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INTRODUCTION |
Melanoma growth-stimulatory activity
(MGSA)1/growth-regulatory
protein (GRO), interleukin-8 (IL-8), neutrophil-activating peptide-2, epithelial derived neutrophil-activating peptide 78, and granulocyte chemotactic protein-2 are members of a family of structurally related
cytokines that induce chemotaxis and respiratory burst in neutrophils
(1-3). These chemokines belong to the CXC chemokine subfamily in which
the first two conserved cysteine residues are separated by an
intervening amino acid (4). Several chemokine receptors have been
cloned that bind MGSA: CXCR2 (formerly called IL-8 RB) (5), Duffy
antigen receptor for chemokine, and two herpesvirus-encoded receptors:
the herpesvirus saimiri ECRF-3 and the human herpesvirus-8 G
protein-coupled receptor (6). IL-8 binds to these four receptors as
well as CXCR1 (formerly called IL-8 RA) (7), which is a receptor for
IL-8 and granulocyte chemotactic protein-2. CXCR2 is a shared receptor
that binds to IL-8, MGSA, neutrophil-activating peptide-2,
epithelial derived neutrophil-activating peptide 78, and
granulocyte chemotactic protein-2 (8).
Like all of the chemokine receptors cloned to date, CXCR2 is a member
of a superfamily of G protein-coupled receptors (GPCRs) that transduce
signals to the interior of the cell through heterotrimeric guanine
nucleotide-binding proteins (G proteins). These receptors share a
common putative structural topology composed of seven-transmembrane domains separated by three extracellular and three intracellular loops.
Upon agonist binding, CXCR2 activates G protein-mediated phosphoinositide hydrolysis to generate diacylglycerol and inositol 1,4,5-trisphosphate, thereby activating protein kinase C and mobilizing Ca2+ to initiate a variety of cellular responses (9).
Receptor activation is followed by receptor phosphorylation on multiple serine residues and subsequent desensitization of the receptor to
further stimulations. These events are usually accompanied by receptor
endocytosis and/or recycling of the receptor (10).
A large body of evidence suggests that the major route of sequestration
of GPCRs is via clathrin-coated pits and into early endosomes (11). The
2-adrenergic receptor (
2-AR), a
prototypical GPCR, becomes phosphorylated upon ligand binding and then
exhibits an increased binding affinity for the adaptor molecules
(either
-arrestin or arrestin 3), which prevent further coupling
between the receptor and G proteins (12, 13). The arrestins are
subsequently involved in
2-AR sequestration by
specifically binding to clathrin, a major component of clathrin-coated
pits (14, 15). It has been suggested that sequestration of GPCRs to
early endosomes and lysosomes might be responsible for receptor
down-regulation and resensitization (16, 17). Within the acidic
environment of the endosomes and lysosomes, the receptors either
undergo dephosphorylation and recycle back to the cell surface and thus
are resensitized or are degraded and down-regulated (11).
At present, much of the information concerning the molecular
mechanisms for GPCR sequestration and down-regulation is derived from
the studies of
2-AR. Due to the enormous number of GPCRs and the diverse cellular function evoked by each individual receptor, it is conceivable that multiple mechanisms may be employed for receptor
sequestration, down-regulation, and resensitization. This is
exemplified by the finding that while clathrin-coated pits are
essential for agonist-promoted
2-AR sequestration,
sequestration of the angiotensin II type 1A receptor does not require
intact function of clathrin-coated pits (18).
In contrast to the
2-AR, little is known regarding the
sequestration, down-regulation, and resensitization mechanisms of chemokine receptors and their roles in mediating cell chemotaxis, which
is a distinct function for chemokine receptors. In order to chemotax
along a gradient of chemotactic cytokine, the cells may employ an
on-off mechanism in which the chemokine receptors undergo a
continuous desensitization and resensitization process. The present
studies were focused to delineate the sequestration and down-regulation
mechanism of CXCR2. To accomplish this, we transiently expressed the
CXCR2 in HEK293 cells and examined the effects of co-expression of the
dynamin I K44A mutant on receptor sequestration, down-regulation, and
resensitization. We also used ligand-mediated MAP kinase activation and
chemotaxis as functional assays to examine the effects of blocking the
endocytosis of CXCR2 on its signal transduction events. The results
demonstrate that clathrin-coated pits are primary routes for CXCR2
sequestration and down-regulation. Blocking the endocytosis of CXCR2
does not affect downstream MAP kinase activation. However, it
does severely attenuate ligand-mediated chemotaxis.
