(Received for publication, April 30, 1996, and in revised form, January 21, 1997)
From the Department of Cell Biology, Vanderbilt
University School of Medicine, Nashville, Tennessee 37232-2175, the
¶ Veterans Affairs Medical Center, Nashville, Tennessee
37212-2637, and § SmithKline Beecham Pharmaceuticals,
Department of Immunology,
King of Prussia, Pennsylvania 19406-0939
We have characterized the ligand-enhanced phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of clonal 3ASubE cell lines expressing receptors truncated or mutated in the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss of melanoma growth stimulatory activity/growth-related protein (MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained based upon altered ligand binding affinity or receptor number. 3ASubE cells expressing 342T or CXCR2 with mutation of Ser-342, -346, -347, and -348 to alanine (4A) exhibit strong mobilization of Ca2+ in response to ligand (interleukin-8 or MGSA/GRO), with a recovery phase significantly slower than that of cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2, which is 93% desensitized by 20 nM ligand, the 331T, 342T, and 4A CXCR2 mutants do not undergo significant ligand-induced desensitization, and respond to a second ligand challenge by mobilizing Ca2+. The 3ASubE cells expressing CXCR2 with mutation of Ser-346, -347, and -348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and are 60-70% desensitized following the initial ligand challenge. WT CXCR2 phosphorylation and desensitization occur in <1 min, while receptor sequestration is a much later event (30-60 min). However, mutant receptors that are neither phosphorylated nor desensitized in response to ligand are <10% sequestered 60 min following ligand challenge. These data demonstrate for the first time that ligand binding to CXCR2 results in receptor phosphorylation, desensitization, and sequestration and that serine residues 342 and 346-348 participate in the desensitization and sequestration processes.
The CXC chemokine,1 IL-8, specifically binds the CXC chemokine receptor, CXCR1, while a second receptor, CXCR2, is shared by IL-8, MGSA/GRO, and several other CXC chemokines (1). In human neutrophils, a 10-min treatment with IL-8 induces the internalization of greater than 90% of the CXC chemokine receptors, CXCR1 and CXCR2 (2). Low MGSA/GRO concentrations (0.2 nM) down-regulated 50% of CXCR2 expressed on neutrophils, while 7-13-fold higher IL-8 concentrations are required to down-regulate 50% of CXCR1 (3). Cell surface expression of CXCR1 is 100% restored after 1.5 h, while only 40% of the initial CXCR2 expression can be detected after 3 h of incubation following ligand treatment (3). We have previously demonstrated in both 3ASubE and 293 stable transfectants expressing CXCR2 that ligand binding results in the phosphorylation of CXCR2 on serine residues (4, 5). Within 1 min after ligand binding, MGSA/GRO stimulates the phosphorylation of CXCR2, and after prolonged treatment with MGSA/GRO ~40% of the receptors are degraded (4). Phorbol ester treatment (100 nM for 2 h) of 3ASubE cells expressing CXCR2 also results in serine phosphorylation, down-regulation, and degradation of the receptor (5). These data suggest that phosphorylation of CXCR2 induces receptor internalization (sequestration) and degradation, as has been observed in certain adrenergic receptors (6-8). Recently, Ben-Baruch et al. (9) demonstrated that 293 cells expressing CXCR2 exhibiting a deletion of amino acids 317-355 from the carboxyl-terminal domain do not exhibit chemotaxis in response to IL-8 (9). Since ligand did induce migration of 293 cells expressing CXCR2 with a deletion of residues 325-355, residues 317-324 appear to be essential for signal transduction and chemotaxis in response to IL-8 (9).
In the present study, we show that CXCR2 mutants exhibiting a loss of residues 331-355 (331T) or 342-355 (342T) no longer undergo ligand-enhanced receptor phosphorylation or desensitization as monitored by Ca2+ mobilization in response to ligand challenge. In contrast, receptors exhibiting deletion of amino acid residues 352-355 (352T) continue to undergo phosphorylation in response to ligand. In an effort to determine which specific serine residues are phosphorylated, we mutated individual serine residues 342, 346, 347, and 348 to alanine (S342A, S346A, S347A, S348A) or groups of serine residues to alanine: 346/7/8 (3A) and 342/6/7/8 (4A). Studies with 3ASubE cells expressing these mutant receptors, as compared with 3ASubE cells expressing wild type CXCR2, revealed that serines 342, 346, 347, and 348 of CXCR2 are involved in the desensitization of the receptor to its ligand. However, when these serine residues are mutated to alanine (4A or 3A CXCR2), other serine residues in the carboxyl-terminal domain can be phosphorylated, and the receptor is partially desensitized in response to ligand. In 3ASubE cells, phosphorylation and desensitization of WT CXCR2 occurs within 1 min after ligand treatment, while receptor sequestration requires >30 min. The 342T CXCR2 receptor is not sequestered in response to ligand. These data demonstrate that for CXCR2, specific residues along the carboxyl-terminal domain of the receptor mediate receptor desensitization and sequestration in response to ligand.
