Cloning and Characterization of a Novel Murine beta  Chemokine Receptor, D6
COMPARISON TO THREE OTHER RELATED MACROPHAGE INFLAMMATORY PROTEIN-1alpha RECEPTORS, CCR-1, CCR-3, AND CCR-5*

(Received for publication, September 12, 1996, and in revised form, March 5, 1997)

Robert J. B. Nibbs , Shaeron M. Wylie , Ian B. Pragnell and Gerard J. Graham Dagger

From the Cancer Research Campaign Laboratories, The Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, United Kingdom

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

The beta -chemokine macrophage inflammatory protein-1alpha (MIP-1alpha ) is chemotactic for many hemopoietic cell types and can inhibit hemopoietic stem cell (HSC) proliferation, effects mediated through G-protein coupled heptahelical receptors. We have isolated cDNAs for seven chemokine receptors, CCR-1 to -5, MIP-1alpha RL1, and a novel cDNA, D6. Chinese hamster ovary cells expressing CCR-1, -3, -5, and D6 bound 125I-murine MIP-1alpha : the order of affinity was D6 > CCR-5 > CCR-1 > CCR-3. Each bound a distinct subset of other beta -chemokines: the order of competition for 125I-murine MIP-1alpha on D6 was murine MIP-1alpha  > human and murine MIP-1beta > human RANTES~JE > human MCP-3 > human MCP-1. Human MIP-1alpha and the alpha -chemokines did not compete. Like other chemokine receptors, D6 induced transient increases in [Ca2+] in HEK 293 cells upon ligand binding. D6 mRNA was abundant in lung and detectable in many other tissues. Bone marrow cell fractionation demonstrated T-cell and macrophage/monocyte expression of D6, and CCR-1, -3, and -5. Moreover, we could detect expression of CCR-3, CCR-5, and to a greater extent D6 in a cell population enriched for HSCs. Thus, we have characterized four murine beta  chemokine receptors that are likely involved in mediating the pro-inflammatory functions of MIP-1alpha and other chemokines, and we present D6, CCR-3, and CCR-5 as candidate receptors in MIP-1alpha -induced HSC inhibition.


INTRODUCTION

Macrophage inflammatory protein-1alpha (MIP-1alpha )1 is a member of the chemokine family, a group of almost 30 small polypeptides related functionally with respect to their ability to act as stimulators of leukocyte chemotaxis, and structurally due to the conservation of four disulfide-forming cysteine residues (reviewed in Refs. 1-5). Variation in this structural motif allows the family to be subdivided into two main branches. The C-X-C or alpha  chemokines, which include interleukin-8 (IL-8), macrophage inflammatory protein-2alpha and -beta (MIP-2alpha and -beta ), and KC, have a single amino acid residue separating the two most amino-terminal cysteines. In the C-C or beta  branch these two cysteines are juxtaposed: macrophage inflammatory protein-1alpha and -beta (MIP-1alpha and -beta ), monocyte chemoattractant proteins 1, 2, 3, and 4 (MCP-1, 2, 3, and 4), C10, eotaxin and RANTES are all members of this subfamily. Lymphotactin, a C chemokine, is unique in containing only a single amino-terminal cysteine residue, and only two in the mature protein (6). This also appears to represent a functional division as there is no sharing of signaling receptors between the subfamilies and while alpha -chemokines are classically viewed as neutrophil chemotaxins, the beta  family stimulate monocyte and macrophage migration and function (1-5). Eosinophils, basophils, and lymphocytes migrate in response to certain members of both families, that in some cases induce more overt inflammatory functions such as a respiratory burst or degranulation (7-13). Thus, these proteins are likely to be crucial during leukocyte trafficking and immune surveillance, and have been implicated in the development of various allergic responses, such as asthma (14), and in inflammatory diseases like rheumatoid arthritis (15) and chronic lung inflammation (16). Mice lacking the MIP-1alpha gene do not exhibit a normal immune response when infected with certain viruses, such as cocksackie virus in which the myocarditis normally associated with this virus is not observed (17). This phenotype attests to the in vivo role of MIP-1alpha as a mediator of inflammatory response.

