Cloning and functional expression of mCCR2, a murine receptor for the C-C chemokines JE and FIC.

The C-C chemokines human monocyte chemoattractant protein-1 and −3 (MCP-1 and MCP-3) and mouse JE and FIC are potent activators of monocytes. Several receptors for MCP-1 and MCP-3 have been cloned from human monocytic cell lines, and one of these receptors, CCR2B, binds both MCP-1 and MCP-3. Thus far, no murine receptors for JE or FIC have been reported. We have cloned a novel murine C-C chemokine receptor, designated mouse CCR2 (mCCR2), from the mouse monocyte cell line WEHI265.1. The predicted 373-amino acid sequence of mCCR2 shows highest identity (80%) with CCR2B. When stably expressed in human embryonic kidney 293 cells, mCCR2 specifically bound I-JE with high affinity. FIC was less potent than JE in competing I-JE binding to mCCR2-expressing cells, while three other mouse chemokines, MIP-1α, C10, and N51/KC, did not compete. mccr2 mRNA expression was detected in elicited peritoneal macrophages as well as in several mouse organs. The cloning of mCCR2 provides an important tool to investigate monocyte/macrophage responses to JE and FIC, to identify other targets for their action, and potentially to study models of CCR2 function in the mouse.

Chemokines are small secreted molecules that chemoattract and activate specific leukocyte subpopulations in vitro and are thought to be important for leukocyte trafficking in vivo (1)(2)(3). The chemokine superfamily has traditionally been divided into two subgroups, C-X-C or C-C, based upon the presence or absence of an amino acid between the first two cysteine residues of a conserved four-cysteine motif. In general, C-X-C chemokines attract neutrophils, while C-C chemokines attract mononuclear cells. Human monocyte chemoattractant protein-1 (MCP-1), 1 MCP-2, MCP-3, mouse JE, and mouse FIC/ MARC are structurally related (50 -72% identity) C-C chemokines that are potent in vitro chemoattractants and activators of monocytes (4 -14). In vivo, MCP-1 has been implicated in monocytic infiltration of tissues during several inflammatory diseases including atherosclerosis (15,16) and rheumatoid arthritis (17), while JE has been implicated in macrophage-mediated tumor growth suppression in mice (18). Recent transgenic mouse models have suggested a role for JE in monocyte/ macrophage recruitment and in host responses to intracellular pathogens (19,20). To further understand the biological functions of MCP-1, MCP-3, JE, FIC, and related chemokines, it will be important to identify and characterize the receptors that bind and mediate their responses.
Three cDNAs, ccr1, ccr2A, and ccr2B, that encode receptors for MCP-1 and/or MCP-3, have been cloned from human monocytic cell lines (21)(22)(23). All three cDNAs encode seven-transmembrane G protein-coupled receptors, and the alternatively spliced CCR2A and CCR2B receptors differ only in their intracellular carboxyl-terminal tails. When expressed in human embryonic kidney 293 (HEK293) cells, CCR2B binds and signals in response to MCP-1 and MCP-3 but not to other C-C chemokines such as MCP-2, RANTES, MIP-1␣, and MIP-1␤ (24,25). When expressed in HEK293 cells, CCR1 receptor binds and signals in response to MCP-3, MIP-1␣, and RANTES, but not to MCP-1 (21,26,27). Because JE and FIC bind some of the same receptors on human monocytes as MCP-1 (13), we predicted that murine receptors for JE and FIC might resemble CCR1 and CCR2A/B. Two murine cDNAs homologous to ccr1 have been cloned, but thus far these receptors have only been reported to bind and signal in response to mouse MIP-1␣ and MIP-1␤ (28,29). We report here the molecular cloning, RNA expression, and functional characterization of the first murine receptor that binds JE and FIC.

EXPERIMENTAL PROCEDURES
Cell Culture-The mouse monocytic cell line WEHI265.1 (ATCC) was grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1% ␤-mercaptoethanol. Human embryonic kidney 293 cells (HEK293; ATCC) were grown in minimal essential medium supplemented with 10% heat-inactivated horse serum and were transfected by calcium phosphate co-precipitation (30). Transfected HEK293 clones were selected by growth in 0.6 mg/ml G418 for 2-3 weeks.