Polymerase chain
reaction strategies were employed to generate truncated CXCR2 mutants
with stop codons introduced at Ser-331, Ser-342, or Ser-352. Polymerase
chain reaction was conducted on the cDNA encoding the entire open
reading frame for the CXCR2, which had been subcloned into BlueScript.
The primer pair for each reaction included a common primer for the 5
end of the open reading frame. Unique primers that would introduce the
desired stop codons were used for the 3
end and were as follows:
ser331(331T), GCGAAGCTTTTAGATCAAGCCATGTATAGC; ser342(342T),
GCGAAGCTTTTAAGGCCTGCTGTCTTTGGG; and ser352(352T),
GCGAAGCTTTTAAGTGTGCCCTGAAGAAGA. The polymerase chain
reaction-generated fragments were isolated, subcloned into BlueScript,
and sequenced. Once the sequences were shown to be correct, the
cDNAs for the truncated receptors were subcloned into the mammalian
expression vector pRc/CMV and subsequently transfected into the human
placental cell line, 3ASubE, or human embryonic kidney 293 cells.
Mutagenesis of
specific serine to alanine (Ser Ala) residues was conducted using
the pALTER site-directed mutagenesis system (Promega). The following
mutations were synthesized using the indicated primers:
ser342ala(S342A), AGACAGCAGGCCGGCCTTTGTTGGC; ser346ala(S346A),
TCCTTTGTTGGCGCCTCTTCAGGGCAC; ser347ala(S347A), CTTTGTTGGCTCAGCTTCAGGGCACA; ser348ala(S348A),
GTTGGCTCTTCTGCCGGGCACACTTCC; ser346/7/8ala(3A),
TCCTTTGTTGGCGCCGCTGCAGGGCACACTT. Receptor mutants were screened
by restriction analysis and then sequenced. The Ser-346/347/348
Ala
mutant was used as a template with the ser342ala primer to generate a
Ser-342/346/347/348
Ala (4A) mutant. Once the mutations were
confirmed, the cDNAs encoding the open reading frame for the CXCR2
mutants were subcloned into the pRc/CMV expression vector and
transfected into the 3ASubE cell line or human embryonic kidney 293 cells.
Immortalized human 3ASubE placental cells were maintained in 5% fetal bovine serum (FBS)/Dulbecco's modified Eagle's medium (DMEM) (Life Technologies, Inc.), and human embryonic kidney 293 cells were maintained in 7.5% FBS/DMEM as described previously (4). Clonally selected, stable 3ASubE transfectants were generated for the truncated receptor mutants as described previously (4). The stable transfectants expressing CXCR2 mutations were selected based upon expression of immunoreactive CXCR2 using antibody to the NH2 terminus of CXCR2 in an indirect immunohistochemical staining protocol. Stable 3ASubE transfectants identified as expressing immunoreactive receptor at the plasma membrane in this screen were analyzed for 125I-MGSA binding activity using our standard binding assay (4). Clones of stable transfectants were generated that bind 125I-MGSA/GRO 3-5-fold above background levels, where the background cpm are calculated as the 125I-MGSA/GRO bound in the presence of 128 ng/ml unlabeled MGSA/GRO (16 nM). Two clones with strong ligand-binding properties were selected for each mutation and analyzed for ligand binding affinity and ligand-induced receptor phosphorylation. One representative clone of each of the mutants analyzed for ligand binding and phosphorylation was selected for analysis of ligand-induced calcium mobilization. Both 3ASubE and 293 clones expressing WT and 342T CXCR2 were analyzed for ligand-induced receptor sequestration. Chemotaxis studies were performed with 293 cells expressing WT or 342T CXCR2, since 3ASubE cells expressing CXCR2 did not undergo MGSA/GRO-induced chemotaxis.
