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Neurobiotechnology Center and Departments of Biochemistry and Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH 43210
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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or CXC
chemokines, chemotax neutrophils and nonhemopoietic cells, whereas
those without an intervening amino acid residue, called ß or CC
chemokines, chemotax monocytes, T cells, eosinophils, and NK cells.
These two groups constitute the majority of known chemokines.
Chemokines specifically bind to seven transmembrane G protein-coupled
receptors present on the target cells and cause chemotaxis and signal
transduction events that are not well understood. A chemokine receptor, CCR5 was recently reported to require tyrosine sulfation for its function as a coreceptor for HIV-1 (5). Tyrosine sulfation of proteins is a modification that widely occurs in multicellular eukaryotic organisms (6, 7). Recently, two protein tyrosine sulfotransferases, PTST-1 and PTST-2, have been cloned (8, 9, 10). The sulfation of tyrosines occurs post-translationally, in the trans part of the Golgi network (6). The most important feature of tyrosine sulfation consensus sequences is the presence of an acidic or neutral amino acid residue directly before a tyrosine to be sulfated, although not all theoretical consensus sequences are sulfated in proteins (6, 11). Several examples are known where sulfation of tyrosines was shown to be important either for protein-protein interaction (12, 13, 14, 15, 16) or for protein precursor processing (11).
Monocyte chemotactic protein-1
(MCP-1)2 receptor
(CCR2B), which is known to play an important role in a variety of
diseases involving inflammation and/or injury such as wound healing,
atherosclerosis, and arthritis (1, 2, 3, 4), also contains a
consensus sulfation domain at the N-terminal region. We noticed that a
hexad of amino acid residues in this region is conserved in several
chemokines, and therefore we suspected it to be functionally important.
To test whether these residues are involved in the biological function
of the receptor, site-directed mutagenesis was used. This mutagenesis
involved the consensus sulfation site, including tyrosine, the
suspected site of sulfation, and the neighboring amino acid residue
that should be a part of the consensus required for enzymatic sulfation
by protein tyrosine sulfotransferases (6, 11). The results
presented here demonstrate that the hexad is important for ligand
binding, lamellipodium formation, chemotaxis, and signal transduction.
Structural alterations in this region of the receptor caused
differential effects on lamellipodium formation and chemotaxis vs
Ca2+ influx and adenylate cyclase inhibition,
indicating that the receptor plays a role beyond the known function of
causing dissociation of the trimeric G protein into
Gi and Gß
. Our results also show that
tyrosine within the hexad is sulfated and that the mutation in the
consensus residue thought to be required for sulfation severely
inhibits sulfation and consequently inhibits the biological function of
the receptor. Some chemokine receptors, such as CXCR4, are known to be
glycoproteins, although no CC chemokine receptor has been shown to be
glycosylated. Our studies show that CCR2B is a glycoprotein and is
sulfated at tyrosine 26.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The expression vector, pcDNA3 was obtained from Invitrogen (San
Diego, CA). Human embryonic kidney (HEK293) cells were obtained from
American Type Culture Collection (ATCC CRL 1573; Manassas, VA).
Lipofectamine, G418, penicillin-streptomycin solution, DMEM/F-12,
BAC-TO-BAC baculovirus expression system, and FBS were purchased from
Life Technologies (Gaithersburg, MD). MEM/Eagle’s medium was obtained
from ICN. Anti-Flag M2 mAb was purchased from Eastman Kodak (Rochester,
NY). Mouse IgG1, 
(MOPC-21), FITC-conjugated sheep anti-mouse
IgG (whole molecule), phalloidin-FITC, chloramine-T,
isobutylmethylxanthine, forskolin, QAE-Sephadex A-25, and Igepal CA-630
were obtained from Sigma (St. Louis, MO). Fura-2 and calcein
acetoxymethyl ester were purchased from Molecular Probes (Eugene, OR).
Na125I and [3H]adenine
were obtained from NEN (Boston, MA); Fluoromount-G mounting medium was
purchased from Southern Biotechnology Associates (Birmingham, AL).
[35S]Na2SO4
was obtained from American Radiolabeled Chemicals (St. Louis, MO).
