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The Journal of Immunology, 2000, 165: 5295-5303.
Copyright © 2000 by The American Association of Immunologists

Monocyte Chemotactic Protein-1 Receptor CCR2B Is a Glycoprotein That Has Tyrosine Sulfation in a Conserved Extracellular N-Terminal Region

Alexander A. Preobrazhensky, Sofya Dragan, Tomonori Kawano, Mikhail A. Gavrilin, Irina V. Gulina, Leena Chakravarty and P. E. Kolattukudy1

Neurobiotechnology Center and Departments of Biochemistry and Molecular and Cellular Biochemistry, Ohio State University, Columbus, OH 43210


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocyte chemotactic protein-1 (MCP-1) binding to its receptor, CCR2B, plays an important role in a variety of diseases involving infection, inflammation, and/or injury. In our effort to understand the molecular basis of this interaction and its biological consequences, we recognized a conserved hexad of amino acids at the N-terminal extracellular domain of several chemokine receptors, including CCR2B. Human embryonic kidney 293 cells expressing Flag-tagged CCR2B containing site-directed mutations in this region, 21–26, including a consensus tyrosine sulfation site were used to determine MCP-1 binding and its biological consequences. The results showed that several of these amino acids are important for MCP-1 binding and consequent lamellipodium formation, chemotaxis, and signal transduction involving adenylate cyclase inhibition and Ca2+ influx into cytoplasm. Mutations that prevented adenylate cyclase inhibition and Ca2+ influx did not significantly inhibit lamellipodium formation and chemotaxis, suggesting that these signaling events are not involved in chemotaxis. CCR2B was found to be sulfated at Tyr26; this sulfation was abolished by the substitution of Tyr with Ala and severely reduced by substitution of Asp25, a part of the consensus sulfation site. The expressed CCR2B was found to be N-glycosylated, as N-glycosidase F treatment of the receptor or growth of the cells in tunicamycin reduced the receptor size to the same level, from 50 to 45 kDa. Thus, CCR2B is the first member of the CC chemokine receptor family shown to be a glycoprotein that is sulfated at the N-terminal Tyr. These post-translational modifications probably have significant biological functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemotactic cytokines (chemokines) recruit leukocytes to sites of injury, infection, and inflammation (1, 2, 3, 4). Chemokines are classified mainly in terms of the patterns of Cys residues, which form structurally important disulfide bonds. Those with an amino acid residue between the N-terminal pair of Cys residues, called {alpha} 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{alpha} and Gß{gamma}. 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.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

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, {kappa} (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 {kappa} 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.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The N-terminal extracellular domain of chemokine receptors is thought to play an important role in ligand binding. Within this region a segment is conserved among CCR1, CCR2, and the CMV-encoded US28. Five residues within the hexapeptide TTFFDY located at positions 21–26 in CCR2B are identical in the three receptors. To test whether this conservation has any functional significance, we used a site-directed mutagenesis approach. We replaced all residues in the hexad with alanine, and each residue was individually substituted by Ala. We expressed the Flag-tagged mutants fused to prolactin leader peptide in pcDNA3 vector in HEK293 cells after confirming by DNA sequencing that each construct did not contain any unintended mutations. Stable transformants were cloned, and the expression of the receptor by each clone was tested by flow cytometry with anti-Flag M2 Ab. The highly expressing clones were selected for producing the cells used for further studies on CCR2B and its mutants; the surface expressions of all the mutants used for our studies were comparable (Fig. 1Go and Table IGo). 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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  		FIGURE 1. Surface expression of CCR2B and its mutants in HEK293 cells. Cloned stably transfected HEK293 cells were grown and examined for expression of the CCR2B and its mutants by flow cytometry after incubation with the Flag mAb conjugated with FITC. Untransfected and transfected HEK293 are denoted by the bold solid line and dotted line, respectively.