125I-MGSA/GRO Binding AssayMGSA/GRO (1 µg), generously provided by R + D Systems and Repligen Corporation, was iodinated using the chloramine T method, yielding a specific activity of ~100 µCi/µg, assuming 100% recovery. For most of the experiments reported here, 125I-MGSA/GRO was purchased from DuPont NEN at a specific activity of 272 µCi/µg. Binding assays were performed on stable transfectants as described previously (4). Each assay was repeated a minimum of two times with n = 3 for each of the representative clones.
In Vivo Phosphorylation AssayIn vivo phosphorylation and receptor immunoprecipitation assays were performed as described previously (4). Briefly, [32P]orthophosphate-labeled cells were then treated with MGSA/GRO (5 nM), 12-O-tetradecanoylphorbol-13-acetate (TPA) (400 nM), or vehicle alone for 10 min at 37 °C. Whole cell lysates were prepared, and the CXCR2 was immunoprecipitated from an equal number of trichloroacetic acid-precipitable counts (5 × 106 cpm), electrophoresed through a 9% SDS-polyacrylamide gel, which was subsequently dried and analyzed by autoradiography (Imaging film, BioMax MR, Eastman Kodak Co.), or transblotted onto nitrocellulose for autoradiography and immunodetection. Each assay was repeated at least twice, and the phosphorylation was quantitated by PhosphorImage analysis (Molecular Dynamics) or densitometric scanning.
Western Blot AnalysisTransblotted membranes were blocked for 1 h at room temperature in 5% milk powder/Tris-buffered saline, incubated overnight with affinity-purified anti-NH2-terminal peptide antibody to CXCR2 (2 µg/ml) in 5% milk powder/Tris-buffered saline and then developed as described previously (4).
Receptor Degradation StudiesConfluent cultures (60-mm dishes) of the 3ASubE transfectants were placed in serum-free DMEM and treated with MGSA (50 nM), TPA (400 nM), or the appropriate vehicle control for 2 h at 37 °C. For some experiments, serum-starved cells were treated with 25 nM MGSA/GRO for 1-6 h. Plates were rinsed on ice with Tris-buffered saline and then scraped in 300 µl of Triton X-100 lysis buffer as described previously (5). The cell lysates were clarified by centrifugation at 15,000 × g at 4 °C for 15 min, the supernatants were transferred to a fresh tube, and protein estimates were performed (BCA, Pierce). Twenty-five micrograms of protein were loaded per lane on a 9% SDS-polyacrylamide gel, electrophoresed, transblotted onto a nitrocellulose membrane, and analyzed as described above.
Calcium FluorimetryTransfected 3ASubE cells expressing
truncation and Ser Ala mutants were grown until confluent. Cells
were released by a short exposure (1-2 min) to Versine (trypsin/EDTA)
and washed once in culture medium containing 5% FBS. Cells were then
washed a second time in Krebs-Ringer solution (118 mM NaCl,
4.56 mM KCl, 25 mM NaHCO3, 1.03 mM KH2PO4, 11.1 mM
glucose, 5 mM HEPES) without Ca2+ or
Mg2+. Cells were resuspended at 2 × 106
cells/ml and incubated with FURA-2 for 30 min (2 µM final
concentration). After 30 min, the volume of buffer was doubled with the
Krebs-Ringer solution (without Ca2+ and Mg2+),
and the cells were incubated for 10 min. Cells were then centrifuged (300 × g, 6 min) and washed once (50 ml) in
Krebs-Ringer solution containing Ca2+ and Mg2+
(1 mM). The cells were finally adjusted to 1 × 106 cells/ml. Ca2+ mobilization experiments
were performed using a single scanning spectrofluorimeter constructed
by the University of Pennsylvania Department of Bioengineering. Data
were collected using an IBM model PS-II computer with custom written
software provided by the Department of Laboratory Automation,
SmithKline Beecham. Data were analyzed using the software program,
Igor, which used the following equation to determine free
Ca2+.