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EXPERIMENTAL PROCEDURES |
Cell Culture and Transfections--
293 human embryonic kidney
cells (HEK293) were cultured in Dulbecco's modified Eagle's medium
supplemented with 10% fetal bovine serum. HEK293 cells cultured to
80% confluence were transfected with the CXCR2 cDNA in the
pRc/cytomegalovirus vector and the dynamin I wild-type or K44A mutant
in the pBJ vector using the LipofectAMINE PLUS reagent (Life
Technologies, Inc.) according to the manufacturer's recommendation.
Before each experiment, an indirect immunfluorescence assay was used to
assess the transfection efficiency. The transfection efficiency was
approximately 80% in each experiment.
Receptor Internalization Assay--
The acid/buffer wash
technique (19) was used to determine the kinetics of MGSA/GRO-induced
internalization of CXCR2.
In Vivo Phosporylation and Western Blot Analysis--
Receptor
phosphorylation assays were performed as described previously (20). In
brief, the transfected cells were plated 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 then labeled by
incubating in [32P]orthophosphate (100 µCi/ml) in the
same medium at 37 °C for 2 h. Cells were then stimulated with
or without 100 ng/ml MGSA/GRO for 10 min. Dephosphorylation was
performed by allowing cells to recover in fresh serum-free media
without ligand at 37 °C for 1 h. The cells were then lysed in
RIPA buffer containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton
X-100, 10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. CXCR2 was
immunoprecipitated with a polyclonal rabbit anti-CXCR2 antibody (20)
from cleared supernatants containing approximately 300 µg of protein
(estimated by the BCA method; Pierce). 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, and the total amount of receptor
was determined by Western blotting using a monoclonal antibody against
CXCR2 to ensure equal loading.
Indirect Immunofluorescence--
HEK293 cells stably expressing
CXCR2 (10) were grown on 10-mm coverslips (Fisher). To compare the
distribution of CXCR2 and the transferrin receptor, cells were washed
once with serum-free medium and then incubated at 37 °C with the
same medium containing 100 µg/ml of fluorescein
isothiocyanate-conjugated transferrin (Molecular Probes, Inc., Eugene,
OR) for 2 h. The cells were washed and then chased for 30 min.
Following MGSA/GRO or IL-8 (100 ng/ml) treatment for the indicated time
periods, cells were processed for indirect immunofluorescence to detect
the subcellular localization of CXCR2 (19).
Receptor Down-regulation Assay--
Cells were collected by
trypsinization 24 h after transfection and plated onto 12-well
plates. The next day, the cells were treated with or without 100 ng/ml
MGSA/GRO for 0.5-8 h. Cells were lysed in 0.5 ml/well RIPA buffer with
proteinase and phosphatase inhibitors described as above. Aliquots of
lysates containing 30 µg of protein were subjected to electrophoresis
(10% SDS-polyacrylamide gel electrophoresis) and then transferred to
nitrocellulose membrane. The amount of CXCR2 was detected by Western
blot using the previously described rabbit anti-CXCR2 antibody.
MAP Kinase Assay--
Agonist-treated cells were lysed by RIPA
buffer. Lysates containing equal amounts of protein were subjected to
SDS-polyacrylamide gel electrophoresis. Phosphorylated MAP kinase
(ERK1/ERK2) was detected by a phosphospecific MAP kinase antibody
(Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Chemotaxis Assay--
Chemotaxis assays were performed on
transiently transfected HEK293 cells as described previously (10) using
a 96-well chemotaxis chamber (Neuroprobe Inc.).
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RESULTS |
Redistribution of CXCR2 to Endosomes following MGSA/GRO
Treatment--
Immunofluoresence microscopy was used to define the
subcellular distribution of CXCR2 in stably transfected HEK293 cells
using rabbit anti-CXCR2 polyclonal antibody. The localization of
endosomes was detected using the transferrin-fluorescein isothiocyanate complex as described previously. The majority of CXCR2 was targeted to
the plasma membrane in untreated cells (Fig.
