant and (C) RBL-2H3 cells overexpressing CXCR1-GFP
and dynamin I-K44A mutant was plotted versus time prior to
and post-agonist treatment for n = 3 (± S.D.)
experiments.
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-Arrestins are thought to act as scaffolding proteins in coupling
GPCRs to CCVs (22, 24-26). Agonist stimulation promotes the formation
of CXCR1-GFP containing vesicles, which are pinched off from the plasma
membrane and translocated into post-endocytic compartments (19, 24,
26). The pinching or sealing off of the vesicles from the plasma
membrane is dependent upon dynamin, a GTPase containing molecule
(27-29). The dominant negative dynamin I-K44A mutant has been utilized
in determining whether GPCRs are internalized via a
dynamin-dependent pathway involving CCVs. We explored
whether CXCR1 required dynamin for agonist-induced receptor internalization by co-expressing CXCR1-GFP with the dynamin I-K44A dominant negative mutant. The expression of dynamin I-K44A successfully blocked redistribution of CXCR1-GFP from the membrane to the cytosol. Vesicles formed in cells expressing the dynamin I-K44A mutant simply
did not pinch off from the inner surface of the plasma membrane (Fig.
4A, iii, 45 min). These results indicate that
agonist-induced internalization of CXCR1 occurs via CCVs and requires
functional
-arrestins and dynamin molecules.
Agonist-induced Colocalization of Transferrin and CXCR1-GFP in
Endosomes and the Presence of CXCR1-GFP Conjugates in the Light
Vesicular Subcellular Fraction--
To further investigate and confirm
the identity of membrane-derived vesicles that translocate
agonist-occupied CXCR1-GFP from the membrane to post-endocytic
compartments, we labeled RBL-2H3 cells transiently expressing the
receptor-GFP construct with a Texas Red-transferrin conjugate.
Transferrin has been shown to undergo receptor-mediated endocytosis
through CCVs upon binding to its cognate transferrin receptor and it
has been described as a significant endosomal marker (37, 38). Agonist
stimulation promoted colocalization of CXCR1-GFP and dye-labeled
transferrin conjugate within CCVs (Fig. 5,
ii) whereas unstimulated
RBL-2H3 cells transiently expressing CXCR1-GFP did not display any
colocalization (Fig. 5, i). These results were further
supported by isolation of the light vesicular (endosomal) subcellular
fraction from transiently transfected HEK 293 cells expressing
CXCR1-GFP and CXCR1-GFP, GRK2, and
-arrestin 1 that were stimulated
or unstimulated with IL-8. A 9.5-fold increase in CXCR1-GFP was found
in the light vesicular subcellular fraction isolated from IL-8
stimulated HEK 293 cells expressing CXCR1-GFP, GRK2, and
-arrestin 1 (Fig. 6, lane 4 versus
lane 2). However, only a modest increase (3-fold) in
CXCR1-GFP was found in the light vesicular subcellular fraction isolated from HEK 293 cells in the absence of transfected GRK2 and
-arrestin 1 (Fig. 6, lane 3 versus lane 1) indicating
that GRK2 and
-arrestin 1 substantially enhance the efficiency of CXCR1 internalization. Together these results support a model for CXCR1
sequestration via clathrin-coated pits that are regulated by GRKs,
-arrestins, and dynamin.

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Fig. 5.
IL-8 stimulates CXCR1-GFP and transferrin
colocalization in endosomes of RBL-2H3 cells. RBL-2H3 cells
transiently expressing CXCR1-GFP fusion protein were stained with Texas
Red-transferrin conjugate and samples processed as described under
"Experimental Procedures." Unstimulated conditions (i)
and agonist-induced CXCR1-GFP-transferrin endosomal colocalization
(ii) are shown. Areas of colocalization are indicated
yellow.
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Fig. 6.
Presence of high CXCR1-GFP amounts in the
endosomal subcellular fraction isolated from HEK 293 cells transiently
expressing CXCR1-GFP, GRK2, and -arrestin
1. A, amounts of CXCR1-GFP in light vesicular
subcellular fractions isolated from HEK 293 cells transiently
expressing CXCR1-GFP (lanes 1 and 3) and
CXCR1-GFP, GRK2, and -arrestin 1 (lanes 2 and
4) pre- and post-stimulation were detected using
GFP-directed polyclonal antibody.
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DISCUSSION |
Agonist-dependent regulation of chemokine receptor
desensitization, internalization, and sequestration is an important
mechanism for regulating leukocyte responsiveness to chemokine
stimulation. Several studies have demonstrated that exposure of
neutrophils to high concentrations of ELR-CXC chemokines renders the
exposed neutrophils unresponsive to additional homologous chemokine
stimulation. The refractory state of neutrophils following stimulation
with high chemokine concentrations appears to be dependent upon
desensitization, internalization, and sequestration of CXCR1, CXCR2, or
both IL-8 receptor subtypes. Initial studies using radiolabeled IL-8
showed that IL-8-binding sites were rapidly lost from the neutrophil surface following stimulation of high concentrations of IL-8 (13). These initial studies were later confirmed by additional work utilizing
monoclonal antibodies directed at the external domains of CXCR1 and
CXCR2, that demonstrated a rapid loss of CXCR1 and CXCR2 following
stimulation of neutrophils with high concentrations of ELR-CXC
chemokines (14, 33).