![]() |
(Eq. 1) |
In the desensitization experiments, cells (6 ml total, at 1 × 106 cells/ml, 37 °C) were stimulated with 20 nM MGSA/GRO, IL-8 (stimulated), or buffer (control) for 5 min before being washed three times with 15 ml of Krebs-Ringer buffer with Ca2+ and Mg2+ (1 mM). Cells were finally resuspended at (1 × 106 cells/ml) and kept on ice until needed. Cells were allowed to warm to 37 °C for 5 min before the second stimulus of IL-8 or MGSA/GRO (5 nM) as described previously. The data were analyzed by both the paired Student's t test and the Kruskall-Wallis test. To measure the time course of desensitization, clones expressing wild type and the 342T CXCR2 were stimulated with MGSA/GRO or IL-8 (20 nM) for 30 s to 30 min before being washed, resuspended, and stimulated with a second concentration of IL-8 (5 nM), and the maximum Ca2+ mobilization was calculated. The data were analyzed by the Wilcoxon rank-sum test.
Effects of Mutation of Carboxyl Terminus of CXCR2 on Receptor SequestrationTo compare the time course of internalization of
the wild type CXCR2 receptor to that of the 342T mutant CXCR2 (4),
confluent, serum-starved 3ASubE cells expressing WT or 342T CXCR2 were
pretreated with saturating concentrations of MGSA/GRO or IL-8 (100 nM) for 1, 5, 15, 30, or 60 min at 37 °C. Free ligand
was removed with three ice-cold (0.5 ml) washes of binding buffer (1 mg/ml ovalbumin in DMEM), and the receptor remaining at the cell
surface was monitored by indirect immunodetection. Briefly, the cells
were incubated with 2 µg of antibody to the NH2 terminus
of CXCR2 in 250 µl of binding buffer for 2 h at 4 °C, excess
antibody was removed by washing with 1 ml of binding buffer at 4 °C,
and then 250 µl of 125I-labeled goat anti-rabbit IgG
(2 × 105 cpm, specific activity 300 µCi/ml) was
added and incubated for 30 min at 4 °C. Excess antibody was
then aspirated; cells were washed twice with 1 ml of binding buffer at
4 °C, lysed in 0.5 ml of lysis buffer (1% SDS, 0.1 N
NaOH); and radioactivity determinations were made by counting.
Nonspecific binding was estimated by preblocking the
NH2-terminal antibody with a 20-fold molar excess of
peptide to which the antibody was generated before adding the antibody
to the cells. The data exhibited a normal distribution and were
analyzed by the paired t test as well as by the
nonparametric Wilcoxon rank-sum test.
Chemotaxis assays were performed on 293 cells expressing WT or mutant CXCR2 according to the protocols of Ben-Baruch et al. (9) with modification. The 96-well chemotaxis chamber (Neuroprobe Inc., Cabin John, MD) was used, and the lower compartment of the chamber was loaded with 360-µl aliquots of 1 mg/ml ovalbumin/DMEM or MGSA/GRO diluted in ovalbumin/DMEM (chemotaxis buffer). The polycarbonate membrane (10-µm pore size) was coated on both sides with 20 µg/ml human collagen type IV for 2 h at 37 °C and stored overnight at 4 °C. The cells were removed by trypsinization and incubated in 10% FBS/DMEM for 1.5 h at 37 °C to allow restoration of receptor, washed in chemotaxis buffer, and then placed into the upper chamber in the ovalbumin medium. The chambers were incubated for 4.5-6 h at 37 °C in humidified air with 5% CO2, and then the filter was removed, washed, fixed, and stained with a Diff-Quik kit. Quantitation of relative chemotaxis indices was by densitometric scanning of the stained filter. Briefly, the filter was scanned using a Epson ES 1200C densitometric scanner and the Adobe Photoshop software. Digitized images were subsequently scanned using the Molecular Dynamics PhosphorImager. Volumes were integrated and normalized to a value of 1 for the control of each set.
We have previously demonstrated that the CXCR2 expressed in nonhematopoietic cells is phosphorylated on serine residue(s) in response to either MGSA/GRO or phorbol ester treatment in a time- and concentration-dependent manner (4, 5). There are eight serine residues within the carboxyl-terminal domain of CXCR2 that may serve as potential phosphorylation sites (Table I). As an initial attempt to localize the site(s) of serine phosphorylation on this receptor, truncated receptors were generated by introducing stop codons at Ser-331, -342, and -352. These truncations were designed to remove all eight serine residues from the carboxyl-terminal domain, the final five serine residues, or the final serine residue, respectively. The truncated receptor constructs were subcloned into the pRc/CMV mammalian expression vector and transfected into the human placental 3ASubE cell line or the human embryonic kidney 293 cell line. Neither of these cells naturally expresses CXCR2 or exhibits specific binding for 125I-MGSA/GRO (9).
