Cloning and Characterization of a Novel Murine Macrophage Inflammatory Protein-1 (cid:97) Receptor*

We have cloned a novel CC chemokine receptor cDNA from mouse thymus. The deduced amino acid sequence shows 74% identity to the human monocyte chemotactic protein (MCP)-1 receptor (CC CKR-2b) and 54% to a recently cloned murine macrophage inflammatory protein (MIP)-1 (cid:97) receptor (Gao, J. L., and Murphy, P. M. (1995) J. Biol. Chem. 270, 17494–17501). Northern blot analysis of mouse tissues showed that the mRNA was also expressed in heart, spleen and liver, and to a lesser extent in lung and brain. The rank order of CC chemokine competition for 125 I-labeled human RANTES (regulated on activation, normal T-cell expressed and secreted) binding to human embryonic kidney (HEK) 293 cells stably transfected with the receptor cDNA was murine MIP-1 (cid:97) >> human MIP-1 (cid:98) > human RANTES > murine RANTES > murine MIP-1 (cid:98) > human MCP-2 > murine MCP-1 (JE) > human MIP-1 (cid:97) > human MCP-3 > human MCP-1. Of the chemokines tested, only murine MIP-1 (cid:97) , human and murine MIP-1 (cid:98) and RANTES, human MCP-2, and JE were able to induce mobilization of intracellular Ca 2 (cid:49) from fura-2-loaded HEK 293 cells expressing the receptor. These results suggest that this receptor func- tions as a high affinity murine MIP-1 (cid:97) Perkin-Elmer Cycler. reaction products purified into 50 (cid:109) l of sterile using a Promega Wizard™ PCR preparations kit. Five-microliter aliquots of the purified reaction then subjected to de- generate oligonucleotide PCR, and the resultant PCR products of the predicted size of 500–550 bp were gel-purified, subcloned into pBlue- script II SK (cid:50) (Stratagene), and sequenced using T3 and T7 primers and on an Applied Biosystems 373 DNA sequencer A 520-bp Hin dIII/ Eco RI cDNA insert from one clone, receptor-like [ 32 P]dCTP, a random-primed to screen 5 (cid:51) 10 plaque-forming units of the murine (cid:108) gt11 library or a murine thymus (cid:108) cDNA library (Stratagene) for the corresponding full-length by plaque hybridization. Several duplicating positive clones identified in the murine thymus library after two rounds of screening. DNA one clone, designated rescued from DNA this was sequenced with T3 and

Chemokines are a superfamily of small proteins involved in leukocyte recruitment and activation. Overall, they show amino acid sequence identity of between 20 and 50%. Their amino acid sequences contain four distinctive conserved cysteine residues. CXC 1 or ␣ chemokines such as interleukin-8 (IL-8), in which the first two cysteines are separated by one amino acid, are generally involved in neutrophil recruitment and activation. CC or ␤ chemokines such as RANTES, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1␣, in which the first two cysteines are adjacent, are generally chemoattractants and activators of other leukocyte populations such as monocytes, T-cells, eosinophils, and basophils (1). In addition, MIP-1␣ is an inhibitor of stem cell proliferation (2).
The specific effects of chemokines on inflammatory cells are mediated by a family of seven-transmembrane (7TM), G-protein-coupled receptors. The cDNAs for four human CC chemokine receptors have been cloned to date. CC CKR-1 and -4 are reported to be receptors for MIP-1␣ and RANTES (3)(4)(5). CC CKR-1 also shows high affinity binding to MCP-3 (6). CC CKR-2 is a high affinity receptor for MCP-1 and MCP-3 (7-9), whereas CC CKR-3 is a receptor for the specific eosinophil chemoattractant eotaxin (10). The overlapping ligand binding specificities observed for these receptors do not cover all of the nine human CC chemokines currently known (11). Other receptors may also exist based on the results of competition binding and functional assays on different leukocyte populations (12,13).