Protein G-agarose and N-glycosidase F deglycosylation kit
were obtained from Roche (Indianapolis, IN). Polyvinylidene difluoride
membrane was purchased from Millipore (Bedford, MA), and GF/C filters
were obtained from Whatman (Clifton, NJ). The enhanced
chemiluminescence kit for immunodetection was obtained from Pierce
(Rockford, IL). Anti-CCR2B Ab CCR2-03 was provided by Prof. M. Mellado
(Universidad Autónoma de Madrid, Madrid, Spain).
Expression of MCP-1
A human MCP-1 cDNA clone (17) was used to express MCP-1 in the Bac-To-Bac baculovirus expression system, and the recombinant protein was purified in a two-step fast protein liquid chromatography protocol using Mono S HR 5/5 and Superdex-75 HR 10/30 columns (American Pharmacia Biotech, Arlington Heights, IL) as described previously (18).
Constructs for CCR2B receptor mutants
CCR2B receptor was cloned using primers that were designed based on the published sequence (19), and the first strand of cDNA was generated from total RNA isolated from the THP-1 monocytic leukemia cell line. We cloned the product into pcDNA3 eukaryotic expression vector at BamHI and XbaI sites that were engineered into the primers. Prolactin leader sequence (20) (from Dr. I. F. Charo, University of Calfornia, San Francisco, CA) and a Flag tag epitope sequence (DYKDDDDK) were introduced at the N terminus immediately following the initiator amino acid, methionine. The presence of an octapeptide Flag tag provides an Ab epitope allowing immunological detection of the recombinant protein (21). The Flag sequence was joined to the receptor by amplification of an N-terminal sequence with both primers. Mutations at the conserved hexapeptide (TTFFDY) sequences located at positions 21–26 were performed by a two-step PCR method (22). Each amino acid at the hexapeptide region was mutated to alanine individually. In addition, Tyr26 was mutated to phenylalanine individually.
Transfections, cloning, and selection of highly expressing stable transformants
HEK293 cells were grown in a 1/1 mixture of DMEM and nutrient
mix F-12 containing 10% FBS and 1% penicillin/streptomycin solution
in a CO2 incubator at 37°C. Lipofectamine was
used at 20 mg/ml to transfect 60–80% confluent cells with 1 µg of
DNA by following the manufacturer’s instructions. Transfected cells
selected in the presence of 0.4 mg/ml G418 were analyzed by flow
cytometry with the anti-Flag mAb (10 µg/ml/0.2 x
106 cells). Untransfected HEK293 cells were mixed
with mouse IgG1 MOPC-21 at 10 µg/ml as a control. Cells were
incubated for 30 min at 4°C, washed with PBS, and labeled with 1/256
diluted FITC-conjugated sheep anti-mouse IgG at 4°C for 30 min in
the dark and washed twice with PBS containing 5% FBS; expression of
the receptor was determined by FACS. Cells were cloned, and the highly
expressing clones were selected for producing the cell cultures used in
the various experiments. Before using each culture, expression of the
receptor was measured by FACS analysis.
MCP-1 binding assay
Radiolabeling of MCP-1 was performed by the chloramine-T method (23) with a modification (18). Stable transformants were washed in PBS and once with HEPES containing binding buffer (1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, and 50 mM HEPES, pH 7.2) (24). The final assay volume (250 µl) contained 5 x 106 transfected and washed cells, various amounts of MCP-1, and 0.02 pmol of iodinated MCP-1 protein. Various transfected cell lines were incubated with labeled and unlabeled MCP-1. Following incubation, the cells were passed through a Whatman GF/C filter, and radioactivity on the filter was measured in a Packard COBRA gamma radiation counter (Downers Grove, IL). Dissociation constant (Kd) values for MCP-1 binding to CCR2B and mutated receptor in various cell lines of HEK293 were determined using the LIGAND program (25).
Measurement of Ca2+ influx induced by MCP-1
Calcium influx into the cytoplasm was measured in HEK293 cell lines expressing CCR2B or its mutants after incubation with 2 µM fura-2/acetoxymethyl ester for 30 min at 37°C in a CO2 incubator. Fluorescence was measured after the addition of different concentrations of MCP-1 in a Perkin-Elmer LS 3B fluorescence spectrometer (Palo Alto, CA) with constant stirring as previously described (18). The maximal fluorescence was measured after treating the cells with 50 µM digitonin, and Ca2+ influx is expressed as a percentage of the maximal fluorescence.