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Selected clones of 3ASubE cells, expressing the WT CXCR2 or various
truncated forms of CXCR2, were characterized for ligand binding and
ligand-induced receptor phosphorylation. Clones were also analyzed to
determine whether these receptors are phosphorylated in response to
TPA, an activator of protein kinase C. As demonstrated in Fig.
1, both the wild-type CXCR2 and the 352T receptor were phosphorylated in response to 10-min treatment with either MGSA/GRO (5 nM) or TPA (400 nM), while the 331T and 342T
receptors were not phosphorylated in response to either of these
treatments (Fig. 1). In contrast, the binding of
125I-MGSA/GRO was not impaired in the 3ASubE cells
expressing CXCR2 truncation mutants relative to cells expressing WT
CXCR2 (data not shown). Western blot analysis of the immunoprecipitates
indicated that approximately equal amounts of CXCR2 protein were
immunoprecipitated from the treated and untreated samples (data not
shown). Similar observations were made when the in vivo
phosphorylation assay was conducted using higher concentrations of
MGSA/GRO (50 nM) (data not shown). These results suggested
that the potential sites of receptor phosphorylation include serine
residues at position 342, 346, 347, and/or 348.
Effects of Ser
Serine to alanine mutations were
then made at individual serine residues (Ser-342 Ala (S342A),
Ser-346
Ala (S346A), Ser-347
ala (S347A), Ser-348
Ala
(S348A)) and at multiple serine residues (Ser-346, -347, and -348 (3A)
or Ser-342, -346, -347, and -348 (4A)) (Table I). Stable 3ASubE
transfectants expressing these Ser/Ala mutations were generated. These
clones were characterized with regard to MGSA/GRO binding and MGSA/GRO
ligand-induced receptor phosphorylation. Four clones expressing CXCR2
exhibited reduced basal receptor phosphorylation as compared with WT:
4A, S342A, S346A, and S348A (Fig. 2 and Table
II). The ligand-induced phosphorylation was diminished
25-75% in the clones expressing mutant receptor (Table II). However,
when these data were normalized to receptor number, we found that the
ligand-induced receptor phosphorylation in the mutant clones was
comparable with that of clones expressing the WT receptor (Table II).
The ligand dissociation constant (Kd) for each
mutant and WT CXCR2 was in the 2-4 nM range (Table II). These results suggested that multiple serine residues were
phosphorylated in response to MGSA/GRO, and altering individual serine
residues did not eliminate ligand-induced receptor phosphorylation.
Replacement of as many as four of the serine residues with alanine (4A)
did not diminish the overall -fold increase for ligand-induction of phosphorylation. (Table II and Fig. 2). Similar results were observed for the two different 4A mutant clones studied. Thus, mutation of
the serine residues, which are the apparent natural substrates for the
ligand-activated serine kinase for CXCR2, did not eliminate ligand-induced receptor phosphorylation.
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In
3ASubE cells, phorbol ester-induced phosphorylation of CXCR2 has been
demonstrated to be accompanied by receptor down-regulation and
degradation (5). MGSA/GRO treatment (50 nM for 2 h) of 3ASubE cells expressing WT CXCR2 also resulted in phosphorylation and
partial degradation of the receptor (5). As an initial approach to
determine whether receptor phosphorylation is required for receptor
degradation in response to these stimuli, the effect of MGSA/GRO (50 nM) or TPA treatment (400 nM) for 2 h at
37 °C on the degradation of 331T, 342T, and 352T CXCR2 was examined in 3ASubE cells. As we have previously demonstrated, Western blot analysis of the lysates prepared from TPA-treated 3ASubE cells expressing WT CXCR2 revealed that continuous TPA treatment resulted in
a significant decrease in immunoreactive CXCR2. MGSA/GRO or TPA
treatment of the 3ASubE cells expressing 352T also decreased CXCR2
protein. Neither MGSA/GRO nor TPA treatment of 3ASubE clones expressing
331T or 342T produced a decrease in CXCR2 protein (Fig. 3A). In response to MGSA/GRO, the reduction
in CXCR2 was less striking, although all of the receptor exhibited the
reduced electrophoretic mobility that is indicative of the
phosphorylated form of the receptor (Fig. 3A). However,
MGSA/GRO treatment (25 nM for 6 h) of 3ASubE cells
expressing WT CXCR2 produces a ~50% diminution in the immunoreactive
CXCR2 (Fig. 3B). Thus, the carboxyl-terminal domain of CXCR2
appears to be required for the receptor degradation that usually
accompanies ligand-induced receptor phosphorylation. Furthermore, the
degradation of CXCR2 that occurs in response to TPA is more extensive
than that which occurs in response to MGSA/GRO (Fig.