1A). A small portion of CXCR2
was located intracellularly as punctate structures that colocalized
with the transferrin receptor (Fig. 1, A and B).
Following MGSA/GRO (100 ng/ml) treatment for 3 h, agonist
stimulation resulted in sequestration of CXCR2, observable as a loss of
cell surface immunofluorescence with a concomitant increase in the
number of intracellular punctate structures. The distribution of the
punctate structures overlapped extensively with those of the
transferrin receptor (Fig. 1, C and D),
suggesting that agonist stimulation enhances CXCR2 redistribution into
endosomes. This phenomenon was observable as early as 10 min with
MGSA/GRO treatment (data not shown). IL-8 treatment induced a similar
redistribution of CXCR2 to the endosomes (data not shown).

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Fig. 1.
Colocalization of CXCR2 with the
transferrin receptor. HEK293 cells expressing CXCR2 were incubated
with fluorescein isothiocyanate-conjugated transferrin for 2 h,
washed, and chased for 30 min. The cells were treated with 100 ng/ml
MGSA/GRO for 0 min (A, B) or 3 h
(C, D). The cells were then fixed, permeablized,
and stained for CXCR2 with a rabbit anti-CXCR2 antibody followed by
rhodamine-conjugated goat anti-rabbit antibody (A,
C). The emission of fluorescein isothiocyanate-transferrin
is shown in B and D. The arrows
indicate the examples of co-localization of CXCR2 and transferrin
receptor. Images were processed using Photoshop software.
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Effect of Dynamin I K44A on CXCR2 Endocytosis--
It has been
shown previously that CXCR2 undergoes a rapid endocytosis within
minutes in response to either MGSA/GRO or IL-8 stimulation. To
determine whether the agonist-promoted CXCR2 endocytosis is a
clathrin-mediated process, HEK293 cells were transiently co-transfected
with cDNAs encoding CXCR2 and dynamin I K44A. Agonist-promoted endocytosis was determined and compared with that observed in cells
transfected only with CXCR2 or cells co-transfected with CXCR2 and
wild-type dynamin I. Cells expressing only CXCR2 showed rapid receptor
internalization after MGSA/GRO treatment (Fig. 2). The MGSA/GRO-stimulated sequestration
of CXCR2 occurred in a time-dependent manner. The
percentage of endocytosed receptor increased progressively from 20% at
1 min to approximately 60% at 20 min. Co-expression of wild-type
dynamin I with CXCR2 seemed neither to promote nor inhibit the rate of
CXCR2 endocytosis. In contrast, in cells co-expressing CXCR2 and
dynamin I K44A, there was a profound inhibition of the
MGSA/GRO-promoted CXCR2 endocytosis, with less than 15% of receptor
endocytosed at 20 min. These data clearly indicate that
agonist-promoted endocytosis of CXCR2 depends on functional expression
of dynamin I and thus is a clathrin-mediated process. This is further
supported by the fact that internalized CXCR2 colocalized with the
transferrin receptor. Based on ligand binding data, we observed similar
CXCR2 cell surface expression level in cells transfected with CXCR2 and
dynamin I K44A as compared with those transfected with only CXCR2. We
also observed a similar effect on receptor endocytosis when the cells
were treated with IL-8 (data not shown).

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Fig. 2.
The effect of the wild-type and K44A mutant
dynamin I on the MGSA/GRO-stimulated CXCR2 internalization. HEK293
cells were transiently transfected with plasmids expressing CXCR2 with
pBluescript (control) (dotted bars), rat dynamin
I (striped bars), or rat dynamin I K44A
(black bars). Two days after transfection, the
cells were preincubated with binding buffer containing the
125I-MGSA/GRO for 30 min at 4 °C. Unbound
125I-MGSA/GRO was removed by washing at 4 °C. The cells
were warmed to 37 °C for the indicated time period (also see
"Expermental Procedures"). 125I-MGSA/GRO remaining at
the cell surface was removed with acetic acid (0.2 M, pH
2.5) containing 0.5 M NaCl, and the internalized
125I-MGSA/GRO was then quantitated on a -counter. Data
are presented as mean ± S.E. from three independent experiments.