The chemokines have also been implicated in the control of the cellular movement during angiogenesis (18-20), and in the control of cell proliferation (21-27). Furthermore, mice lacking the alpha chemokine stromal cell-derived factor have severely reduced numbers of B-cell and bone marrow myeloid progenitor cells (28), while mice lacking the murine IL-8 type B receptor homologue have an increased number of B-cells (29), implicating these two proteins in the control of blood cell development. We have demonstrated that MIP-1alpha , and to a lesser extent MIP-1beta , are able to inhibit the proliferation of transiently engrafting hemopoietic stem cells both in vivo and in vitro (26). MIP-1alpha is also active as a reversible inhibitor of clonogenic epidermal cell proliferation, with the intradermal source of the chemokine being the epidermal Langerhans cells (27). Binding sites have been identified on the surface of purified human hemopoietic stem cells and on a number of immature myeloid progenitor cell lines that appear to have a higher affinity for MIP-1alpha than those present on more mature cell types (30-32). We have shown that the hemopoietic stem cell receptor is distinct from the major monocyte receptor by using a mutated form of MIP-1alpha , HepMut, which is still active in stem cell inhibition assays but is unable to induce monocyte chemotaxis (33).

A number of chemokine receptors, all members of the G-protein-coupled heptahelical receptor superfamily, have now been cloned which are able to bind selected subsets of chemokines from either the alpha  or beta  branches (reviewed in Refs. 4 and 5). Human CCR-1, binds MIP-1alpha , MIP-1beta , MCP-1, MCP-3, and RANTES and is expressed on nearly all mature hemopoietic cell types (34-36). Human CCR-2, which is expressed on monocytes and some T-cells, can be produced as two alternatively-spliced forms both of which bind and signal in response to MCP-1 and MCP-3 with the altered COOH terminus affecting G-protein partner selection (36-38). Degenerate oligonucleotide-primed PCR has resulted in the identification of human and murine CCR-3, -4, and -5 (4, 5). Human CCR-5, which acts as a high affinity site for MIP-1alpha , -beta , and RANTES, is a central component of the protein complex required for the infection of macrophages and T-cells by many isolates of the human immunodeficiency virus: CCR-2 and -3, and the stromal cell-derived factor receptor fusin (CXCR-4) also act as co-receptors for different viral isolates (39-44). The CCR-5 ligands are able to prevent the infection of cells with human immunodeficiency virus (45), revealing that these proteins may play an important role in controlling the progression of AIDS. Furthermore, cells from individuals homozygous for a natural mutant of CCR-5 are resistant to infection with certain human immunodeficiency virus isolates (46).

To further understand the mode of action of MIP-1alpha , we have cloned seven murine members of the beta -chemokine receptor family, six previously reported and one novel sequence, D6. Four of these genes, including D6, produce proteins capable of binding to murine MIP-1alpha when expressed in Chinese hamster ovary (CHO) cells with dissociation constants ranging from 110 pM to 12 nM. D6 appears to be the highest affinity murine MIP-1alpha receptor cloned to date. The differential binding properties of murine and human MIP-1alpha , and murine HepMut, suggests that the interaction with each receptor is mediated by different amino acid residues in the ligand. Also, the spectrum of other chemokines that are able to interact with the four MIP-1alpha receptors is highly variable and demonstrates the high degree of receptor sharing that occurs within the beta -chemokine family. We have also obtained expression profiles for each of the receptors and demonstrate their expression in many hemopoietic cell types. In addition, we present evidence implicating a subset of these receptors in the processes of stem cell inhibition.