3A).
Effects of Ser
To determine the requirement for
serine phosphorylation in signal transduction, Ca2+
mobilization and desensitization were investigated in 3ASubE clones
expressing WT and mutant CXCR2. All 3ASubE clones studied mobilized
Ca2+ in response to IL-8 comparable with that observed in
neutrophils or differentiated HL60 cells (EC50, 2-5
nM), except that the maximum Ca2+ response was
diminished in magnitude. The transfected 3ASubE cells expressing WT
CXCR2 responded to IL-8 (5 nM) with an increase in
intracellular Ca2+, which peaked approximately 25 s
following agonist addition (Fig. 4). The level of
intracellular Ca2+ returned to base line rapidly. 3ASubE
clones expressing 331T or 4A CXCR2 exhibited a markedly slower removal
of ligand mobilized Ca2+ with a t80
of 179 and 135 s, respectively, while the clone expressing WT
CXCR2 required only 71 s to remove the mobilized Ca2+
(Fig. 4 and Table III). These data suggest that serines
342-, 346-, 347-, and 348- participate in the regulation of
Ca2+ homeostasis. 3ASubE clones expressing S342A CXCR2
exhibited removal of Ca2+, which was not significantly
different from the WT. Since intracellular Ca2+ as
determined by FURA-2 in the presence or absence of EGTA (5 mM) has both a component of influx and efflux (data not
shown), we cannot rule out the possibility that the various mutants
differ in the removal of influxed versus effluxed
Ca2+.
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To determine if mutation or truncation of the receptor was associated
with desensitization to ligand, FURA-2-loaded clones were stimulated
with buffer, 20 nM IL-8, or 20 nM MGSA/GRO.
Ligand was removed by repeated washing, and the clones were treated a second time with a lower concentration (5 nM) of either
MGSA/GRO or IL-8. As expected, WT clones were substantially
desensitized by the first addition of IL-8, with the second addition
(noted by the arrow on the chart) promoting a
Ca2+ mobilization response that was only 7.1% of the
original response (Fig. 5, A and
B). In contrast, the clones expressing the 342T- (Fig. 5,
C and D) and 331T (Table III) truncation mutants
failed to be desensitized by the high concentration of ligand
(p < 0.05) and exhibited a second response to ligand
that was equivalent to the original response (Fig. 5 and Table III).
Clones expressing the 4A mutant of CXCR2 exhibited a second calcium
response, which was 79% of the initial response (only 21%
desensitization). The clones expressing the 3A mutant or the single Ser
Ala mutants were 60-70% desensitized (Fig. 5, E and
F, and Table III). These data strongly suggest that the
residues beyond Ser-342 are responsible for desensitization of the
receptor to its ligand, and serine residues 342, 346, 347, and 348 appear to be involved. Statistical analysis of the data comparing
ligand-induced desensitization of mutant CXCR2 with WT CXCR2
demonstrated the mutant clones were significantly different from WT in
their desensitization properties (Table III).