Binding to duplicate samples varied generally by less than 10%.
The data were analyzed using Student's paired t test (*,
p < 0.05; **, p < 0.005 (compared
with control cells transfected with CXCR2 only)).
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The Effect of Dynamin K44A on CXCR2 Phosphorylation and
Dephosphorylation--
We have previously shown that CXCR2 undergoes a
rapid phosphorylation and desensitization after MGSA/GRO treatment
(10). To further investigate the effects of the inhibition of receptor endocytosis on CXCR2 dephosphorylation and resensitization, transiently transfected 293 cells were metabolically labeled with
[32P]orthophosphate, treated with MGSA/GRO, and allowed
to recover from ligand. In the absence of dynamin I K44A expression,
phosphorylation of CXCR2 was detected after 10-min treatment of 100 ng/ml MGSA. After a 1-h recovery period following removal of ligand,
CXCR2 underwent dephosphorylation to 44% of the stimulated level.
However, the dephosphorylation of CXCR2 in dynamin I K44A
co-transfected cells was greatly attenuated. After the same recovery
period, about 80% of receptor still remained in the phosphorylated
form as compared with ligand-stimulated cells without recovery (Fig. 3, A and D). The
equal loading of CXCR2 and expression of dynamin I K44A were confirmed
by Western blots using antibodies against CXCR2 (Fig. 3B)
and dynamin I (Fig. 3C). The rate of CXCR2 dephosphorylation with co-expression of wild-type dynamin is comparable with that which
occurred in cells transfected with CXCR2 alone.

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Fig. 3.
The effect of the dynamin I K44A mutant on
CXCR2 phosphorylation and dephosphorylation. HEK293 cells were
transiently transfected with plasmids expressing CXCR2 with or without
rat dynamin I K44A. A, a representative autoradiograph from
two independent experiments showing the phosphorylation and
dephosphorylation of CXCR2 in the whole cell lysates. Cells were
treated with 100 ng/ml MGSA/GRO for 0 min (lanes
1 and 4) or 10 min (lanes
2, 3, 5, and 6). Cells were
then washed, incubated in fresh medium without ligand for 1 h
(lanes 3 and 6), or kept on ice
(lanes 2 and 5). B, the
equal amount of receptor loading was confirmed by Western blot using a
monoclonal anti-CXCR2 antibody. C, the expression of dynamin
I K44A on the transfected cell lysates was detected by Western blot
using anti-dynamin antibody. D, quantification of the
mean ± S.E. of two different experiments performed in duplicate.
The data were normalized to MGSA-stimulated CXCR2 phosphorylation.
Dotted bars, phosphorylated; striped
bars, dephosphorylated.
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Effect of Dynamin K44A on CXCR2 Down-regulation--
Previously,
we have observed that MGSA/GRO treatment resulted in a
concentration-dependent decrease in the level of CXCR2 in
stably transfected 3ASubE P-3 and HEK293 T2 cells (10, 20). Based on
information from a
2-AR down-regulation experiment (16), we hypothesized that a portion of the internalized CXCR2 may be subject
to proteolytic degradation in the endosome or lysosome following
receptor endocytosis. Moreover, reducing this portion of receptors by
blocking endocytosis should have a concomitant effect on receptor
degradation and down-regulation. To test this hypothesis, we determined
the effect of the dynamin I K44A mutant on CXCR2 degradation and
down-regulation in HEK293 cells. Transiently transfected cells were
stimulated with MGSA/GRO for various time periods and lysed. The
relative amount of CXCR2 was assessed by Western blot using whole cell
lysates. As demonstrated in Fig. 4,
MGSA/GRO treatment promoted progressive CXCR2 degradation in a
time-dependent fashion. After 2 h of treatment, cells
lost 14% of receptor. The percentage of CXCR2 loss was increased to
49% following 8 h MGSA/GRO incubation. In contrast, the dynamin I K44A mutant effectively reduced the agonist-promoted CXCR2 degradation, with only 15% of CXCR2 being degraded after 8 h of MGSA/GRO
treatment (Fig. 4). These data suggest that a portion of endocytosed
CXCR2 undergoes degradation and down-regulation via a clathrin-mediated event.

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Fig. 4.