 

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  		Table I. Effects of mutation in the conserved hexad of amino acids in the N-terminal domain of CCR2B on its biological activity

 
Substitution of all conserved amino acid residues with Ala abolished MCP-1 binding and Ca2+ influx into the cytoplasm caused by MCP-1 addition (data not shown). When mutants with single substitutions were compared, F24A caused a 5-fold decrease in binding, whereas F23A and D25A showed only minimal changes in binding (Table IGo). 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. 2Go). 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. 3Go). 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. 3Go), 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. 3Go). 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. 3Go and Table IGo). 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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  		FIGURE 2. Confocal microscopic image of lamellipodia induced by MCP-1 treatment of HEK293 cells expressing CCR2B and its mutants. HEK293 cells transfected with wild-type (Wt) CCR2B and its mutants were left resting (control) or were stimulated with 50 nM MCP-1 for 15 min on plastic slides, and actin-based morphological changes were visualized with phalloidin-FITC. Experiments were repeated three times, and most representative images are presented.

 


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  		FIGURE 3. Correlation between lamellipodium formation and chemotactic migration by MCP-1 treatment of HEK293 cells expressing CCR2B and its mutants. HEK293 cells untransfected or transfected with CCR2B or its mutants were left resting or were stimulated with 50 nM MCP-1 for 15 min on plastic slides. The number of cells forming lamellipodia was counted before and after addition of 50 nM MCP-1. Cells forming lamellipodia (>12 µm) were counted under the confocal microscope, and this number was plotted against migration (percentage).

 
Chemotaxis of HEK293 cells expressing the wild-type CCR2B and its mutants showed that chemotactic capability drastically decreased by the substitution of F24 or D25 by Ala and moderately decreased by substitution of T22 with Ala (Table IGo). 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. 4GoA).



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  		FIGURE 4. Measurement of calcium influx and adenylate cyclase inhibition by MCP-1 in HEK293 cells expressing CCR2B or its mutants. A, The MCP-1 concentration dependence of fluorescence enhancement in fura-2-labeled HEK293 cells expressing CCR2B or its mutants was measured. The calcium flux is expressed as a percentage of the maximum fluorescence elicited by treating the cells with digitonin. B, MCP-1-dependent inhibition of adenylate cyclase was measured as described in Materials and Methods. IC50 values are shown in Table IGo.

 
Since CCR2B is a G protein-coupled receptor that functions with Gi, MCP-1 binding causes adenylate cyclase inhibition (Fig. 4GoB). 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. 4GoB). 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. 5GoA). 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. 5GoB). 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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  		FIGURE 5. Immunoprecipitation of [35S]sulfate-labeled CCR2B and its mutants from HEK293 cells expressing CCR2B. A, Indirect precipitation using mAb M2 and protein G-agarose (lane 3), control Ab (lane 2), and total cell extract before immunoprecipitation (lane 1). B, Direct precipitation of 35S-labeled proteins with M2-agarose gel.

 
Sulfation experiments showed that the 35S-labeled receptor and its mutant D25A migrated in SDS-PAGE as a protein considerably larger in size (50 kDa) than the size (41 kDa) expected from its amino acid composition. CCR2B immunoprecipitated from the detergent extract with anti-Flag mAb (M2) and protein G-agarose was subjected to SDS-PAGE and immunoblotted with the anti-Flag Ab or anti-CCR2B Ab (CCR2-03) (30). In both cases, a major 50-kDa species and a minor 38-kDa species were found (Fig. 6Go, 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. 6Go, 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. 7GoA). 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. 7GoB). 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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  		FIGURE 6. Immunoprecipitation of CCR2B expressed in HEK 293 cells by M2-agarose gel. Immunoblots were performed with mAb M2 (lane 1), mAb CCR2-03 (lane 2), and control Ab (lane 3).

 


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  		FIGURE 7. Decrease in the molecular size of CCR2B expressed in HEK293 cells by treatment with N-glycosidase F (A and C) and by treatment of the cells with tunicamycin (B). +, Treatment; -, control. In all cases, immunodetection was with mAbM2. C, The transfected cells were grown in 35SO4-2.