Adenylate cyclase assay
Stably transfected, confluent HEK293 cells were labeled overnight with 2 µCi/ml [3H]adenine (31.7 Ci/mmol) in HEPES buffer containing DMEM/F-12 medium supplemented with 10% FBS, 1% penicillin-streptomycin, and 0.4 mg/ml of G418 in 12-well tissue culture plates. The cells were washed in the presence of 1 mM isobutylmethylxanthine and treated with different concentrations of MCP-1 in the presence or the absence of an activator, forskolin, for 30 min at room temperature. Intracellular [3H]ATP and [3H]cAMP, separated through Dowex 50W and neutral alumina columns, respectively (26, 27, 28), were assayed for 3H in a scintillation counter. The amount of cAMP was normalized to its own ATP pool for each fraction and estimated by their ratios (cpm of cAMP/ATP x 100). In each experiment a full MCP-1 dose-response curve was generated and expressed as a percentage of the forskolin control. All data points were determined in duplicate, and results were plotted as a percentage of adenylate cyclase inhibition.
Chemotaxis assay
Chemotaxis of transfected HEK293 cell lines was measured as described previously (18). Transfected cells (107/ml) were suspended in Gey’s balanced cell solution containing 0.2% BSA, incubated with 2 µM calcein acetoxymethyl ester at 37°C for 30 min, washed with PBS buffer, and resuspended in the Gey’s BSA solution at 1 x 106 cells/ml. Various amounts of MCP-1 were placed in a 96-well Polytronic view plate (UniPlate 350; Neuroprobe, Cabin John, MD), and 200 µl of cell suspension was added to the top wells of the chamber. Following 2-h incubation at 37°C, the number of migrating cells was determined by measuring fluorescence with a Cytofluor 2300 plate reader (Millipore). The quantitation of HEK293 cell migration is useful only to detect major changes in cell mobility; therefore, we used the measured comparative chemotactic activities only in this context.
Confocal microscopic analysis
HEK293 cells expressing wild-type and mutant forms of CCR2B were stained with phalloidin-FITC to visualize polymerization of actin as previously described (29). The cells in culture (70% confluent) were harvested and suspended in PBS containing 10 mM EDTA, washed twice with fresh culture medium, spread on plastic chamber slides, and grown for 24–36 h. The cells were incubated with or without MCP-1 (50 nM) for 15 min on the slides, fixed by soaking the slides in ice-chilled PBS containing 4% paraformaldehyde for 15 min, and washed twice in PBS. The slides were incubated with 0.05 mg/ml phalloidin-FITC for 40 min at room temperature in a humidified chamber in the dark, washed three times in PBS for a total of 30 min, and mounted with glass coverslips using Fluoromount-G mounting medium. Actin polymerization in the cells was visualized using a Bio-Rad MRC 1024 laser scanning confocal microscope (Hercules, CA). FITC fluorescent signals were detected using a 480-nm excitation filter. Cells forming lamellipodia (>12 µm) were counted under the confocal microscope. The frequency of lamellipodium formation (percentage) before the addition of MCP-1 was subtracted from that measured after MCP-1 treatment. Samples were triplicated, and approximately 50 cells were examined in each replicate.
Preparation of detergent extract
HEK293 cells stably transfected with wild-type or mutant receptors were cultured in 75-cm2 flasks in DMEM/F-12 medium until 50–75% confluence. In some experiments tunicamycin was added to the cells at 1, 3.3, or 10 µg/ml for 2 days, with a change in medium after 24 h. The cells were removed with 10 mM EDTA solution in PBS, washed twice with PBS, and lysed in a buffer (50 mM Tris-HCl buffer, pH 7.5) containing 150 mM NaCl, 1% Igepal CA-630, and 0.5% deoxycholate. The following proteinase inhibitors were added to the buffer just before use: PMSF (up to 1 mM), leupeptin (6.8 µg/ml), pepstatin (6.9 µg/ml), and aprotinin (5 µg/ml). Usually 1 ml of buffer was used to lyse 5 x 106 cells. The homogenate was centrifuged in an Eppendorf microfuge at 14,000 rpm for 15 min.
Cell labeling with 35SO4-2
The monolayers of cells were washed three times with MEM/Eagle’s medium supplied with nonessential amino acids, glutamine, and CaCl2 at 0.2, 10, and 1 mM, respectively. Then, 500 µCi of Na235SO4 was added to the cell culture in 10 ml of the same medium, and the culture was incubated for 24 h. After immunoprecipitation, labeled protein preparations were subjected to SDS-PAGE, polyvinylidene difluoride blotted, and visualized by a phosphorimager.