Library Construction-mRNA was prepared from WEHI265.1 cells by the QuickPrep mRNA purification kit (Pharmacia Biotech Inc.), and cDNA was synthesized from the mRNA using the ZAP-cDNA synthesis kit (Stratagene). The cDNA was cloned into the EcoRI-XhoI polylinker sites of pcDNAI-Amp (InVitrogen), and a plasmid library of Ϸ1.25 ϫ 10 6 clones was generated by electroporation into the bacterial strain DH10B. The average insert size of the cDNA clones is 1.6 kb.
PCR Amplification and cDNA Cloning-2 g of the cDNA library was used as template for PCR with the following degenerate primers: sense, 5Ј-GGCAGGATCCAACCTGGCCAT(C/T)TCTGA(T/C)CTGCT-3Ј; and antisense, 5Ј-CGGTGAATTCTAGGG(A/G)GTCCA(A/G)AA-GAGAAA-3Ј. The PCR was performed with 0.5 M each of sense and antisense primers under the following amplification conditions: 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C, 35 cycles. Amplified bands of the expected sizes were digested with the restriction endonucleases BamHI and EcoRI, gel-purified (Qiagen), and cloned into pBS-KSϩ (Stratagene) for sequencing. Candidate receptor fragments were labeled by random priming to screen the WEHI265.1 cDNA library by colony hybridization. Filters were hybridized overnight at 65°C in hybridization solution (5 ϫ SSPE, 0.1% pyrophosphate (PPiESS buffer); 1 ϫ Denhardt's solution, 1% SDS) and washed once for 15 min at room temperature and twice at 65°C for 20 min in 0.2 ϫ PPiESS, 0.5% SDS.
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The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) U51717.
Four positive cDNA inserts contained all or part of the mccr2 coding region by DNA sequencing.
Northern Blotting-Total RNA was prepared from mouse organs and cultured cells using RNAzol (Cinna/BioTecx). 10 g of total RNA was electrophoresed through a 1% agarose, 2 M formaldehyde gel, the electrophoresed RNA was stained with ethidium bromide to check for equivalent loading, and the RNA transferred in 10 ϫ SSC to Gene-Screen Plus (DuPont NEN) membranes. Northern blots were hybridized and washed in an identical manner to colony hybridizations except for the addition of 100 g/ml sonicated salmon sperm DNA to the hybridization. To generate the mccr2 probe, a 1.1-kb mccr2 coding region fragment was PCR-amplified from cDNA with the primer pair: sense, 5Ј-GCGGAAGCTTATGGAAGACAATAATATGTTACCT-3Ј; and antisense, 5Ј-GCGGTCTAGATTACAACCCAACCGAGACCTCTTG-3Ј. The 0.6-kb PCR-amplified ␤-actin probe (31) was used as a control.
Macrophage Preparation-Mice were injected intraperitoneally with 5 ml of 3% brewer's thioglycollate (Difco) in phosphate-buffered saline, and 4 days post-injection, peritoneal cells were lavaged with Dulbecco's modified Eagle's medium, 5% fetal bovine serum. Cells were collected by centrifugation, resuspended at 1 ϫ 10 6 cells/ml, and plated in tissue culture dishes for 1 h at 37°C. Nonadherent cells were rinsed by three washes with phosphate-buffered saline, and total RNA was immediately extracted from adherent macrophages.