EXPERIMENTAL PROCEDURES

Materials

All DNA modifying enzymes were purchased from Life Technologies, Inc. Ltd., Paisley, United Kingdom. Plasmid DNA and DNA fragments were purified using the appropriate products from Qiagen, Dorking, UK. Agarose gels were buffered with 1 × TAE (40 mM Tris acetate, 1 mM EDTA). PCR and RT-PCR was performed using AmpliTaqTM polymerase and buffers (Perkin-Elmer, Roche Molecular Systems, Inc., Branchburg, NJ) except where indicated. Oligonucleotides were synthesized on an Applied Biosystems 392 DNA/RNA synthesizer. DNA was sequenced on an Applied Biosystems 373A DNA sequencer.

Chemokines

All chemokines were purchased in 10-µg aliquots from R&D Systems, Abingdon, UK, except human (h) lymphotactin and MIP-1alpha , and murine (m) eotaxin (Peprotech, London, UK). Due to the tendency of wild type murine MIP-1alpha to aggregate, we have previously designed a number of fully active non-aggregating variants of the molecule which are more appropriate for binding studies of the type described here (47). One of these mutants, PM2, is used throughout this work and will be referred to as mMIP-1alpha hereafter.

Cell Culture

All cell culture reagents were purchased from Life Technologies, Inc. unless otherwise stated. CHO cells were maintained in special liquid medium supplemented with 4 mM glutamine and 10% fetal calf serum (FCS) (TCS Biologicals, Buckingham, UK). HEK 293 cells were maintained in Dulbecco's modified Eagle's medium with 10% FCS and 4 mM glutamine. XS52 Langerhan's cells were grown as described previously (48).

Cloning of Receptors

The following oligonucleotides were synthesized from regions of similarity between human CCR-1 and -2, and cytomegalovirus US28 in the case of TEFDY: sense GALPY: 5'-GGGGCICA(A/G)CT(G/C)CT(G/C)CCICC-3'. Sense DIYLLN: 5'-AT(T/C)TA(T/C)CTICTIAACCT(C/G)GC-3'. Sense TEFDY: 5'-ACCAC(A/C)ITITTTGA(T/C)TATG-3'. Antisense FIILL: 5'-GTIAG(G/C)AGGATGAT(A/G)AA(A/G)AAAAT-3'. Antisense DRYLA: 5'-GACIAT(A/G)GCCAGGTACC(G/T)GTC-3'. Genomic DNA was isolated from the homogenized spleen of a C3H mouse according to Sambrook et al. (49). PCR was performed using variable amounts of MgCl2 (from 1.25 to 2.25 mM), 0.3 mM of each dNTP, and 6 ng/µl of one sense and one antisense oligonucleotide. Reactions were incubated for 35 cycles at 94 °C for 1 min, 50-52 °C for 1 min, and 72 °C for 2 min. Products of expected size were cloned into pCRScript (Stratagene, La Jolla, CA) and sequenced. 5' and 3' rapid amplification of cDNA ends (RACE) was performed using RACE kits from Life Technologies, Inc. according to their instructions on tissues or cell lines positive for each receptor in RT-PCR reactions. No alternatively spliced forms of the type seen with human CCR-2 (37) were detected. Pfu polymerase (Stratagene) was used on cDNA or genomic DNA according to the manufacturers' instructions, with reactions containing 10% dimethyl sulfoxide and 5% glycerol and oligonucleotides specific to the 5' and 3' ends of the gene. Three separate reactions were performed, and all products were cloned into pCRScript and fully sequenced in both orientations. Sequences were analyzed using GCG software (50): phylogenic relationships were determined using the Distances (Kimura method) and Growtree (Neighbor-joining method) programs.