The identification of murine homologues of chemokine receptors based on sequence, tissue and cellular distribution, and functional similarities will allow the construction of knockout mice, enabling us to determine the biological relevance of chemokines and receptors in health and disease. Although binding of MCP-1 and MIP-1␣ to mouse leukocytes has been reported (14,15), only three murine CC chemokine receptors have been described so far, all of which bind human or murine MIP-1␣: mMIP-1␣R or CCR-1, which shows 80% amino acid identity to human CC CKR-1; mMIP1␣RL2 or CCR-3, which shows 68% amino acid identity to human CC CKR-3 (16,17); and a murine homologue of CC CKR-4, which shows 86% identity to the human receptor (18). No sequence homologue of the MCP-1 receptor, CC CKR-2, has been reported to date in mouse. To identify novel murine CC chemokine receptors, we have used an orphan receptor cloning strategy with degenerate oligonucleotide polymerase chain reaction (PCR) primers based on the conserved sequences found in human chemokine receptors (5).
Here we describe the cloning and functional characterization of a distinct high affinity receptor for murine MIP-1␣.

EXPERIMENTAL PROCEDURES
Materials-Restriction enzymes and DNA-modifying enzymes were purchased from New England Biolabs, except for Amplitaq™, which was from Perkin-Elmer. All cell culture reagents were from Life Technologies, Inc. except geneticin (G418), which was purchased from Sigma. MIP-1␣, RANTES, and MCP-1 were expressed in Escherichia coli and prepared at the Glaxo Institute for Molecular Biology as described (19). All other recombinant chemokines used in this study were purchased from R&D Systems. Murine MCP-1 (JE) and murine RAN-TES (mRANTES) were kindly provided by Dr. Barrett Rollins (Harvard University) and Dr. Ian Clark-Lewis (Vancouver), respectively. Radiolabeled 125 I-human RANTES (RANTES) and MIP-1␣ were prepared by Amersham to a specific activity of 2000 Ci/mmol. 125 I-human MCP-1 was purchased from DuPont NEN.
cDNA Library Screening-A murine spleen gt11 cDNA library was purchased from Clontech. cDNA inserts were amplified from 2 l of phage stock by 40 cycles of PCR (95°C, 2 min; 55°C, 2 min; and 72°C, * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) X94151.
2 min) in 10 mM Tris-HCl, pH 8.3, buffer, 50 mM KCl, 1.5 mM MgCl 2 , 0.2 mM dNTPS, and 2.5 units of Amplitaq™ using 1 M of gt11 primers (GT11 PCR-1, 5Ј GAT TGG TGG CGA CGA CTC CT and GT11 PCR-2, 5Ј CAA CTG GTA ATG GTA GCG AC) in a Perkin-Elmer DNA Thermal Cycler. PCR reaction products were purified into 50 l of sterile water using a Promega Wizard™ PCR preparations kit. Fivemicroliter aliquots of the purified reaction were then subjected to degenerate oligonucleotide PCR, and the resultant PCR products of the predicted size of 500 -550 bp were gel-purified, subcloned into pBluescript II SKϪ (Stratagene), and sequenced using T3 and T7 primers and on an Applied Biosystems 373 DNA sequencer exactly as described previously (5). A 520-bp HindIII/EcoRI cDNA insert from one clone, MS13, which appeared to encode a novel receptor-like sequence, was gel-purified, labeled with [ 32 P]dCTP, using a random-primed labeling kit (Boehringer Mannheim), and used to screen 5 ϫ 10 5 plaque-forming units of the murine spleen gt11 library or a murine thymus ZAP cDNA library (Stratagene) for the corresponding full-length cDNA by plaque hybridization. Several duplicating positive clones were identified in the murine thymus library after two rounds of screening. Bluescript plasmid DNA for one clone, designated AM4-7, was rescued from ZAP according to the manufacturer's instructions (Stratagene). CsCl gradient-purified plasmid DNA from this clone was sequenced with T3 and T7 primers and several internal primers based on the previous sequencing results.