Immunoprecipitation of CCR2B by anti-Flag Ab-agarose
Anti-Flag M2 affinity gel (100 µl) was added to 1–3 ml of cell extract in a test tube, and the suspension was slowly rotated overnight at 4°C. The gel was washed once with the lysis buffer and twice with 50 mM Tris-HCl buffer, pH 7.5, containing 150 mM NaCl and 0.1% Igepal CA-630. The proteins were eluted either by incubation of the gel with an equal volume of 2x Laemmli sample buffer for 5 min at 100°C or twice by 0.15 ml of 0.1 M glycine (pH 2.5) and 0.1% Igepal CA-630 for 10 min each elution, followed by neutralization with 1 M Tris and precipitation with ethanol. Immunodetection was performed using the enhanced chemiluminescence method.
Indirect immunoprecipitation of CCR2B
After centrifugation of the homogenate, 25 µg of mAb M2 or a control mAb was added to the extract, and the mixture was incubated on ice for 1–3 h. Then, 100 µl of protein G-agarose preincubated with 5% FBS in lysis buffer was added to the extract, and the test tube with the mixture was slowly rotated overnight at 4°C. The gel was washed several times according to the manufacturer’s protocol, and the proteins were eluted from the gel by adding equal volume of 2x Laemmli sample buffer.
Treatment CCR2B with N-glycosidase F
The N-glycosidase F deglycosylation kit was used according to the manufacturer’s (Roche) instructions for treatment of CCR2B isolated by M2 affinity gel precipitation.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Table I). The wild-type CCR2B and its mutants
were tested for ligand binding, induction of filopodia and
lamellipodium formation by MCP-1, chemotaxis in an MCP-1 gradient,
induction of Ca2+ influx into the cytoplasm
induced by this chemokine, and adenylate cyclase inhibition caused
by it.
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At position 26, substitution of Tyr with Ala caused only a 10-fold
decrease in binding, whereas substitution with Phe virtually abolished
binding.
Effects of mutations in the conserved segment of CCR2B on the
actin-based cytoskeletal changes induced by MCP-1 were analyzed by
confocal microscopy. MCP-1 binding to wild-type CCR2B caused formation
of very thin, finger-like protrusions of plasma membrane, called
filopodia, in about 5 min (data not shown). In MCP-1-treated CCR2B
cells, number of filopodia formed per cell reached 3.2, which is
2.6-fold greater than that in the control. After filopodia were formed,
another type of actin-based membrane protrusion, known as membrane
ruffles or lamellipodia, was formed within about 10 min (Fig. 2). The percentage of cells forming
lamellipodia extending >12 µm was used as the measure of the
frequency of lamellipodium formation. To determine whether this
frequency of lamellipodium formation correlated with chemotactic
migration, the frequency observed in the cells expressing wild-type
CCR2B and its mutants were plotted against their score for migration
(Fig. 3). Y26A cells showed the highest
level of MCP-1-induced formation of lamellipodia among all mutant
receptors used in this study, and this level was similar to that
observed with the wild-type CCR2B-expressing cells. This mutant also
showed chemotactic activity somewhat comparable to that of the wild
type. On the other hand, Y26F showed only about 3% lamellipodium
formation (Fig. 3), and this mutant showed very little chemotactic
activity. Untransfected cells and all mutants with drastically
decreased chemotactic activity induced by MCP-1 did not show much
MCP-1-induced lamellipodium formation (Fig. 3). On the other hand,
cells expressing wild-type CCR2B and its mutants that were active in
MCP-1-induced chemotactic migration showed high frequency of
lamellipodium formation (Fig. 3 and Table I). Lamellipodium formation
showed good correlation with chemotaxis. Neither the frequency of
filopodium formation nor the length of the filopodia showed correlation
with chemotaxis (data not shown). Thus, formation of lamellipodia is
probably involved in MCP-1-induced chemotactic migration
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). T21A and F23A showed moderate
chemotactic activity. Unexpectedly, substitution of Y26 with Phe caused
a drastic decrease in chemotaxis, whereas substitution with Ala showed
only a small decrease in chemotaxis. In all these cases, lamellipodium
formation correlated with chemotaxis.