mRNA Analysis

Total RNA was extracted using Trizol (Life Technologies, Inc.). Northern blots onto Hybond N+ (Amersham, Little Chalfont, UK) were performed according to Nibbs et al. (51). DNA probes were labeled with [alpha -32P]dCTP (Amersham) using the Ready-to-go kit (Pharmacia), unincorporated nucleotides were removed using NICK Sephadex G-50 columns (Pharmacia) and the probe denatured for 3 min at 100 °C prior to hybridization. Filters were exposed to Kodak X-Omat x-ray film and stripped in 0.1% sodium dodecyl sulfate (SDS) at 100 °C prior to reprobing. RT-PCR was performed using the RNA PCR core kit (Perkin-Elmer). For each RNA sample reverse transcription was done in two tubes, one with and one without reverse transcriptase. 1 µg of heat-denatured total RNA was used per PCR reaction, which had been previously treated with DNase I to remove genomic DNA contaminants (according to Life Technologies, Inc. RACE kits), followed by phenol/chloroform extraction, ethanol precipitation, and 70% ethanol wash. Specific PCR primers used were (AS: antisense, S: sense): D6; AS: 5'-GGAAGAGACAGTAATGAGTAAGGC-3' and S: 5'-GTGACAGAGAGCCTGGCCTTC-3' for tissue PCR (expected size, 345 base pairs). PCR proceeded for 30 cycles of 94 °C for 1 min, 58 °C for 1 min, 72 °C for 1 min.

Generation of Stably Transfected Cell Lines

Full-length cDNAs were excised as BamHI/NotI fragments from the pCRScript vectors and cloned into pcDNA3 (Invitrogen, Abingdon, UK). These vectors were transfected into CHO cells and single cell clones selected in 1.6 mg/ml geneticin (Life Technologies, Inc.) as described previously (33). Total RNA was extracted from approximately 12 separate clones, Northern blots performed to assess expression levels and three of the highest expressors selected for ligand binding experiments. pcDNA3 containing D6 cDNA was transfected into HEK 293 cells using Transfectam (Promega, Southampton, UK) according to the manufacturers' instructions. Stably transfected pools of cells were selected in 700 µg/ml geneticin.

Receptor Binding Studies

5-µg aliquots of mMIP-1alpha were labeled on a regular basis with Na125I (DuPont NEN) using IODO-GEN (Pierce) as described previously (30). Stably transfected CHO cells were plated at 105 cells/well in 24-well plates and incubated overnight at 37 °C. After washing with warm PBS, the cells were incubated in 250 µl of binding buffer (special liquid medium plus 10% fetal calf serum, 4 mM glutamine, 0.2% sodium azide, 25 mM HEPES (pH 7.4)) containing variable concentrations of radioiodinated MIP-1alpha and cold competitor chemokine. Binding proceeded for 90 min at 22 °C, then each well was washed three times in ice-cold PBS and the cell monolayer was then solubilized by the addition of 0.5 ml of 1% SDS. Lysed cells were transferred to a counting vial and bound radioactivity counted for 1 min in a Beckman Gamma 5500B counter. Each data point was assayed in triplicate and each experiment performed at least twice for accuracy. Data was analyzed using Scahot and Scafit programs in LIGAND software (52). Initially, each transfected cell line, and the parental cell line, was tested for binding at 40 and 4 nM 125I-mMIP-1alpha . Transfected cells that exhibited a significant increase in binding above background were subjected to a full Scatchard analysis with 12 data points in triplicate with varying concentrations of 125I-mMIP-1alpha . Kd values were then confirmed by varying the amount of unlabeled mMIP-1alpha while maintaining the 125I-mMIP-1alpha at approximately the Kd value. Competition experiments with other unlabeled chemokines were similarly performed.