Recent studies by Richardson et al. (32) have demonstrated
that phosphorylation of critical serine residues in the C-terminal region of CXCR1 is important for internalization of the receptor following agonist stimulation. Our work here compliments these observations by demonstrating that GRK2 is necessary for
internalization of CXCR1 in HEK 293 cells (Fig. 2A,
iv and v). GRK2 is a serine-threonine kinase and
a member of a multigene family whose members regulate GPCR
function and internalization by phosphorylating serine/threonine residues located within the cytoplasmic regions of various receptors (17). GRK2 is abundantly expressed in human peripheral leukocytes (Ref.
31 and data not shown) and may represent the endogenous kinase
responsible for CXCR1 phosphorylation in neutrophils. Alternatively one
of the other members of the GRK family (GRK1 and 3-5) may serve the
same function in regulating phosphorylation of CXCR1 in neutrophils.
CCR5, a member of the CC chemokine family of receptors (4), is
preferentially phosphorylated by GRK2 and GRK3 indicating that GRK
phosphorylation likely represents a common feature of both CC and CXC
chemokine receptor regulation (30).
While GRK phosphorylation represents a critical step in regulating
the desensitization and internalization of a subset of GPCRs, it is the
-arrestin proteins which facilitate the translocation of GPCRs from
the plasma membrane to CCVs. Our data in the present study places
-arrestins as central regulators of CXCR1 internalization in
response to agonist stimulation. This has been substantiated using two
separate cells lines displaying two different phenotypes. HEK 293 cells
which have low expression of
-arrestins require expression of
-arrestin 1 or
-arrestin 2 for agonist induced internalization.
In sharp contrast to HEK 293 cells, RBL-2H3 cells express higher levels
of
-arrestins and do not require additional expression of
-arrestins for CXCR1 internalization (Fig. 3A). However,
agonist-induced internalization of CXCR1 could be blocked by
co-expressing the dominant negative
-arrestin 1-V53D mutant in
RBL-2H3 cells (Fig. 4, A, ii, and B).
These experiments provide strong evidence for
-arrestin regulation
of agonist-induced CXCR1 internalization.
Additionally, it is also clear from our studies that cellular factors
other than GRK2 and
-arrestin are involved in the CXCR1 internalization machinery. Dynamins have been previously described as
key proteins involved in the pinching off or sealing of CCVs from the
membrane by stimulating GTP/GDP exchange which facilitates endocytic
vesicle release (27, 28). In contrast to the angiotensin II type 1A
receptor, which is able to undergo dynamin-independent endocytosis
(23), our studies indicate that in the presence of dynamin I-K44A
mutant, CCVs saturated with agonist-occupied receptor are not released
from the membrane into the cytosolic compartment (Fig. 4A,
iii). Thus CXCR1 appears to undergo internalization and
sequestration through a dynamin-driven and
clathrin-dependent internalization pathway similar to
several other GPCRs (20, 26, 30). This is supported by two additional
pieces of data, agonist-promoted colocalization of transferrin and
CXCR1-GFP in endosomal vesicles of RBL-2H3 cells (Fig. 5) and
redistribution of CXCR1-GFP to the light vesicular fraction following
IL-8 stimulation in HEK 293 cells transiently expressing CXCR1-GFP,
GRK2, and
-arrestin 1 (Fig. 6). Even though CXCR1 and CXCR2 display
77% amino acid identity and elicit several similar functional
responses they appear to have divergent pathways for internalization
and recycling. Chuntharapai et al. (14) showed that CXCR1
but not CXCR2 was recycled back to the plasma membrane following
agonist-induced internalization. The mechanism for the divergence of
CXCR1 and CXCR2 recycling is presently unknown, although differences in the C-terminal region may play a role in how the two proteins undergo
intracellular trafficking (15). In this context CXCR1 and CXCR2 may
have differential requirements for
-arrestin proteins, which target
CXCR1 and CXCR2 to different intracellular compartments. Preliminary
data in our laboratory suggests that CXCR2 internalization is regulated
differently from CXCR1 in HEK293 cells. The precise role of
-arrestins in regulating CXCR1 and CXCR2 recycling awaits further
investigations. It is interesting to note that
-arrestins can
function as signaling molecules since studies have implicated a role
for
-arrestins in the activation of tyrosine kinases (36). Thus, it
is conceivable that some of the functional responses elicited by CXCR1
are due to signals transduced by
-arrestins coupling to the CXCR1 receptor.
While there are at least five independent mechanisms for endocytotic
internalization including the clathrin- and non-clathrin-coated pits,
micropinocytosis, caveolae, and phagocytosis, GPCRs appear to utilize
only two: clathrin-dependent and dynamin-independent endocytotic pathways (23, 34, 35). We present here a previously undescribed model for CXCR1 chemokine receptor internalization whereby
we have demonstrated that GRK2,
-arrestin, and dynamin are necessary
molecules for the entry of CXCR1 into the cell. Upon IL-8 binding, GRK2
phosphorylates C-terminal serine-threonine residues on CXCR1 allowing
-arrestins to couple phosphorylated receptor to cytoplasmic
complexes containing clathrin. Furthermore, our data demonstrates that
dynamin is required to pinch off CCVs containing CXCR1 and allow
vesicular entry of the activated receptor into the cell. The importance
of chemokine receptor internalization may be to serve as a mechanism of
reducing the chemotactic activity of leukocytes under conditions of
high exposure to inflammatory stimuli thereby preventing their
continued migration and departure from the site of inflammation. These
studies provide insight into the biochemical factors involved in
chemokine receptor entry into the cell and thus may further our
understanding of the inflammatory process.