 
To test whether the N-glycosidically attached carbohydrates on the receptor are sulfated, 35S-labeled CCR2B, immunologically isolated from HEK293 cells expressing this receptor in 35SO4-2 containing medium, was treated with N-glycosidase, and the products were subjected to SDS-PAGE and phosphorimaging (Fig. 7GoC). 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. 8Go). 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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  		FIGURE 8. Inhibition of the surface expression of CCR2B by tunicamycin. HEK293 cells transfected with CCR2B were incubated with 3.3 µg/ml tunicamycin for 2 days and stained with anti-Flag M2 and FITC-conjugated anti-mouse IgG Abs. The bold curve indicates staining in untreated (control) cells, and the light curve represents CCR2B expression in the presence of tunicamycin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The extracellular N-terminal domain of chemokine receptors is thought to play a significant role in binding the ligand (31, 32). In our effort to explore possible functions of this N-terminal region, we noted that a segment within this region is conserved in CCR1, CCR2, and the virally encoded US28 that also binds MCP-1 and RANTES (33, 34, 35). To test for a possible functional role of this segment, site-directed mutagenesis was used. This approach indicated that some of the residues in this segment are important for the productive interaction of MCP-1 with CCR2B. One of the functions we tested involves the signaling elicited by MCP-1 binding on the receptor CCR2B. Chemokines, like a large number of hormones and neurotransmitters, exert their effect on cells by binding to a heterotrimeric G protein-coupled receptor (G{alpha}ß{gamma}) (36, 37). Interaction of the ligand with the seven-transmembrane receptor alters GTP binding on the G{alpha} subunit, with subsequent dissociation of the receptor-G protein complex and release of G{alpha} and Gß{gamma} 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 {alpha}i subunit of Gi proteins is involved in the rapid rise in cytoplasmic calcium and the potent inhibition of adenylate cyclase. Gß{gamma} 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{alpha} 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{gamma} function, whereas adenylate cyclase inhibition and Ca2+ influx are linked to Gi{alpha}. 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{gamma} from Gi{alpha}. 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{alpha} 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 {alpha}-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.


   Acknowledgments
 
We thank Drs. Cliff Beall and Janice Liu for assistance with preliminary experiments. We thank Dr. I. Charo for providing us with the prolactin leader construct and Dr. M. Mellado for providing us with CCR2 Abs.


   Footnotes
 
1 Address correspondence and reprint requests to Dr. P. E. Kolattukudy, Neurobiotechnology Center, Ohio State University, 206 Rightmire Hall, 1060 Carmack Road, Columbus, OH 43210. Back