For comparison, 3ASubE cells expressing WT and 342T CXCR2 were treated
with 20 nM MGSA/GRO for 5 min, and then cells were washed and then warmed and stimulated with a second concentration of
MGSA/GRO
(5 nM) as described under "Experimental
Procedures." The experiment was performed in triplicate. The
percentage of response remaining with the second stimulus of MGSA/GRO
(WT CXCR2, 18.3%; 342T CXCR2, 79.8%) was not remarkably different
from that which occurred in response to IL-8 (WT CXCR2, 7.1%; 342T
CXCR2, 100.8%)
To compare the time course of desensitization of the WT CXCR2 to that of the 342T CXCR2, we examined the time required for recovery following ligand induction of CXCR2 desensitization in 3ASubE clones expressing these receptors. Desensitization of WT CXCR2 occurred within 1 min after ligand treatment and remained >70% desensitized after 30 min. In contrast, the 342T CXCR2 underwent only 18.5% desensitization by 5 min and was almost fully recovered after 30 min. (Table IV). To determine whether desensitization occurred as a result of receptor internalization, we monitored the time course for CXCR2 sequestration in 3ASubE cells expressing mutant or WT CXCR2. After a 15-min incubation with saturating concentrations of ligand (37 °C), approximately 25% of the WT receptors were sequestered (Table V). By 30 min, approximately 30% of the WT receptors were sequestered, and by 60 min, 33% were sequestered (Table V). IL-8 was about 10-15% more efficient than MGSA/GRO in the WT receptor sequestration assay, p < 0.01 (Table V). In contrast, for 3ASubE cells expressing the 342T mutant, nearly 100% of the immunoreactive receptor remained at the membrane after treatment with saturating concentrations of ligand for up to 30 min. At 60 min, 92% of the receptor remained at the membrane (Table V). IL-8 was not more efficient than MGSA/GRO in sequestering 342T CXCR2 in 3ASubE cells. (Table V). This was the expected result, based upon the failure of these receptors to phosphorylate or desensitize in response to ligand. The failure to sequester 100% of CXCR2 was probably due to the dynamics of the display of receptors at the cell membrane at 37 °C, where new receptors would be cycling to the membrane to replace those that were sequestered.
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To determine whether loss of ligand-induced CXCR2 phosphorylation, desensitization, and sequestration resulted in a chemokine receptor that exhibited loss of function, we examined the ability of 293 cells and 3ASubE cells expressing WT and 342T CXCR2 to chemotax toward a gradient of MGSA/GRO. We did not observe chemotaxis in response to MGSA/GRO in the 3ASubE cells expressing WT or 342T CXCR2 using filters coated with collagen type I, fibronectin, or Matrigel or filters with no coating (data not shown). However, 293 cells expressing both WT and 342T CXCR2 exhibited chemotaxis toward MGSA/GRO, and the cells expressing 342T CXCR2 showed a chemotaxis response that at the higher concentrations (40 ng/ml) was equivalent to that of cells expressing WT CXCR2 (data not shown). Thus, disruption of the carboxyl-terminal domain of CXCR2 involved in phosphorylation, desensitization, and sequestration, does not ablate the chemotactic functions of this receptor.
There is now considerable evidence that ligand binding to transmembrane-spanning receptors stimulates receptor phosphorylation on the carboxyl-terminal domain of the receptor and that this event coincides with receptor desensitization, sequestration, and in some instances degradation or recycling (3-8). Both CC and CXC chemokine receptors are phosphorylated in response to ligand binding, and multiple serine or serine and threonine residues are phosphorylated (4, 10, 11). Richardson et al. (11) have shown that the phosphorylation of CXCR1 is accompanied by receptor desensitization. CXCR2 expressed in CHO cells is reported to be internalized more rapidly than CXCR1, and MGSA induces this internalization more slowly than does IL-8 (12). Moreover, truncation of CXCR1 eliminates ~60% of the ligand-induced internalization of the receptor (12). In contrast, neutrophil CXCR1 and CXCR2 are both rapidly down-modulated by ligand (5 min), although considerably more ligand is required to down-modulate CXCR1 than CXCR2 (3). CXCR2 recycles more slowly than the CXCR1; moreover, only about 40% of the CXCR2 recycles during a 3-h culture period (3), while CXCR1 fully recovers 1.5 h after treatment with the IL-8 ligand in neutrophils. Our studies with 3ASubE cells show that desensitization and phosphorylation of WT CXCR2 occurs within 30-60 s after ligand treatment, while sequestration occurs much more slowly, requiring 30-60 min. Thus, CXCR2 clearance in 3ASubE cells is different from that reported for CHO cells (12). We have also observed that after 6 h of treatment with 25 nM MGSA/GRO, 3ASubE cells expressing WT CXCR2 exhibit only ~50% reduction in the total receptor detected by Western blot, suggesting that either only a percentage of the receptors that bind ligand undergo degradation after sequestration or, alternatively, newly translated CXCR2 rapidly replenishes the receptor pool. A recent study of the N-formyl peptide receptor demonstrates a similar dynamic state of receptor sequestration, counterbalanced by the reappearance of receptors at the membrane (13).