Intracellular Ca2+ Measurements

HEK 293 cells stably transfected with D6 were harvested by trypsinization, washed in HACM buffer (125 mM NaCl, 5 mM KCl, 1 mM MgCl2, 0.5 mM glucose, 0.025% bovine serum albumin, 20 mM HEPES (pH 7.4)), and loaded in HACM containing 10 µM Fura-2-AM (Sigma) for 60 min at 37 °C in the dark. Cells were washed twice in HACM warmed to 37 °C and resuspended to 2-4 × 106 cells/ml in HACM. A 2-ml aliquot was placed in a continuously stirred cuvette at 37 °C in a Perkin-Elmer LS50 Spectrometer, CaCl2 added to 100 nM and fluorescence monitored at 340 nm (lambda ex) and 500 nm (lambda em). Data were collected every 100 ms. Chemokines were added to a final concentration of 100 nM.

Generation of cDNA Fragments from Sorted Bone Marrow Cells

Preparation of cDNA

Bone marrow cells from the femurs of 20 mice were isolated into PBS containing 2% fetal calf serum (PBS/FCS). 5 × 106 cells were used directly for poly(A)+ mRNA preparation (see below). The remaining cells were diluted to 2.5 × 107 cells/ml, layered onto an equal volume of 1.077 g/ml Nycodenz solution (Nycomed, Oslo, Norway) and spun at 1,000 × g for 30 min at 22 °C. The low density cells at the interface were removed, spun for a second time through the density gradient, washed twice in PBS/FCS, and resuspended to 1 × 106 cells/ml in PBS/FCS. Different cell types were then sorted sequentially in the following order by addition of rat antibodies, Gr-1, B220, CD5/CD8, and Mac-1 (Pharmingen, San Diego, CA, except Mac-1, Caltag, San Francisco, CA) followed by magnetic separation with sheep anti-rat M-450 beads (Dynal, Oslo, Norway), according to the manufacturers' instructions. After the four antibody positive cell fractions were purified, remaining unbound cells were designated lineage negative (lin-). Poly(A)+ mRNA was then isolated from the six cell populations (total bone marrow, Gr-1+, B220+, CD5/CD8+, Mac-1+, and lin-) using the Micro-FastTrackTM kit (Invitrogen) and cDNA prepared, according to the suppliers instructions, with the Copykit (Invitrogen). cDNA was digested with AluI, the enzyme was heat-inactivated and the cDNA fragments were phenol/chloroform extracted and ethanol precipitated.

Preparation of Linkers

Specific pairs of oligonucleotide linkers, as shown, were used for each cDNA sample to minimize carry over contamination: bone marrow: A, 5'-ACAGTCCCATGGTACAAGTTCAGCA-3' and B, 5'-GAACTTGTACCATGGGACTGT-3'; granulocyte (Gr-1): A, 5'GAACTCGGATCCCATGTGAAGGTGT-3' and B, 5'-CTTCACATGGGATCCGAGTTC-3'; B-cells (B220): A, 5'-AACCGTAGATCTGCTGCACACCAAC-3' and B, 5'-GTGTGCAGCAGATCTACGGTT-3'; T-cells (CD5/CD8): A, 5'-TAGACCAAGCTTAGACTGACATCCT-3' and B, 5'-TGTCAGTCTAAGCTTGGTCTA-3'; monocytes/macrophages (Mac-1): A, 5'-AGCCATTCTAGACGTGTAACTGATA-3' and B, 5'-AGTTACACGTCTAGAATGGCT-3'; lineage negative (lin-): A, 5'-TAGTCCGAATTCAAGCAAGAGCACA-3' and B, 5'-CTCTTGCTTGAATTCGGACTA-3'. Each set of linker pairs (6 µg of each oligonucleotide, heat denatured at 90 °C for 2 min and cooled to 4 °C) were kinased in 1 × polynucleotide kinase buffer (NBL Gene Sciences Ltd., Cramlington, UK) containing 1 mM dATP and 1 µl of [gamma -32P]dATP (Amersham) with 20 units of polynucleotide kinase (NBL) at 37 °C for 1 h. The reaction was placed 70 °C for 5 min, allowed to cool over 2 h to 22 °C to anneal the complementary linkers, and stored at -20 °C.