Creation of Stably Transfected Cell Lines-An 1.8-kb XhoI/BamHI fragment containing the coding sequence of AM4-7 was subcloned into the mammalian cell expression vector pcDNA1neo (Invitrogen). Human embryonic kidney (HEK) 293 cells maintained in Dulbecco's modified Eagle's medium-F-12 containing 10% heat-inactivated fetal calf serum, 2 mM glutamine, and 100 units/ml penicillin/streptomycin were harvested by trypsinization and resuspended at 2 ϫ 10 7 cells/ml in 20 mM HEPES buffer, pH 7.3, containing 150 mM NaCl. Five-hundredmicroliter aliquots of cells were electroporated with 30 g of AM4-7p cDNA1neo at 260 V, 960 F using a Bio-Rad Gene Pulser. After electroporation cells were transferred to fresh medium and allowed to recover for 48 h before addition of 600 g/ml geneticin (G418). Fourteen days post-electroporation, individual G418-resistant colonies were isolated by ring cloning and maintained in complete medium containing G418. Individual clones were then tested for ligand binding specificity as described below. One clone HEKAM4-7/6, which showed high specific binding to 125 I-human RANTES was further purified by FACS using a fluorescent derivative of RANTES, 7-nitrobenz-2-oxa-1Ј3-diazol-4-yl (NBD)-RANTES. This was made by coupling the NBD group to the oxidized amino-terminal serine of RANTES using similar methodology to that described previously for IL-8 (20). Cells were harvested by trypsinization, resuspended in complete medium at 5 ϫ 10 6 cells/ml, and incubated with 200 nM NBD-RANTES for 1 h at 37°C in the dark. Cells were then washed twice in phosphate-buffered saline to remove unbound NBD-RANTES, resuspended in complete medium, and sorted on a Becton-Dickinson FACS star plus, gated to select cells expressing high levels of the receptor.
mRNA Analysis-A multiple tissue Northern blot of mouse poly(A) ϩ mRNA was purchased from Clontech and sequentially probed with the 520-bp HindIII/EcoRI insert from clone MS13, and a ␤-actin cDNA probe according to the manufacturers' instructions. DNase-I-treated, total RNA was purified from isolated mouse leukocyte populations (21-23) using the Trizol reagent (Life Technologies, Inc.). Mouse neutrophil and J774 cell line RNA were kindly provided by Dr. David Greaves (William Dunn School of Pathology, Oxford). Reverse transcriptase PCR analysis of full-length AM4-7 mRNA expression was performed using the following primers sense 5Ј GGC TCT TGC A GG ATG GAT CT and antisense 5Ј GTC ATA AAC CAG TAG AAA C on 1 g of total RNA as described previously (5). As a control for the quality of the input RNA, the mouse housekeeping gene hypoxanthine phosphoribosyltransferase was also amplified using hypoxanthine phosphoribosyltransferase-specific primers sense 5Ј GTT GGA TAC AGG CCA GAC Intracellular [Ca 2ϩ ] Mobilization-HEKAM4-7/6 cells were harvested by trypsinization, resuspended at 2 ϫ 10 6 cells/ml in Krebs-Ringer solution containing 0.4% bovine serum albumin, and loaded with 2 M fura-2-AM (Fluka) for 30 min at 37°C in the dark. Cells were then washed twice and resuspended in Krebs-Ringer solution at 2 ϫ 10 6 cells/ml. A 0.5-ml aliquot of the cell suspension was placed in a continuously stirred cuvette at 37°C, in a Jasco FP777 spectrofluorimeter, and the fluorescence monitored at 340 nm ( ex ) and 505 nm ( em ) on application of 100 nM recombinant chemokines at 1-min intervals.

Cloning of a Novel Mouse MIP-1␣
Receptor-In order to identify novel chemokine receptor-like sequences, we used a PCR cloning strategy as described previously (5). Degenerate oligonucleotide primers based on the conserved sequences found in the second intracellular loop (between transmembrane domains 3 and 4), and in transmembrane domain 7, of the human chemokine receptors, were used to screen a human spleen gt11 cDNA library by PCR. Amplification products of the predicted size range between 500 and 550 bp were subcloned into pBluescript II and sequenced. Of 28 clones sequenced, 20 encoded the murine homologue of the orphan receptor BLR1 (25), 2 encoded the murine homologue of the orphan receptor LESTR (26); 2 clones encoded the recently identified murine orphan receptors MIP-1␣RL2 or CCR3, presumed to be the homologue of human CC CKR-3 (16, 17); 1 clone encoded the murine homologue of the IL-8 receptor B (27); 1 clone (MS23) encoded a human CC CKR-2 homologue (70% DNA sequence identity) and 2 clones (MS13 and MS28) showed 75% DNA sequence identity to CC CKR-2, but were distinct from MS23. A 520-bp HindIII/EcoRI fragment from clone MS13 was used to screen a murine spleen gt11 cDNA library for the full-length cDNA. As we were unable to obtain any full-length clones from this library, we also screened a murine ZAP thymus library. This resulted in the isolation of a clone AM4-7 which contained the same sequence as MS13.