The effect of dynamin I K44A mutant on
MGSA-stimulated CXCR2 degradation and down-regulation.
A, HEK293 cells were transiently transfected with plasmids
expressing CXCR2 with (lanes 5-8) or without
(lanes 1-4) rat dynamin I K44A. The cells were
harvested and plated equally onto 12-well plates. The cells were then
treated with 100 ng/ml MGSA/GRO for 0 min (lanes
1 and 5), 0.5 h (lanes
2 and 6), 2 h (lanes 3 and 7), and 8 h (lanes 4 and
8). 30 µg of protein from clarified RIPA lysates were
loaded per lane, and Western blot analysis was performed with rabbit
anti-CXCR2 polyclonal antibody and visualized by horseradish
peroxidase-conjugated secondary antibody. The arrow
indicates CXCR2 epitope. B, the density of the bands
(mean ± S.E.) representing CXCR2 was determined by densitometric
scanning. The results represent one of two experiments performed in
duplicate. Dotted bars, CXCR2; striped
bars, CXCR2 plus dynamin I K44A.
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Effect of Dynamin K44A on CXCR2-mediated MAP Kinase
Activation--
Previous studies demonstrated that IL-8 stimulation on
its receptors in neutrophils resulted in activation of MAP kinase
pathway (21). We also confirmed the activation of MAP kinase following MGSA/GRO stimulation in HEK293 T2 cells. To study the downstream CXCR2
signal transduction pathway, we next examined the effect of dynamin I
K44A expression on MAP kinase activation. The
MGSA/GRO-dependent activation of MAP kinase in cells
expressing both CXCR2 and dynamin I K44A was compared with that of
cells expressing only CXCR2 by using a phosphospecific ERK1/ERK2
antibody. As demonstrated in Fig. 5,
A and C, dynamin I K44A cells exhibited an
identical MAP kinase activation response following 5 min of MGSA/GRO or
IL-8 (100 ng/ml) stimulation. Equal loading was confirmed by
immunoblotting with an antibody against ERK-2.

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Fig. 5.
The effect of dynamin I K44A mutant on MGSA
or IL-8-stimulated ERK activation in HEK293 cells. A,
HEK293 cells were transiently transfected with plasmids expressing
CXCR2 with (lanes 1-3) or without
(lanes 4-6) dynamin I K44A. The cells were
harvested and plated equally onto 60-mm plates. The cells were then
treated with buffer (lanes 1 and 4) or
100 ng/ml MGSA/GRO (lanes 2 and 5) or
IL-8 (lanes 3 and 6) for 5 min. 30 µg of protein from clarified RIPA lysates were loaded per lane, and
Western blot analysis was performed with a phosphospecific MAP kinase
antibody and visualized by an horseradish peroxidase-conjugated
secondary antibody. B, the blot from A was
striped and reprobed with an antibody against ERK-2 to ensure the equal
loading. C, the density of the bands (mean ± S.E.)
representing phosphorylated ERK1 and ERK2 was determined by
densitometric scanning. The results were obtained from three
independent experiments. Dotted bars, CXCR2;
black bars, CXCR2 plus dynamin I K44A.
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Effect of Dynamin K44A on CXCR2-mediated
Chemotaxis--
Ligand-stimulated CXCR2-mediated chemotaxis is a
direct and effective functional test to access chemokine receptor
signal transduction. We used chemotaxis assays to determine if blocking of receptor endocytosis by co-expression of dynamin I K44A could abolish the CXCR2-mediated chemotaxis toward a gradient of IL-8 (Fig.
6) or MGSA/GRO (data not shown). We
observed an IL-8 concentration-dependent chemotactic
response in cells expressing wild-type receptor, with a peak migration
occurring at a concentration around 25-50 ng/ml IL-8. Also, the
cellular migration response followed a typical bell-shaped curve in
which the chemotaxis was inhibited at higher concentrations of IL-8
(Fig. 6). Strikingly, overexpression of dynamin I K44A resulted in
marked attenuation of CXCR2-mediated chemotaxis. These data suggest
that agonist-mediated endocytosis and resensitization are crucial for
CXCR2-mediated chemotaxis.

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Fig. 6.