2 Abbreviation used in this paper: MCP-1, monocyte chemotactic protein-1. Back

Received for publication March 14, 2000. Accepted for publication August 9, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Murphy, P. M.. 1994. The molecular biology of leukocyte chemoattractant receptors. Annu. Rev. Immunol. 12:593.[Medline]
  2. Rollins, B. J.. 1997. Chemokines. Blood 90:909.[Free Full Text]
  3. Luster, A. D.. 1998. Chemokines: chemotactic cytokines that mediate inflammation. N. Engl. J. Med. 338:436.[Free Full Text]
  4. Baggiolini, M.. 1998. Chemokines and leukocyte traffic. Nature 329:565.
  5. Farzan, M., T. Mirzabekov, P. Kolchinsky, R. Wyatt, M. Cayabyab, N. P. Gerard, C. Gerard, J. Sodroski, H. Choe. 1999. Tyrosine sulfation of the amino terminus of CCR5 facilitates HIV-1 entry. Cell 96:667.[Medline]
  6. Huttner, W. B.. 1988. Tyrosine sulfation and the secretory pathway. Annu. Rev. Physiol. 50:363.[Medline]
  7. Huttner, W. B.. 1984. Determination and occurrence of tyrosine O-sulfate in proteins. Methods Enzymol. 107:200.[Medline]
  8. Ouyang, Y.-B., W. S. Lane, K. L. Moore. 1998. Tyrosylprotein sulfotransferase: purification and molecular cloning of an enzyme that catalyzes tyrosine O-sulfation, a common posttranslational modification of eukaryotic proteins. Proc. Natl. Acad. Sci. USA 95:2896.[Abstract/Free Full Text]
  9. Beisswanger, R., D. Corbeil, C. Vannier, C. Thiele, U. Dohrman, R. Kellner, K. Ashman, C. Niehrs, W. B. Huttner. 1998. Existence of distinct tyrosylprotein sulfotransferase genes: molecular characterization of tyrosylprotein sulfotransferase-2. Proc. Natl. Acad. Sci. USA 95:11134.[Abstract/Free Full Text]
  10. Ouyang, Y.-B., K. L. Moore. 1998. Molecular cloning and expression of human and mouse tyrosylprotein sulfotransferase-2 and a tyrosylprotein sulfotransferase homologue in Caenorhabditis elegans. J. Biol. Chem. 273:24770.[Abstract/Free Full Text]
  11. Bundgaard, J. R., J. Vuust, J. F. Rehfeld. 1997. New consensus features for tyrosine O-sulfation determined by mutational analysis. J. Biol. Chem. 272:21700.[Abstract/Free Full Text]
  12. Stone, S. R., J. Hofsteenge. 1986. Kinetics of the inhibition of thrombin by hirudin. Biochemistry 25:4622.[Medline]
  13. Hofsteenge, J., S. R. Stone, A. Donella-Deana, L. A. Pinna. 1990. The effect of substituting phosphotyrosine for sulphotyrosine on the activity of hirudin. Eur. J. Biochem. 188:55.[Medline]
  14. Leyte, A., H. B. van Schijndel, C. Niehrs, W. B. Huttner, M. P. Verbeet, K. Mertens, J. A. van Mourik. 1991. Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor. J. Biol. Chem. 266:740.[Abstract/Free Full Text]
  15. Pouyani, T., B. Seed. 1995. PSGL-1 recognition of P-selectin is controlled by a tyrosine sulfation consensus at the PSGL-1 amino terminus. Cell 83:333.[Medline]
  16. Wilkins, P. P., K. L. Moore, R. P. McEver, R. D. Cumming. 1995. Tyrosine sulfation of P-selectin glycoprotein ligand-1 is required for high affinity binding to P-selectin. J. Biol. Chem. 270:22677.[Abstract/Free Full Text]
  17. Li, Y.-S., Y.-J. Shyy, J. G. Wright, A. J. Valente, J. F. Cornhill, P. E. Kolattukudy. 1993. The expression of monocyte chemotactic protein (MCP-1) in human vascular endothelium in vitro and in vivo. Mol. Cell. Biochem. 126:61.[Medline]
  18. Beall, C. J., S. Mahajan, D. E. Kuhn, P. E. Kolattukudy. 1996. Site-directed mutagenesis of monocyte chemoattractant protein-1 identifies two regions of the polypeptide essential for biological activity. Biochem. J. 313:633.
  19. Charo, I. F., S. J. Myers, A. Herman, C. Franci, A. J. Connolly, S. R. Coughlin. 1994. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc. Natl. Acad. Sci. USA 91:2752.[Abstract/Free Full Text]