Chemokine Binding to HEK 293 Cells Stably Expressing AM4-7-To determine if AM4-7 encoded a chemokine receptor, the full-length cDNA was subcloned into the mammalian cell expression vector pcDNA1neo and stably expressed in HEK 293 cells. We subsequently tested G418-resistant clones for their ability to specifically bind radiolabeled, recombinant human CC chemokines 125 I-RANTES, MIP-1␣, and MCP-1, since radiolabeled murine chemokines were not available. Of 16 clones tested, 13 showed high specific binding to radiolabeled hRANTES with little or no binding to hMCP-1 and hMIP-1␣, respectively, when compared with HEK 293 cells transfected with empty pcDNA1neo, which were unable to bind any of the chemokines tested (Fig. 3). One clone, HEKAM4-7/6, which showed high specific binding to 125 I-hRANTES, was further purified by FACS to remove cells expressing only low levels of the receptor or nonexpressors, using the fluorescent RANTES derivative NBD-RANTES. Displacement of NBD-RANTES binding could be achieved by incubation with a 10-fold excess of unlabeled RANTES (Fig. 4). We then tested the ability of AM4-7 to bind other human and murine chemokines in competition binding assays using clone HEKAM4 -7/6 in which the binding of 125 I-hRANTES to AM4-7 was competed with varying concentrations of unlabeled human or murine chemokines. Binding of 125 I-hRANTES to AM4-7 could be displaced by all unlabeled CC chemokines tested (Fig.  5). The rank order with IC 50 was as follows mMIP-1␣ (6.8 Ϯ 9. Chemokine-induced Intracellular Ca 2ϩ Mobilization in AM4-7 Transfectants-Ca 2ϩ mobilization on ligand stimulation is a characteristic of most chemokine receptors so far described. We therefore examined the ability of various human and murine CC chemokines to induce intracellular Ca 2ϩ mobilization in HEK 293 cells stably expressing AM4-7. pcDNA1neo-transfected HEK 293 cells were unable to respond to any of the chemokines tested, whereas HEKAM4-7/6 cells gave a robust Ca 2ϩ response on application of 100 nM mMIP-1␣, hRANTES, mRANTES, hMIP-1␤, and mMIP-1␤, a weak response to hMCP-2 and JE, and no response to human MCP-1, hMIP-1␣, or IL-8. The EC 50 for calcium mobilization by mMIP-1␣ was 0.5 nM (Fig. 6). In addition, while mMIP-1␣, human and murine RANTES, and MIP-1␤ were all able to mutually desensitize each other (data not shown), prior stimulation of the receptor with 500 or 400 nM JE or hMCP-2, respectively, did not completely desensitize the receptor to subsequent application of 1 nM mMIP-1␣. Tissue Distribution of AM4-7 mRNA-High stringency Northern blot analysis of poly(A) ϩ RNA from mouse peripheral tissues indicated that the major mRNA transcript for AM4-7 mRNA was approximately 3.1 kb and was expressed predominantly in heart, spleen, and liver and to a lesser extent in lung and brain (Fig. 7A). A minor transcript of 4 kb was also detected in heart, spleen, and lung and may represent an alternatively spliced form of the receptor mRNA. Reverse transcriptase PCR analysis of mouse leukocyte populations indicated that AM4-7 mRNA was expressed in macrophages and was also detectable in mouse thymic lymphocytes, notably CD8ϩ T cells, but was not observed in eosinophils, neutrophils or B cells (Fig. 7B). Consistent with theses findings we were also able to detect AM4-7 message in the mouse macrophage cell line J774 (Fig. 7B) and in the mouse T cell line CTLL (data not shown). DISCUSSION A wide range of biological activities have been described for MIP-1␣, such as chemotaxis and the activation of monocytes, eosinophils, basophils, and T cells (12,28,29). MIP-1␣ is an inhibitor of stem cell proliferation and suppresses immature progenitor cells (2,30). In addition, MIP-1␣ has been implicated in the pathology of a number of inflammatory diseases (31)(32)(33)(34). The existence of specific binding sites for MIP-1␣ in mouse cells has been reported (14,15). Two human receptors for MIP-1␣ have been cloned (3)(4)(5), and recently, the cloning and characterization of their murine counterparts mMIP-1␣R or CCR-1 and mCC CKR-4 has been reported (16 -18). Here, we describe the cloning and characterization of a novel murine MIP-1␣ receptor AM4-7, which also functions as a high affinity receptor for several other CC chemokines.