Calcium influx into the cytoplasm caused by MCP-1 binding to the
receptor expressed on HEK293 cells was affected by mutation in the
conserved N-terminal region. Ca2+ influx was
dependent on the MCP-1 concentration; with the cells expressing CCR2B,
6 nM MCP-1 produced the maximal fura-2 fluorescence, which was 38% of
the maximal fluorescence observed when the membrane was disrupted with
digitonin. T21A, T22A, and F24A showed only small decreases in
Ca2+ influx compared with that elicited by CCR2B,
whereas F23A, D25A, Y26A, and Y26F showed no calcium influx (Fig. 4A).
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B). A 50% inhibitory dose
(IC50) of <1.0 nM was found for the wild-type
CCR2B, and a similar IC50 was manifested by the
mutants, T21A and T22A. With T24A, a somewhat higher concentration of
MCP-1 (8 nM) was required to achieve 50% inhibition, whereas F23A and
D25A showed some inhibition, but the 50% level could not be achieved
or calculated from the data (Fig. 4B). With Y26A and Y26F,
absolutely no adenylate cyclase inhibition could be observed with any
concentration of MCP-1.
Since the conserved N-terminal region studied includes a consensus
sequence as a sulfation site, we tested whether CCR2B is sulfated at
Y26. Incubation of the HEK293 cells that express CCR2B in
[35S]sulfate-containing medium followed by
immunological isolation of the solubilized receptor and SDS-PAGE showed
that a 50-kDa protein was labeled with 35S (Fig. 5A). To determine whether this
sulfation is located at Y26, mutants in which this Y is substituted
with F or A were tested for sulfation (Fig. 5B).
Immunologically recovered mutant receptors contained little label,
showing that Y26 is the site of sulfation. To determine whether the
mutation in the neighboring position affects tyrosine sulfation, the
D25A mutant of CCR2B was tested for sulfation. The immunologically
isolated D25A mutant of CCR2B showed a very low level of label,
demonstrating that this consensus acidic amino acid is required for
optimal sulfation at Y26. In all these cases, immunological examination
of the cells expressing the Flag-tagged CCR2B and its mutants showed
that they were all equally expressed on the surface; thus, the
difference in labeling represented the level of sulfation rather than
any differences in expression of the receptor.
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, lanes 1 and 2).
On a control blot treated with an mAb against another Ag, only heavy
and light chains of M2 Ab could be detected with the anti-mouse HRP
conjugate (Fig. 6, lane 3). To test whether
N-glycosidically attached carbohydrates contributed to this
larger size, the immunologically isolated receptor preparation was
treated with N-glycosidase, and the products were examined
by SDS-PAGE (Fig. 7A). The
untreated samples showed a major band at 50 kDa and a minor band at 38
kDa. The N-glycosidase-treated samples showed
immunologically cross-reacting major species at 45 kDa and a minor one
at a slightly lower Mr. We also tested
the size of the receptor produced in the presence of
N-glycosylation inhibitor. CCR2B was immunoprecipitated from
cells growing for 2 days in the presence of 10 µg/ml of tunicamycin
(Fig. 7B). After SDS-PAGE, the major receptor species from
control cells appeared at 50 kDa, with a minor one at 38 kDa, whereas
in tunicamycin-treated cells, the major receptor was found at 45 kDa
with a minor one at a slightly lower
Mr than 38 kDa, exactly as seen after
N-glycosidase treatment. The lowering of the molecular size
by N-glycosidase and tunicamycin treatments shows that CCR2B
contained N-glycosidically attached carbohydrates.
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C). The molecular size of the
35S-labeled CCR2B was shifted from 50 to 45 kDa,
with no indication of any loss in 35S. This
result confirmed the glycoprotein nature of CCR2B and showed that the
sulfation is not on the carbohydrate.