  20. Franci, C., J. Gosling, C.-L. Tsou, S. R. Coughlin, I. F. Charo. 1996. Phosphorylation by a G protein-coupled kinase inhibits signaling and promotes internalization of the monocyte chemoattractant protein-1 receptor: critical role of carboxyl-tail serines/threonines in receptor function. J. Immunol. 157:5606.[Abstract]
  21. Prickett, K. S., D. C. Amberg, T. P. Hopp. 1989. A calcium-dependent antibody for identification and purification of recombinant proteins. BioTechniques 7:580.[Medline]
  22. Higuchi, R., B. Krummel, R. K. Saiki. 1988. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 16:7351.[Abstract/Free Full Text]
  23. Hunter, W. M., F. C. Greenwood. 1962. Preparation of iodine-131 labeled human growth hormone of high specific affinity. Nature 194:495.[Medline]
  24. Van Riper, G., S. Siciliano, P. A. Fischer, R. Meurer, M. S. Springer, H. Rosen. 1993. Characterization and species distribution of high-affinity GTP-coupled receptors for human rantes and monocyte chemoattractant protein-1. J. Exp. Med. 177:851.[Abstract/Free Full Text]
  25. Munson, P. J., D. Robard. 1980. Ligand: a versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220.[Medline]
  26. Hung, D. T., Y. H. Wong, T.-K. H. Vu, S. R. Coughlin. 1992. The cloned platelet thrombin receptor couples to at least 2 distinct effectors to stimulate phosphoinositide hydrolysis and inhibit adenylyl cyclase. J. Biol. Chem. 267:20831.[Abstract/Free Full Text]
  27. Solomon, Y., C. Loudos, M. Rodbell. 1974. A highly sensitive adenylate cyclase assay. Anal. Biochem. 58:541.[Medline]
  28. Myers, S. J., L. M. Wong, I. F. Charo. 1995. Signal-transduction and ligand specificity of the human monocyte chemoattractant protein-1 receptor in transfected embryonic kidney-cells. J. Biol. Chem. 270:5786.[Abstract/Free Full Text]
  29. Wulf, E., A. Deboben, F. A. Bautz, H. Faulstich, T. H. Weiland. 1979. Fluorescent phalloidin, a tool for the visualization of cellular actin. Proc. Natl. Acad. Sci. USA 76:4498.[Abstract/Free Full Text]
  30. Frade, J. M. R., M. Mellado, G. del Real, J. C. Gutierrez-Ramos, P. Lind, C. Martinez-A. 1997. Characterization of the CCR2 chemokine receptor: functional CCR2 receptor expression in B cells. J. Immunol. 159:5576.[Abstract]
  31. Monteclaro, F. S., I. F. Charo. 1996. The amino-terminal extracellular domain of the MCP-1 receptor, but not the RANTES/MIP-1{alpha} receptor, confers chemokine selectivity: evidence for a two-step mechanism for MCP-1 receptor activation. J. Biol. Chem. 271:19084.[Abstract/Free Full Text]
  32. Monteclaro, F. S., I. F. Charo. 1997. The amino-terminal domain of CCR2 is both necessary and sufficient for high affinity binding of monocyte chemoattractant protein 1: receptor activation by a pseudo-tethered ligand. J. Biol. Chem. 272:23186.[Abstract/Free Full Text]
  33. Kuhn, D. E., C. J. Beall, P. E. Kolattukudy. 1995. The cytomegalovirus US28 protein binds multiple CC-chemokines with high-affinity. Biochem. Biophys. Res. Commun. 211:325.[Medline]
  34. Bodaghi, B., T. R. Jones, D. Zipeto, C. Vita, L. Sun, L. Laurent, F. Arenzana-Seisdedos, J. L. Virelizier, S. Michelson. 1998. Chemokine sequestration by viral chemoreceptors as a novel viral escape strategy: withdrawal of chemokines from the environment of cytomegalovirus-infected cells. J. Exp. Med. 188:855.[Abstract/Free Full Text]
  35. Billstrom, M. A., G. L. Johnson, N. J. Avdi, G. S. Worthen. 1998. Intracellular signaling by the chemokine receptor US28 during human cytomegalovirus infection. J. Virol. 72:5535.[Abstract/Free Full Text]