Ligation of Linkers and PCR Amplification of cDNA Fragments

The duplexed, kinased linkers were ligated onto the corresponding cDNA according to Sambrook et al. (49), in 1 × ligase buffer (NBL) containing 1 mM hexamminecobalt chloride and 0.5 mM spermidine, at 16 °C for 20 h. Linkers were separated from cDNA by adding glycerol to 20% and briefly electrophoresing each sample on a 2% low melting point agarose TAE gel. DNA separate from the unincorporated linkers (~100-1000 base pairs) was excised in a gel volume of approximately 0.5 ml. PCR was performed directly on 5 µl of this slice melted at 70 °C in a reaction containing 0.4 mM of each dNTP, 16 ng/µl of the cell type-specific B oligonucleotide (see above), and 7.5 units of AmpliTaqTM. Reactions were incubated for 35 cycles at 94 °C for 1 min, 52 °C for 1 min, and 72 °C for 3 min.

Southern Blotting

Visually estimated equal quantities (approximately 5 µg) of the separate PCR products from each population of sorted cells were separated on a 1.2% agarose gel that was subsequently treated for 2 × 15 min with 1.5 M NaCl, 0.5 M NaOH and then neutralized for 2 × 30 min in 1.5 M NaCl, 0.5 M Tris (pH 7.5), 1 mM EDTA. DNA was transferred to Hybond N+, fixed, prehybridized, hybridized, washed, and exposed as described for Northern blots. Filters were stripped in 0.1% SDS at 100 °C prior to reprobing. Full-length cDNA was used as a probe for each of the chemokine receptors. Other probes used were murine T cell receptor Cbeta cDNA and murine immunoglobulin H cDNA (provided by Dr. M. Stewart, University of Glasgow), murine lysozyme M (provided by Dr. M. Cross, CRC Paterson Institute, Manchester), v-fms (provided by Dr. A. Ford, LRF Chester Beatty Labs., London), murine CD34 cDNA (provided by Dr. G. May, LRF Chester Beatty Labs., London), and murine beta -actin (D. Jarmin, Beatson Institute, Glasgow).


RESULTS

Identification of Murine Chemokine Receptors from Genomic DNA Using PCR

Utilizing combinations of the three 5' and two 3' primers described above, we identified fragments of seven members of the murine G protein-coupled receptor family which were subsequently used to isolate full-length cDNAs (see "Experimental Procedures"). Six of our sequences (Fig. 1) are essentially identical, with a few amino acid changes, to previously cloned murine chemokine receptors, i.e. RN31 is CCR-1, FG15 is CCR-2 (homologous to human CCR-2b form), G17 is CCR-3, D2 is CCR-4, RN3 is CCR-5, and ME is the CCR-1-like orphan receptor MIP-1alpha RL1 (Refs. 53-57). In addition, we have also identified a novel receptor, D6, which shows homology to these and other members of the subfamily of G-protein-coupled chemokine receptors.


Fig. 1. Alignment of the predicted amino acid sequences of the seven cloned murine chemokine receptors. Each is named with their clone name designated upon isolation, followed by the published nomenclature. Dots indicate gaps that were inserted to optimize the alignment. The location of the predicted membrane-spanning segments I to VII are bracketed with a dashed line and termination codons are shown with a asterisk (*). Arabic numbers correspond to the full protein length. Underlined italicized amino acids in bold represent residues which varied between our predicted amino acid sequence and the data base sequence. Thus, FG15 was identical to CCR-2 (GenBank/EMBL Accession number, U47035[GenBank]: Ref. 56); RN3 had Phe for Leu at position 3 and Leu for Phe at 90 compared with CCR-5 (X94151[GenBank]: Ref. 57); ME had Leu for Pro at 38, Val for Glu at 334, and TP for RL at 343-4 compared with MIP1alpha RL (U28405[GenBank]: Ref. 53); RN31 had Gly for Ala at 141, Phe for Leu at 149, and Gln for His at 278 compared with CCR-1 (U28404[GenBank] and U29678[GenBank]: Refs. 53 and 54); Gly-17 was identical to CCR-3 with Ser at 270 (U28406[GenBank]: Ref. 53), and D2 had Cys for Trp at 221, Ala for Gly at 246, and Ala for Gly at 293 compared with CCR-4 (X90862[GenBank]: Ref: 55).
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Characterization of the D6 cDNA, a Novel Seven Transmembrane Spanning Receptor