Surface expression of CCR2B decreased 2-fold after treatment with
tunicamycin (Fig. 8). However, this
treatment did not alter Ca2+ influx into the
cytoplasm and adenylate cyclase inhibition at saturating concentrations
of MCP-1 (data not shown). Thus, some of the signaling events caused by
MCP-1 treatment are not affected by the absence of glycosylation. Since
tunicamycin is a general inhibitor of protein
N-glycosylation that can affect many parameters, we did not
perform extensive studies on functional changes that may be caused by
tunicamycin treatment. Specific effects of glycosylation of CCR2B on
its biological function must await site-directed mutagenesis of the
receptor that selectively prevents its glycosylation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß) (36, 37). Interaction of the ligand with the seven-transmembrane
receptor alters GTP binding on the G subunit, with subsequent
dissociation of the receptor-G protein complex and release of G and
Gß subunits (36). Both these complexes can then
activate or inhibit signaling pathways by interacting with effectors
such as adenylate cyclase, phospholipases, cyclic nucleotide
phosphodiesterases, and ion channels (37). The
i subunit of Gi proteins
is involved in the rapid rise in cytoplasmic calcium and the potent
inhibition of adenylate cyclase. Gß is an essential mediator of
chemotaxis (38). In the present study the rapid influx of
calcium into the cytoplasm in response to MCP-1 was compared among the
cells transfected with the wild-type and mutant CCR2B receptors. Our
data suggest that CCR2B and its mutants, T21A, T22A, and F24A, elicit
robust calcium influx and adenylate cyclase inhibition when treated
with MCP-1 in a dose-dependent manner. The other mutants, F23A, D25A,
Y26A, and Y26F, showed neither calcium influx elicitation nor adenylate
cyclase inhibition in response to MCP-1. We found a good correlation
between calcium influx and adenylate cyclase inhibition elicited by all
the receptor mutants.
Among the mutants in the conserved N-terminal region, only one mutant,
Y26F, did not show ligand binding, signaling, and chemotaxis. Other
mutations retain ligand binding, but differentially affect signaling
and chemotaxis. Migration of cells transfected with CCR2B receptor
showed maximal chemotactic activity at 1 nM MCP-1. Cells transfected
with F24A, D25A, and Y26F mutant receptors showed a loss in their
chemotactic activity. Four mutants (F23A, D25A, Y26A, and Y26F) were
found to be defective in adenylate cyclase inhibition and calcium
signaling, and three (F24A, D25A, and Y26F) were defective in cell
migration. Only D25A and Y26F were inactive in signaling and
chemotaxis. F23A and Y26A mutations caused a loss in signaling, but had
only moderate effects on chemotaxis. However, F24A, which retained
calcium elicitation and adenylate cyclase inhibition, showed no
chemotaxis in response to MCP-1. These results show that
Ca2+ influx and chemotaxis elicited via MCP-1
binding to CCR2B are not necessarily coupled. Even though we cannot
rule out the possibility that Ca2+ influx, too
low to detect by the methods we used, is involved in chemotaxis, we
consider it unlikely. Whether the Ca2+ influx
associated with chemokine binding to cell surface receptors is
necessary for chemotaxis has been uncertain (39). Cell
migration has been previously observed in the absence of calcium
influx, and calcium influx has been observed without chemotaxis
(40). Neutrophil chemotaxis toward a formylated peptide
(41) and macrophage-derived chemokine-induced chemotaxis
of eosinophils (42) were reported to occur without
significant changes in Ca2+ signaling.
Arachidonic acid release (43) and phosphoinositol 3-kinase
activity (44), which are thought to be involved in
chemotaxis of monocytes, were not correlated with the
Ca2+ influx induced by MCP-1. Reports that G
is not required for chemotaxis by Gi-coupled
receptors (45) and recent observations that inhibition of
neutrophil chemotaxis was not accompanied by intracellular
Ca2+ levels (46) are consistent with
our results. All these results are consistent with the idea that
lamellipodium formation and chemotaxis are linked to Gß function,
whereas adenylate cyclase inhibition and Ca2+
influx are linked to Gi. However, our
observation that CCR2B structural alterations by a mutation in it
(F24A) barely affects the adenylate cyclase
inhibition/Ca2+ influx, but prevents
lamellipodium formation and chemotaxis, suggests that the receptor
influences chemotaxis in ways other than by merely causing dissociation
of Gß from Gi. One possibility is that
the structural alteration affects association with a kinase necessary
for chemotaxis. However, such possibilities have not been tested.