  36. Hamm, H. E.. 1998. The many faces of G protein signaling. J. Biol. Chem. 273:669.[Free Full Text]
  37. Berman, D. M., A. G. Gilman. 1998. Mammalian RGS proteins: barbarians at the gate. J. Biol. Chem. 273:1269.[Free Full Text]
  38. Neptune, E. R., H. R. Bourne. 1997. Receptors induce chemotaxis by releasing the ß{gamma} subunit of Gi, not by activating Gq or Gs. Proc. Natl. Acad. Sci. USA 94:14489.[Abstract/Free Full Text]
  39. Sozzani, S., M. Locati, D. Zhou, M. Rieppi, W. Luini, G. Lamorte, G. Bianchi, N. Polentarutti, P. Allavena, A. Mantovani. 1995. Receptors, signal transduction, and spectrum of action of monocyte chemotactic protein-1 and related chemokines. J. Leukocyte Biol. 57:788.[Abstract]
  40. Needham, M., N. Sturgess, G. Cerillo, I. Green, H. Warburton, R. Wilson, L. Martin, D. Barratt, M. Anderson, C. Reilly, et al 1996. Monocyte chemoattractant protein-1: receptor interactions and calcium signaling mechanisms. J. Leukocyte Biol. 60:793.[Abstract]
  41. Laffafian, I., M. B. Hallett. 1995. Does cytosolic free Ca2+ signal neutrophil chemotaxis in response to formylated chemotactic peptide?. J. Cell Sci. 108:3199.[Abstract]
  42. Bochner, B. S., C. A. Bickel, M. L. Taylor, Jr D. W. MacGlashan, P. W. Gray, C. J. Raport, R. Godiska. 1999. Macrophage-derived chemokine induces human eosinophil chemotaxis in a CC chemokine receptor 3- and CC chemokine receptor 4-independent manner. J. Allergy Clin. Immunol. 103:527.[Medline]
  43. Locati, M., D. Zhou, W. Luini, V. Evangelista, A. Mantovani, S. Sozzani. 1994. Rapid induction of arachidonic acid release by monocyte chemotactic protein-1 and related chemokines: role of Ca2+ influx, synergism with platelet-activating factor and significance for chemotaxis. J. Biol. Chem. 269:4746.[Abstract/Free Full Text]
  44. Yen, H.-H., Y. Zhang, S. Penford, B. J. Rollins. 1997. MCP-1-mediated chemotaxis requires activation of non-overlapping signal transduction pathways. J. Leukocyte Biol. 61:529.[Abstract]
  45. Neptune, E. R., T. Iiri, H. R. Bourne. 1999. G{alpha}i is not required for chemotaxis mediated by Gi-coupled receptors. J. Biol. Chem. 274:2824.[Abstract/Free Full Text]
  46. Konishi, K., M. Urada, I. Adachi, T. Tanaka. 2000. Inhibitory effect of sanguiin H-11 on chemotaxis of neutrophil. Biol. Pharm. Bull. 23:213.[Medline]
  47. Sham, R. L., P. D. Phatak, T. P. Ihne, C. N. Abboud, C. H. Packman. 1993. Signal pathway regulation of interleukin-8-induced actin polymerization in neutrophils. Blood 82:2546.[Abstract/Free Full Text]
  48. Hall, A.. 1998. Rho GTPases and the actin cytoskeleton. Science 279:509.[Abstract/Free Full Text]
  49. Parent, C. A., P. N. Devreotes. 1999. A cell’s sense of direction. Science 284:765.[Abstract/Free Full Text]
  50. Small, J. V., K. Rottner, I. Kaverina. 1999. Functional design in the actin cytoskeleton. Curr. Opin. Cell Biol. 11:54.[Medline]
  51. Waterman-Storer, C. M., E. D. Salmon. 1999. Positive feedback interactions between microtubule and actin dynamics during cell motility. Curr. Opin. Cell Biol. 11:61.[Medline]
  52. Huttenlocher, A., R. R. Sandborg, A. F. Horwitz. 1995. Adhesion in cell migration. Curr. Opin. Cell Biol. 7:697.[Medline]
  53. Svitkina, T. M., A. V. Verkhovsky, K. M. McQuade, G. G. Borisy. 1997. Analysis of the actin-myosin II system in fish epidermal keratocytes: mechanism of cell body translocation. J. Cell Biol. 139:397.[Abstract/Free Full Text]
  54. Azuma, T., W. Witke, T. P. Stossel, J. H. Hartwig, D. J. Kwiatkowski. 1998. Gelsolin is a downstream effector of rac for fibroblast motility. EMBO J. 17:1362.[Medline]