Binding Assays-A 3.8-kb cDNA fragment containing the entire mccr2 coding region was excised with PvuII and XhoI restriction enzymes and cloned into the EcoRV-XhoI sites of pcDNA3 (InVitrogen). 10 -20 g of pcDNA3 mccr2 was transfected into HEK293 cells, and individual G418-resistant clones expressing mCCR2 (293/mCCR2) were identified by Northern blotting. 293/mCCR2 cells were seeded into 24-well plates 48 -72 h before binding experiments to attain a final density of 5 ϫ 10 5 cells/well. For saturation binding, medium was aspirated and cells incubated for 15 min at 37°C in 200 l of assay buffer (Hank's balanced salt solution, 25 mM Hepes, pH 7.4, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.01% NaN 3 , 0.2% bovine serum albumin) containing 0.2-7.5 nM 125 I-JE in the presence or absence of 0.75 M unlabeled JE. Binding was terminated by aspiration, and cells washed three times with 500 l of assay buffer to separate bound and free 125 I-JE. Cellbound radioactivity was recovered by the addition of 500 l 1% Triton X-100, 0.1% bovine serum albumin and transferred to tubes for ␥ counting. The binding data were curve-fitted with the SigmaPlot (Jandel Scientific) computer program, and linear transformation of data was conducted as described by Scatchard (32). For competition binding, cells were incubated for 15 min at 37°C in 200 l of assay buffer containing 0.5 nM 125 I-JE and increasing concentrations of unlabeled chemokine. Cell were washed and cell-bound radioactivity counted as described for saturation binding assays.
Recombinant JE, FIC, and N51/KC proteins were expressed using a baculovirus system and purified as described previously (13,33). JE (20 g) was radiolabeled by Bolton-Hunter to a specific activity of 2200 mCi/mmol (DuPont NEN Custom Iodination Laboratory). Recombinant mouse MIP-1␣ and C10 were purchased from R&D Systems.

RESULTS AND DISCUSSION
Degenerate Cloning of a Murine cDNA Homologous to ccr2B-Receptors for the murine C-C chemokine FIC were previously identified on human monocytes, and cross-desensitization studies indicated that FIC, JE, and MCP-1 can signal through common receptors (13). These results suggested that murine receptors for FIC and JE might be structurally similar to CCR1, CCR2A, and CCR2B. Since specific FIC binding sites had been identified on the mouse monocytic cell line WEHI265.1 (13), we utilized a PCR strategy using WEHI265.1 cDNA template and degenerate primers based on conserved sequences in ccr1 and ccr2B to clone potential murine receptors for FIC and JE.
Similar to other cloned chemokine receptors, the mCCR2 sequence contains seven hydrophobic regions suggesting a Gprotein-coupled receptor. Several other hallmarks of C-C chemokine receptors are also contained in the mCCR2 sequence, including conserved extracellular cysteine residues, the conserved sequence IFFIILLTIDRYLAIVHAVFAL from the middle of transmembrane domain 3 to intracellular loop region 2, an extremely basic intracellular loop region 3, and a serine/ threonine-rich COOH terminus (23,34). The predicted NH 2terminal extracellular region of mCCR2, like that of mCCR1 and mCCR3, contains no predicted N-glycosylation sites, unlike human CCR1, CCR2A, and CCR2B.
RNA Expression of mccr2-To characterize mccr2 RNA expression, Northern blots were performed on total RNA isolated from WEHI265.1 cells and stable HEK293 clones (293/mCCR2) transfected with an expression plasmid containing a 3.8-kb cDNA fragment from pCA-4A ( Fig. 2A and data not shown). In both WEHI265.1 cells and 293/mCCR2 cells, a predominant mRNA of Ϸ3.8 kb was detected, indicating that the 3.8- kb   FIG. 1. Sequence alignment of murine CCR2 (mCCR2) and human CCR2B (hCCR2B). The numbering of residues for the respective sequences is indicated on the right. Identical amino acids are shaded, and the seven hydrophobic domains are indicated by solid lines above the alignment. Conserved cysteines thought to be involved in extracellular disulfide bonding are indicated (*) above the respective residues. FIG. 2. Northern blot analysis of mccr2. A, total RNA (10 g) from WEHI265.1 cells and 293/mCCR2 cells, and the indicated mouse organs was blotted and hybridized with a probe specific for mccr2 (top). The blot was stripped and reprobed with a probe specific for mouse ␤-actin (bottom). The mccr2-specific blot was exposed for 4 days at Ϫ70°C, and the ␤-actin blot was exposed overnight at Ϫ70°C. B, total RNA (10 g) from WEHI265.1 cells and thioglycollate-elicited macrophages was similarly blotted and hybridized. cDNA insert of pCA-4A represents approximately a full-length cDNA clone. A smaller mRNA species was also detected in both WEHI265.1 and 293/mCCR2 cells. Since the smaller mRNA was not detected in control or vector-transfected 293 cells (data not shown), it is likely to be an alternatively spliced version of the 3.8-kb mccr2 mRNA rather than a cross-hybridizing mRNA.