The regulation of leukocyte migration to the sites of inflammation involves the coordinated action of surface receptors, second messengers, and the cytoskeleton (4, 40, 47). Actin-based morphological changes in various motile cells have been studied in many biological systems (48, 49, 50, 51). Cells need to make certain types of contacts to extracellular matrix on which to crawl; therefore, the degree to which cells anchor themselves to a substrate is directly correlated with their motile activity (50, 52). It has been proposed that in the leading lamellipodia, orthogonally cross-linked actin filaments are contracted by myosin II, pulling the cell body forward (53). With gelsolin-null fibroblasts, evidence was presented indicating that the lamellipodia are necessarily involved in cell motility (54). Lamellipodia protrusion produced by actin polymerization that involves Rac1 activation was suggested to be required for migration of fibroblasts (55). The signaling events in chemotaxis in other cell types involve actin-based morphological changes. For example, stimuli such as IL-8 (47), fMLP (56), and phosphatidic acid (57) induce actin-based morphological changes and chemotaxis in neutrophils. Recent results suggested that a symmetrical establishment of sites of actin polymerization produce directional migration of neutrophils in response to chemotactic gradients (58). Very little is known about the role of actin polymerization in MCP-1-induced chemotaxis of its target cells. During trans-endothelial migration of monocytes, polymerized actin and cell adhesion molecules are colocalized at the leading edge of membrane protrusion (59). Our results show correlation between lamellipodium formation and chemotaxis mediated by MCP-1 binding of CCR2B. They suggest that aa 24, 25, and 26 (F-D-Y), including the putative site of sulfation in the MCP-1 receptor, are important for function of the receptor with respect to lamellipodium formation and chemotaxis. However, Y26A showed the highest responsiveness to MCP-1 in both morphological changes and chemotaxis among all mutants tested here, suggesting that tyrosine sulfation at position 26 is not essential in MCP-1-induced lamellipodium formation and cell migration in vitro. In addition, F23A, which is completely defective in induction of cytosolic Ca2+ influx, showed moderate responsiveness to MCP-1 in the induction of filopodia and lamellipodium formation. This finding suggested that the actin-mediated membrane protrusion induced by MCP-1 is not regulated by the increase in cytosolic Ca2+.
It was shown in a recent paper that the chemokine receptor CCR5, a
principal HIV-1 coreceptor, is post-translationally modified by
sulfation of tyrosines in the N-terminal part of the molecule
(5). Sulfated tyrosines were shown to contribute to the
binding of CCR5 to macrophage inflammatory protein-1 and -1ß and
HIV-1 gp120/CD4 complexes and to the ability of HIV-1 to enter cells
expressing CCR5 and CD4. CXCR4, another important HIV-1 coreceptor, was
also shown to be sulfated. In the present study we show that one of the
N-terminal tyrosines of chemokine receptor CCR2B, Y26, is sulfated, and
this modification may be critical for the interaction of the receptor
with MCP-1. In this segment of the receptor, there is another potential
sulfation site at Y28. Since substitution of Y26 with F or A virtually
abolished sulfation of the receptor under experimental conditions, it
is likely that Y28 is not sulfated. Substitution of one of three
sulfated tyrosines of P-selectin glycoprotein ligand-1 did not lead to
lack of sulfation of two other tyrosines taking part in interaction
with P-selectin, and only substitution of all these tyrosines prevented
binding of the ligand (60).
According to the amino acid sequence of CCR2B, it should be a 41-kDa protein. Previously, an Mr of 38 kDa had been reported for this receptor on the basis of SDS-PAGE (61). In our experiments two molecular species of CCR2B were detected after SDS-PAGE, the major at 50 kDa and the minor one at 38 kDa; both species were visualized with anti-Flag Ab and anti-CCR2B Ab. Another group reported a Mr of 50 kDa for chemokine receptor CXCR4, which has a theoretical Mr of 40 kDa according to the amino acid sequence (62). The same Mr has been reported for chemokine receptor CCR5 (63), which has a very similar number of amino acids with a calculated Mr of 40.6 kDa. When CCR2B was indirectly immunoprecipitated with anti-Flag mAb M2, the main band of receptor was clearly resolved from the heavy chain of the mAb, which was at about 55 kDa.
Examination of the amino acid sequences of chemokines of CC and CXC groups shows that most of the chemokines contain potential sites for N-glycosylation in their N-terminal segments. There is only one consensus N-glycosylation site in the amino acid sequence of CCR2B (residues 14–17). Our results from N-glycosidase treatment of CCR2B and tunicamycin treatment of cells expressing CCR2B very strongly suggest that this receptor contains an N-bound glycan. The loss of this glycan converted the major 50-kDa receptor to a species migrating as 45 kDa on SDS gels. A similar shift in mobility was observed after N-glycosidase treatment in the case of 35S-labeled receptor, showing that the sulfated receptor was also glycosylated. It is known that both modifications occur in trans-Golgi, just before export of the protein to the cell surface, consistent with the idea that the 50-kDa form is probably surface expressed. We did not observe any decrease in 35S in the receptor band after treatment with N-glycosidase. This observation and the lack of 35S labeling of the receptor with Y26 substitutions show that virtually all the sulfate label incorporated into CCR2B was located on Tyr26. The minor immunologically cross-reacting component found at 38 kDa may be a degradation product. Since N-deglycosylation also reduced the size of this species, it also is a glycosylated moiety. In our 35S labeling studies, we did not find a sulfated 38-kDa species, possibly because it is only a minor component. Since it is likely to be a minor degradation product, we did not pursue its identity.