The relative binding affinities observed for the MIP proteins to AM4-7 are interesting. Murine MIP-1␣ bound the receptor approximately 12,000 times stronger than hMIP-1␣. Murine and human MIP-1␤ also bound the receptor at significantly higher affinity (40 and 700 times stronger, respectively) than hMIP-1␣ despite the observation that mMIP-1␣ is more closely related to hMIP-1␣ than to hMIP-1␤ based on sequence identity or sequence similarity alone. An alignment of the predicted amino acid sequences for the four MIP cDNAs (Fig. 8) shows that there are 7 residues common to human and murine MIP-1␣ that are conservatively changed in the MIP-1␤s. However, hMIP-1␣ and mMIP-1␤ differ significantly from mMIP-1␣ and hMIP-1␤ at position 19, where Gln becomes Lys, and again at position 37, where Lys becomes Gln, thereby introducing a charge change at these positions. There are also two further charge changes in the carboxyl-terminal of hMIP-1␣: residue 57 is Glu but is Thr, Ser, or Pro in mMIP-1␣, hMIP1␤, and mMIP-1␤, respectively; residue 61 is Lys in hMIP-1␣, which is Glu in the other MIPs. Residues 57 and 61 are located in a predicted carboxyl-terminal helix, and based on the three-dimensional structures of hMIP-1␤, hRANTES, and mMIP-1␣ (35)(36)(37), they are in close proximity to residue 19. These observations suggest that subtle changes in sequence might confer differences in the range of MIP-1␣ and MIP-1␤ activities across the species and also stress the importance of defining murine homologues of human chemokines on functional data, not simply on the basis of sequence alone. However the importance of these residues in the receptor interaction needs to be verified by site-directed mutagenesis.
AM4-7 also functioned as a high affinity receptor for RAN-TES. The differences in ligand binding specificity between the human and mouse homologues of RANTES was not as marked as that observed for MIP-1␣. The affinity for hRANTES was only three times higher than for mRANTES, to which it shows 85% amino acid identity. JE, hMCP-3, and hMCP-1 were able to displace 125 I-RAN-TES from this receptor with IC 50 values of 20, 117, and 676 nM, respectively. In addition, this is the first report of binding of hMCP-2 to a specific CC chemokine receptor. Of the MCP's tested, only JE and hMCP-2 were able to induce a slight intracellular Ca 2ϩ mobilization in HEK 293 cells stably expressing AM4-7. So although the deduced amino acid sequence of AM4-7 predicts an MCP-1-like receptor (74% amino acid identity to CC CKR-2), our results suggest that the MCPs are unlikely to be important agonists of AM4-7 in vivo. Previous studies on the IL-8 receptors have indicated that the NH 2 -terminal extracellular domain is an important determinant of ligand binding specificity (38). The marked divergence in sequence of AM4-7 from CC CKR-2B, and the two murine MIP-1␣ receptors, mMIP-1␣R and mCC CKR-4, in the predicted NH 2 -terminal extracellular domain of the receptors may contribute to the observed differences in ligand binding specificities.
An analysis of the tissue distribution AM4-7 mRNA indicated the presence of a major 3.1-kb transcript in heart, spleen, and liver. A second transcript of 4 kb was also detectable in heart, spleen, and lung and probably represents an alternatively spliced form. The specific mouse leukocyte populations expressing the receptor mRNA appeared to be macrophages, and T-cells, most likely of the CD8ϩ subset. In humans at least both of these cell types are known to be responsive to MIP-1␣ (39).
In summary, we have cloned a novel high affinity receptor for mMIP-1␣ from mouse thymus. The receptor also binds other CC chemokines at high affinity but failed to bind the CXC chemokine IL-8. In addition, AM4 -7 stably expressed in HEK 293 cells was able to mobilize intracellular Ca 2ϩ in response to mMIP-1␣, hRANTES, mRANTES, hMIP-1␤, mMIP-1␤, hMCP-2, and JE. Despite the high level of sequence homology of AM4-7 to CC CKR-2B, which is a receptor for human MCP-1, the murine homologue of MCP-1, known as JE, only binds to AM4-7 at low affinity. These observations suggest that AM4-7 is functionally distinct from any of the known human or murine CC chemokine receptors cloned to date. Targeted gene disruption studies will therefore be important in determining the role of this receptor in vivo.