To determine the distribution of mccr2 expression in the mouse, Northern blot analyses were performed on total RNA extracted from multiple organs ( Fig. 2A). Out of 12 organs analyzed by Northern blotting, mccr2 expression was detected in the kidney, lung, spleen, and thymus, although the mRNA levels were significantly lower than in WEHI265.1 cells. The low levels of mccr2 mRNA in the organs may represent expression in a specific subset of cells within the particular organs or in contaminating leukocytes such as monocytes/macrophages. Since mccr2 was cloned from the monocytic cell line WEHI265.1, we determined if mccr2 mRNA was also expressed in mouse mononuclear cells. When Northern blot analysis was performed on total RNA from thioglycollate-elicited peritoneal macrophages, mccr2 mRNA was detected at lower levels than in WEHI265.1 cells (Fig. 2B). Unlike the WEHI265.1 cells, the predominant mccr2 mRNA form in the elicited peritoneal macrophages is the smaller 2.8-kb species. The preferential expression of larger or smaller mRNAs in the WEHI265.1 cells and peritoneal macrophages suggests differential transcriptional regulation of mccr2 in different cell types.
Chemokine Binding to 293/mCCR2 Cells-To investigate chemokine binding to mCCR2, several stable HEK293 lines expressing high levels of mCCR2 mRNA were cloned ( Fig. 2A). Since mCCR2 is most homologous to the MCP-1/MCP-3 receptor CCR2B, 293/mCCR2 cells were tested for their ability to bind 125 I-JE. In two independent 293/mCCR2 lines, 75-80% specific binding of 1.0 nM 125 I-JE was detected that reached equilibrium within 15-20 min at 37°C, while no specific binding was detected to control HEK293 cells (data not shown). Equilibrium binding of increasing concentrations of 125 I-JE to 293/mCCR2 cells was saturable and approached maximal binding at 5 nM (Fig. 3A). Scatchard transformation of the binding data revealed a single class of 125 I-JE binding sites with a K d of 2.1 nM (Fig. 3B). This K d value is representative of high affinity chemokine binding to chemokine receptors expressed in HEK293 cells.
To determine the ligand binding specificity of mCCR2, competition binding analyses were performed on 293/mCCR2 cells with 0.5 nM 125 I-JE and increasing concentrations of unlabeled chemokines (Fig. 4). Both JE and FIC competed for 125 I-JE binding to mCCR2, with JE competing approximately ten times more effectively than FIC. Two other mouse C-C chemokines, MIP-1␣ (35) and C10 (36), and the mouse C-X-C chemokine N51/KC (37, 38) did not effectively compete for 125 I-JE binding to mCCR2. These results suggest that mCCR2 preferentially binds JE and FIC among the mouse chemokines tested so far and that mCCR2 is yet another chemokine receptor that binds more than one ligand.
The sequence and binding specificity of mCCR2 make it the most likely mouse species analog of CCR2B. mCCR2 binds JE with high affinity and FIC with lower affinity, while CCR2B binds MCP-1 with high affinity and MCP-3 with lower affinity. Interestingly, it has been proposed by sequence similarities that JE is the mouse MCP-1 analog and FIC the mouse MCP-3 analog (39,40). Since mCCR2 does not bind MIP-1␣, while murine MIP-1␣R/mCCR1 and murine MIP-1␣RL2/mCCR3 have not been reported to bind JE or FIC, mCCR2 most likely mediates distinct functions from the other two cloned murine C-C receptors. mccr2 expression was detected in both the WEHI265.1 monocytic cell line and in elicited peritoneal macrophages, suggesting that mCCR2 will be important for mediating mononuclear cell responses to JE and FIC. The generation of mice with a targeted deletion of the mccr2 gene will be useful for testing the role of mCCR2 in JE-mediated monocyte/macrophage recruitment and host defense and may be useful for developing mouse models for MCP-1/CCR2 functions.