  55. Waterman-Storer, C. M., R. A. Worthylake, B. P. Liu, K. Burridge, E. D. Salmon. 1999. Microtubule growth activates Rac1 to promote lamellipodial protrusion in fibroblasts. Nat. Cell. Biol. 1:45.[Medline]
  56. Arcaro, A., M. P. Wymann. 1993. Wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses. Biochem. J. 296:297.
  57. Siddiqui, R. A., D. English. 1997. Phosphatidic acid elicits calcium mobilization and actin polymerization through a tyrosine kinase-dependent process in human neutrophils: a mechanism for induction of chemotaxis. Biochim. Biophys. Acta 1349:81.[Medline]
  58. Weiner, O. D., G. Servant, M. D. Welch, T. J. Mitchison, J. W. Sedat, H. R. Bourne. 1999. Spatial control of actin polymerization during neutrophil chemotaxis. Nat. Cell. Biol. 1:75.[Medline]
  59. Sandig, M., E. Negrou, K. A. Rogers. 1997. Changes in the distribution of LFA-1, catenins and F-actin during transendothelial migration of monocytes in culture. J. Cell Sci. 110:2807.[Abstract]
  60. Liu, W.-J., V. Ramachandran, J. Kang, T. K. Kishimoto, R. D. Cummings, R. P. McEver. 1998. Identification of N-terminal residues on P-selectin glycoprotein ligand-1 required for binding to P-selectin. J. Biol. Chem. 273:7078.[Abstract/Free Full Text]
  61. Rodriguez-Frade, J. M., A. J. Vila-Coro, A. M. de Ana, J. P. Albar, C. Matrinez-A., M. Mellado. 1999. The chemokine monocyte chemoattractant protein-1 induces functional responses through dimerization of its receptor CCR2. Proc. Natl. Acad. Sci. USA 96:3628.[Abstract/Free Full Text]
  62. Zhou, H., H.-H. Tai. 1999. Characterization of recombinant human CXCR4 in insect cells: role of extracellular domains and N-glycosylation in ligand binding. Arch. Biochem. Biophys. 369:267.[Medline]
  63. Zhao, J., L.-H. Ben, Y.-L. Wu, W. Hu, K. Ling, S.-M. Xin, H.-L. Nie, L. Ma, G. Pei. 1999. Anti-HIV agent trichosanthin enhances the capabilities of chemokines to stimulate chemotaxis and G protein activation, and this is mediated through interaction of trichosanthin and chemokine receptors. J. Exp. Med. 190:101.[Abstract/Free Full Text]
  64. Chakravarty, L., L. Rogers, T. Quach, S. Breckenridge, P. E. Kolattukudy. 1998. Lysine 58 and histidine 66 at the C-terminal {alpha}-helix of monocyte chemoattractant protein-1 are essential for glycosaminoglycan binding. J. Biol. Chem. 273:29641.[Abstract/Free Full Text]
  65. Fuentes, M. E., S. K. Durham, M. R. Swergel, A. C. Lewin, D. C. Barton, J. R. Megill, R. Bravo, S. A. Lira. 1995. Controlled recruitment of monocytes and macrophages to specific organs through transgenic expression of monocyte chemoattractant protein-1. J. Immunol. 155:5769.[Abstract]
  66. Grewal, I. S., B. J. Rutledge, J. A. Fiorillo, L. Gu, R. P. Gladue, R. A. Flavell, B. J. Rollins. 1997. Transgenic monocyte chemoattractant protein-1 (MCP-1) in pancreatic islets produces monocyte-rich insulitis without diabetes: abrogation by a second transgene expressing systemic MCP-1. J. Immunol. 159:401.[Abstract]
  67. Gunn, M. D., N. A. Helken, X. Liao, L. T. Williams. 1997. Monocyte chemoattractant protein-1 is sufficient for the chemotaxis of monocytes and lymphocytes in transgenic mice but requires an additional stimulus for inflammatory activation. J. Immunol. 158:376.[Abstract]
  68. Kolattukudy, P. E., T. Quach, S. Bergese, S. Breckenridge, J. Hensley, R. Altschuld, G. Gordillo, S. Klenotic, C. Orosz, J. Parker-Thornburg. 1998. Myocarditis induced by targeted expression of the MCP-1 gene in murine cardiac muscle. Am. J. Pathol. 152:101.[Abstract]



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