Tyrosine sulfation is probably not directly involved in lamellipodium
formation and chemotaxis, as shown by our finding that cells expressing
Y26A mutant, which is not sulfated, respond to MCP-1 binding by
lamellipodium formation and are chemotaxed. It is noteworthy, however,
that Y26F was inactive in MCP-1 binding, signaling, lamellipodium
formation, and chemotaxis, although it was surface expressed, and its
sequence was reconfirmed to be correct. Substitution of the highly
polar sulfated Y26 in the receptor with a highly hydrophobic F probably
causes significant abnormalities in the conformation of the N terminus
that is known to be important for the function. Substitution with a
much less hydrophobic A would cause much less conformational
perturbation, and thus Y26A retains some of the functions of the
receptor. In any case, the signaling involved in lamellipodium
formation does not require Tyr26
sulfation in CCR2B. When free MCP-1 binds to the receptor on the cell
surface in the in vitro chemotaxis assays, sulfation of
Tyr26 of the receptor is not necessary,
as Y26A forms lamellipodia and is active in chemotaxis. In such assays,
addition of heparin does not inhibit chemotaxis because the heparin
bound at the C-terminal basic amino acids (64), distal to
the receptor binding domains of MCP-1, does not interfere with binding
to the receptor. On the other hand, Tyr26
probably plays an important role in the trans-endothelial
migration of monocytes in vivo. This process would involve the binding
not of free MCP-1, but of MCP-1 that is tethered to the endothelial
cells via interaction with the sulfates of the proteoglycans on the
endothelial surface. We have previously identified two basic amino acid
residues in the C-terminal -helix of MCP-1 that are involved in this
ionic interaction (64). Probably, the sulfated N-terminal
domain of the receptor initially grabs the tethered MCP-1 by ionic
interaction for the other extracellular interacting domains of CCR2B to
complete the ligand receptor binding that involves the other amino acid
residues of MCP-1. This interaction with the other domains of CCR2B
probably thermodynamically promotes transfer of the tethered MCP-1 to
CCR2B.
Our results show that mutants of CCR2B that allow lamellipodium formation retain chemotactic capability even when such mutants do not elicit detectable Ca2+ influx and adenylate cyclase inhibition. Our finding that the actin-mediated membrane protrusions induced by MCP-1 and chemotaxis are not regulated by the increase in cytosolic Ca2+ suggests that the biological consequences of MCP-1 binding to its receptor can be viewed as two distinct steps 1) chemotaxis that involves lamellipodium formation, and 2) the other signaling events involving Ca2+ influx and adenylate cyclase inhibition and subsequent events that probably lead to changes in gene expression resulting in activation of the target cells. In the recent transgenic experiments in which MCP-1 production was targeted at specific tissues, chemotaxis of monocytes/macrophages to the location of MCP-1 production was observed. However, these infiltrating cells were apparently not activated to cause any pathology (65, 66, 67), except in one case where targeted expression of MCP-1 in the cardiomyocytes caused heart failure in older animals as a result of the events initiated by the infiltrating monocytes (68). The general conclusion from the transgenic experiments has been that MCP-1 causes infiltration of target cells, but their activation requires additional signals. The molecular events initiated in the monocytes/macrophages as a result of MCP-1 binding, the nature of the additional signals that are required to trigger activation, and the molecular events involved in this activation remain to be elucidated.
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Acknowledgments |
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Footnotes |
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2 Abbreviation used in this paper: MCP-1, monocyte chemotactic protein-1.
Received for publication March 14, 2000. Accepted for publication August 9, 2000.
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receptor, confers chemokine selectivity: evidence for a two-step mechanism for MCP-1 receptor activation. J. Biol. Chem. 271:19084. subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA 94:14489.i is not required for chemotaxis mediated by Gi-coupled receptors. J. Biol. Chem. 274:2824.-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding. J. Biol. Chem. 273:29641.This article has been cited by other articles:
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