The effect of the dynamin I K44A mutant on
IL-8-stimulated CXCR2-mediated chemotaxis of HEK293 cells. HEK293
cells were transiently transfected with plasmids expressing CXCR2 with
(striped bars) or without (dotted
bars) rat dynamin I K44A. Two days after transfection, cells
were compared for chemotaxis in response to IL-8 stimulation as
described under "Experimental Procedures." Values represent the
means ± S.E. of three independent experiments performed on
different days. The data were analyzed using Student's paired
t test (*, p < 0.05).
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DISCUSSION |
CXCR2 is internalized into vesicular compartments following
agonist stimulation and phosphorylation at multiple serine residues in
the carboxyl-terminal domain in a manner similar to that observed for
several other GPCRs. However, the mechanisms responsible for receptor
endocytosis and the functional significance of this event regarding
receptor signal transduction are largely unknown. Our studies provide
direct biochemical evidence for the requirement of a dynamin-mediated
formation of clathrin-coated pits in CXCR2 sequestration,
resensitization, and down-regulation. More importantly, our data
suggest that endocytosis of receptor is prerequisite to its ability to
continuously sense the agonist stimulation that is unique for chemokine
receptor-mediated chemotaxis.
Previous studies have identified dynamin as a major component of
clathrin-mediated endocytosis (22). Dynamin associates with the adaptor
protein AP2 complex (23). Once localized to the clathrin-coated pits,
dynamin promotes endocytosis by catalyzing the pinching off process of
coated vesicles (24, 25). Dynamin GTPase activity is essential for the
formation of clathrin-coated pits. This is confirmed by the observation
that expression of a GTPase-defective dynamin mutant prevents formation
of the endocytic clathrin-coated vesicles (25, 26).
Using a dominant-negative mutant of dynamin I (K44A), we have
demonstrated inhibition of ligand-induced CXCR2 endocytosis. This
observation suggests that CXCR2 internalization is mediated by a highly
conserved mechanism that is dynamin- and
clathrin-dependent. Our immunofluorescence experiments
revealed that CXCR2 internalization in HEK293 cells reached to
endosomal compartments following MGSA/GRO treatment. Colocalization of
CXCR2 with the transferrin receptor in transfected HEK293 cells is
consistent with the concept that both receptors utilize the same
clathrin-dependent pathway. In the absence of agonist, a
small but detectable portion of CXCR2 seems to be localized in the
endosomes (Fig. 1A). This may be due to constitutive
receptor internalization and recycling. This idea is supported by the
finding that overexpression of dynamin I K44A reduces basal
intracellular localization of CXCR2 (data not shown).
After internalization to the endosome, the receptor will be
dephosphorylated and either recycled back to cell surface or
degraded by proteolytic enzymes (11, 27). Using the dynamin I K44A mutant, we have shown that clathrin-mediated receptor internalization is a critical initial step for CXCR2 resensitization and
down-regulation. Co-expression of dynamin I K44A resulted in
significant diminution of both CXCR2 dephosphorylation and
down-regulation. Although the exact mechanism responsible for
dephoshorylation of GPCRs remains unclear, it is postulated that the
conformational change of the receptor in the acidic environment
facilitates dephosphorylation by a G protein-coupled receptor
phosphatase (28). Recycling and resensitization of dephosphorylated
receptor is believed to allow the chemokine receptors to sense and
respond to a continuous gradient of chemokine stimulation.
The functional role of agonist-promoted CXCR2 endocytosis has long been
elusive in the field of CXCR2 signal transduction. The result of
MGSA/GRO and IL-8-mediated MAP kinase experiments suggests that CXCR2
endocytosis is not required for its immediate downstream signaling.