A full-length D6 cDNA, isolated from brain RNA revealed a single open reading frame, encoding a protein of 378 amino acids (Fig. 2A). 5' RACE PCR performed from heart, brain, and liver, gave three identical products that may represent three potential transcriptional initiation sites for this gene (indicated with a bracket in Fig. 2A): one may initiate translation at the second methionine to produce an amino-terminally truncated protein. This potential variant would be similar to an alternatively spliced form of BLR-1, an orphan receptor related to the IL-8 receptors, in which two distinct 5' exons result in the generation of either full-length or amino-terminally truncated forms of the receptor (58). No similar 5' RACE products starting prior to the first ATG were seen with the six other receptors. However, we cannot exclude the possibility that these D6 variants are the result of PCR artifact. The full-length D6 protein contains the predicted seven-transmembrane spanning regions, the four conserved cysteine residues thought to maintain the receptor in a cylindrical structure and in addition has a single putative N-linked glycosylation site at the amino terminus. In common with other chemokine receptors, the amino terminus is highly acidic although the first 13 amino acids are predicted to form a hydrophobic domain that is not seen in the related chemokine receptors. A phosphorylation site for cAMP-dependent protein kinase is present on the first cytoplasmic loop and further phosphorylation sites for protein kinase C and casein kinase II are found in the carboxyl-terminal tail which possesses a total of 13 serine and threonine residues. The strongly conserved DRYLAIV motif in the second intracellular domain which is involved in G-protein docking is altered to DKYLEIV in D6, a change which likely affects the selection of G-protein partners. Furthermore, D6 lacks the putative G protein-binding site present in many heptahelical receptors at the carboxyl-terminal end of the third intracellular loop, i.e. BBXXB or BBXB (where B is a basic amino acid and X is any amino acid) (59). It does, however, contain a similar site at the amino-terminal end of the long cytoplasmic tail which is not present in many of the other chemokine receptors (Fig. 2A). Analysis of the homologies between D6 and the other chemokine receptors indicate that D6 displays some 30-37% identity and around 60% similarity to each of the cloned chemokine receptors (data not shown). Phylogenic analysis suggests that this receptor lies between the C-C and C-X-C receptor subfamilies, most closely related to CCR-4 and two murine IL-8 receptor-like genes, mIL-8R (29) and mIL-8RL2 (Fig. 2B). However, comparison of extracellular amino termini, believed to be important in ligand binding, shows that in this domain D6 is more closely related to the C-C chemokine receptors, especially CCR-1 (data not shown).