3ASubE cells expressing wild type and mutant CXCR2 exhibit only subtle differences in the time course for desensitization or receptor sequestration produced by IL-8 and MGSA/GRO. However, larger differences in IL-8- versus MGSA/GRO-induced calcium signaling in 293 cells expressing transfected CXCR2 were recently observed by Damaj et al. (21), who suggested that MGSA/GRO elicits a stronger influx of Ca2+ than does IL-8. Differences in IL-8 versus MGSA/GRO induction of sequestration of CXCR2 were observed by Prado et al. in transfected CHO cells (11). The basis for the different results between these two studies and our study is unclear but could reflect differences in the specific activity or potency of the ligand preparations or differences in the manner in which the various cell types process the receptors. More in keeping with our own findings, Ahuja et al. (22, 23) reported similar profiles of calcium mobilization for IL-8 and MGSA/GRO proteins in stably transfected 293 cells expressing CXCR2, although MGSA/GRO was unable to totally desensitize receptors to a second weaker response to IL-8.
The -adrenergic receptors are phosphorylated along the carboxyl tail
by
-adrenergic receptor kinase and then assume a conformation that
allows association with an arrestin (14). This association with
arrestin is thought to be necessary for both desensitization and for
sequestration. In the case of the
2-adrenergic receptor, tyrosine phosphorylation of Tyr-326 is involved in facilitation of
sequestration of this receptor (14). We observe no tyrosine phosphorylation of CXCR2 in response to ligand or phorbol-ester stimulation. While some investigators have reported that sequestration is not dependent upon phosphorylation, others have shown that overexpression of
-adrenergic receptor kinase-1 can facilitate the
sequestration of the M2-muscarinic receptor. Moreover, for the M2
receptor, desensitization has been shown to proceed in the absence of
sequestration (15, 16). A number of seven-transmembrane G
protein-coupled receptors show loss of both phosphorylation and
sequestration when the carboxyl-terminal domain is deleted (17-19).
Our studies on CXCR2 phosphorylation, desensitization, and
sequestration demonstrate that the loss of four specific serine residues involved in ligand-stimulated receptor phosphorylation results
in markedly diminished receptor desensitization and loss of receptor
sequestration but no loss of ligand-induced chemotaxis. However, we are
unable to assign significance to any one specific phosphorylation site
regarding a role for desensitization or sequestration. Multiple sites
appear to be phosphorylated. A similar cluster of serine and threonine
residues regulates CXCR1 desensitization in the RBL-2H3 leukemia cell
line (11). However, it is important to note that desensitization does
not always require receptor phosphorylation. Richardson et
al. (20) have recently shown that activation of an epitope-tagged
fMLP receptor can cross-desensitize and cross-phosphorylate both the
epitope-tagged C5a receptor and CXCR1. C5a and IL-8 also
cross-desensitized the epitope-tagged fMLP receptor based upon
Ca2+ mobilization and phosphoinositide hydrolysis, but this
is not accompanied by phosphorylation of the epitope-tagged fMLP
receptor. Richardson et al. (20) concluded that these data
indicate that activation of phospholipase C, independent of receptor G
protein coupling, might be involved in cross-desensitization in the
absence of receptor phosphorylation (20).
We have observed only serine phosphorylation of CXCR2 (4, 5). Moreover, phosphoamino acid analysis of the mutant forms of CXCR2 studied here revealed only serine phosphorylation (data not shown). Truncation and serine mutants of CXCR2 that exhibited a loss of ligand-induced CXCR2 phosphorylation also exhibited a loss of TPA-induced CXCR2 phosphorylation. Thus, in 3ASubE cells CXCR2 is regulated very differently from CXCR1 in RBL-2H3 leukemia cells, where threonine phosphorylation may also be important, and phorbol-ester regulation of receptor phosphorylation appears to differ from that initiated by ligand (11). Like the CXCR1, phosphorylation of CXCR2 along the carboxyl-terminal domain appears to be important in the desensitization process, and loss of amino acid residues involved in this phosphorylation results in diminution of the receptor desensitization as well as receptor sequestration in response to ligand. Moreover, these two receptors appear to exhibit differences in the time course of sequestration and recycling (3). These events are likely to be important in the regulation of the inflammatory response, where an acute response to injury is required to quickly turn on and gradually shut off in an environmental milieu where excess ligand may persist for some time.
We are grateful to Rita Perry Davis and Hamid Haghnegahdar for excellent technical assistance, to Judithann M. Lee for assistance with the cell culture work throughout the calcium studies, to Paul Matrisian for help with the mutant receptor constructs, to Brandon Mosely for excellent help with the photography, and to Rebecca Shattuck-Brandt and Lee Limbird for helpful discussions.