These data are supported by the previous observation that a mutant
CXCR2 deleting 31 carboxyl-terminal amino acids exhibited significantly
impaired agonist-induced receptor endocytosis but not inhibition of
adenylyl cyclase or MAP kinase activation (29). Further evidence
suggesting the separation between CXCR2 endocytosis and immediate
downstream signal transduction events came from the studies of another
CXCR2 deletion mutant (30). Richardson et al. (30)
demonstrated that a CXCR2 mutant (331T), exhibiting a deletion of the
last 25 amino acid residues of the carboxyl-terminal domain, lost
agonist-stimulated phosphorylation and sequestration when expressed in
the placental cell RBL-2H3 cells. However, the immediate downstream
signal transduction events such as GTPase activity,
phosphatidylinositol hydrolysis,
-hexosaminidase release, and
Ca2+ mobilization were not impaired. Studies of the
2-AR indicate that the capacity of the receptor to
signal through adenylyl cyclase remains intact following total
inhibition of receptor endocytosis by dynamin K44A (17). However,
others demonstrate that in HEK293 cells, the
2-AR
mediated activation of MAP kinase is inhibited by blocking receptor
internalization although tyrosine phosphorylation-mediated activation
of Shc and Raf kinase are unaffected (31). The reasons for the
discrepancy in ligand-mediated receptor endocyrosis and downstream MAP
kinase activation between CXCR2 and
2-AR are not clear.
The chemotaxis assay is believed to be the most useful assay to
evaluate the signal transduction capability of chemokine receptors, since it measures the ultimate result of a cascade of intracellular events that are activated by ligand-receptor interaction. More importantly, this assay also provides a functional read out for a
process composed of sequential desensitization and resensitization events with respect to receptor activation. Our results clearly demonstrate that overexpression of dynamin I K44A abolished the CXCR2-mediated HEK293 cell chemotaxis toward both IL-8 and MGSA/GRO. The equal or similar cell surface expression of receptors has been
confirmed in each experiment by immunofluorescence assay to exclude the
possibility that the reduced chemotaxis in cells expressing dynamin I
K44A is due to reduced receptor expression or cell surface targeting.
The specific signal pathway responsible for chemotaxis is poorly
understood. In human neutrophils, phosphatidylinositol 3-kinase
activity is required for both induction of chemotaxis and MAP kinase
activation (32). When phosphatidylinositol 3-kinase is blocked with
wortmannin, IL-8-induced MAP kinase is also blocked (21). The fact that
MAP kinase activation in cells expressing dynamin I K44A is normal
makes it unlikely that phosphatidylinositol 3-kinase activity is
affected in these cells. Considering the fact that endocytosis
permits CXCR2 dephosphorylation and resensitization, we
therefore postulate that endocytosis, dephosphorylation,
resensitization, and recycling of CXCR2 is required for graded response
to ligand stimulation.
Blocking receptor internalization does not have major effects on the
generation of immediate downstream signals but does alter the ability
of cells to respond to a continuous signal generated over a
concentration gradient, based upon the loss of receptor dephosphorylation and the subsequent receptor recycling. Another possible explanation for the blocking effects of dynamin I K44A on cell
chemotaxis may be due to the inhibition of the redistribution of cell
surface chemokine receptor and receptors for matrix attachment. One model of cell migration suggests that there is an increased exocytosis at the leading edge of moving cells (33, 34). This polarized
exocytosis may be involved in delivery of fresh integrins and chemokine
receptors to the cell front. This process enables the migrating cells
to attach to the extracellular matrix and to sense the chemokine stimulation.
Following ligand-stimulated receptor phosphorylation, GPCRs
bind to adaptor molecules that directly inhibit the interaction between receptor and G-protein (12, 13). In the case of the
2-AR, the binding of
-arrestin and arrestin-3 is also
an initial event required for receptor internalization (14, 15). Both
-arrestin and arrestin-3 can function as adaptors. The association of these molecules with phosphorylated receptor and clathrin is a key
event in the formation of clathrin-coated pits. Recently, Aragay
et al. (35) reported that
-arrestin associates with CCR2
shortly after agonist stimulation, suggesting that arrestins might play
a universal role in mediating GPCR desensitization and internalization.
Studies are currently in progress to test the role of arrestins and
other adaptor molecules in CXCR2 desensitization and internalization.
In summary, we have established that agonist-promoted CXCR2 proceeds
through the formation of clathrin-coated pits to endosomes. Furthermore, we demonstrated that the CXCR2 internalization is required
for receptor dephosphorylation, resensitization, and down-regulation
but not for immediate downstream signaling. More importantly, our
chemotaxis experiments indicate that agonist-stimulated receptor
endocytosis and dephosphorylation is essential for normal chemokine
receptor-mediated chemotaxis in HEK293 cells.