Fig. 2. A, nucleotide and predicted amino acid sequence of D6. Regions underlined in bold indicate the position of the seven putative membrane-spanning domains. Brackets show the position of the three different 5'-RACE products identified in heart, brain, and liver. Three small squares denote the position of a potential N-linked glycosylation site in the amino-terminal extracellular domain. # shows the four conserved cysteine residues and the stop codon is indicated with a asterisk (*). The box indicates the position of a potential cAMP-dependent protein kinase site, the triple underlined residues potential protein kinase C sites, and the dashed underlined residues potential casein kinase II sites found in the intracellular domains. The bracketed sequences, DKYLEIV and RRYLK, are putative G-protein docking sites. B, phylogenic relationship of chemokine receptors. Amino acid sequences (Fig. 1) were compared using the GCG software Pileup and Distances (Kimura method) programs, and then displayed graphically with Growtree (Neighbor-joining method). The distances between the receptors indicate the number of amino acid changes between them.
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Initial analysis of D6 receptor expression patterns using tissue blots (Fig. 3A) indicated that expression of a single approximately 3.2-kilobase transcript for D6 was detectable in lung, and at much lower levels in liver and spleen. In addition, RT-PCR analysis revealed expression of D6 in heart, brain, thymus, ovary, muscle, liver, and kidney, but not mammary gland or testes (Fig. 3B). Transcripts were also detectable by Northern blot analysis in murine solid tissues for CCR-1 (spleen > lung, heart > skeletal muscle), CCR-3 (spleen), CCR-4 (liver, thymus > lung > heart, brain > striated muscle), and CCR-5 (low levels in spleen, liver), but not CCR-2 or ME-MIP-1alpha RL1 (data not shown). It must be emphasized, however, that the contribution of resident leukocytes to the signal in many of these tissues cannot be ruled out and definitive assessment of the cell type specific expression patterns in each of these tissues awaits in situ hybridization studies.


Fig. 3. A, Northern blot analysis of the tissue distribution of mRNA for D6. Top panel: Each lane contains 20 µg of total RNA blotted onto a Hybond N+ filter, hybridized for 20 h to full-length D6 cDNA labeled with [alpha -32P]dCTP and washed to a final stringency of 0.1 × SSC, 0.1% SDS at 65 °C for 20 min. The blot was exposed to Kodak X-Omat film for 80 h at -70 °C in a cassette containing Hi-Speed-X intensifying screens. Lane 1, brain; lane 2, heart; lane 3, testes; lane 4, ovary; lane 5, gut; lane 6, lung; lane 7, thymus; lane 8, spleen; lane 9, liver; lane 10, mammary gland; lane 11, skeletal muscle. RNA molecular weight markers (in kilobase pairs) are indicated to the right of the panel, and the D6 transcript is indicated with an arrow. Bottom panel, the blot was stripped in boiling 0.1% SDS and reprobed with a similarly labeled beta -actin (Actin) probe, washed at the same stringency and exposed for 20 h. B, RT-PCR analysis of D6 tissue expression. 1 µg of DNase I-treated total RNA was used per reaction, and identical reactions were performed in the absence (-) or presence (+) of reverse transcriptase as indicated above each lane. PCR was performed for 30 cycles and conditions and oligonucleotide primers used are under "Experimental Procedures." 20 µl from the 100-µl final reaction volume was electrophoresed on a 1.5% agarose, 1 × TAE gel at 150 V for 90 min and the DNA visualized by ethidium bromide staining of the gel. The sizes (in base pairs) of the DNA molecular weight markers electrophoresed in lanes on either side of the samples are shown to the right of the panel. Tissues tested are indicated at the top of the panel.
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Ligand Binding Profiles of the Murine Chemokine Receptors

As our primary interest lies in the identification of MIP-1alpha receptors, each of the seven receptors described above was stably transfected into CHO cells and three clones expressing each receptor were tested for their ability to bind radioiodinated mMIP-1alpha . CCR-1, CCR-3, CCR-5, and D6 bound 125I-mMIP-1alpha and we have thus concentrated our subsequent binding studies on these four receptors. Murine CCR-4 has been reported to bind human MIP-1alpha (55) and our inability to detect an interaction with murine MIP-1alpha is surprising and may imply that CCR-4 can distinguish between MIP-1alpha from these two species. However, we have not demonstrated cell surface expression of CCR-4 in the CHO cell transfectants. The affinities of 125I-mMIP-1alpha for the receptors (expressed as dissociation constants Kd, Table I) allows the subdivision of the receptors into high affinity (D6 and CCR-5) and low affinity (CCR-1 and CCR-3) groups. These results also present D6 as the highest affinity receptor for MIP-1alpha identified to date. Displacement curves (Fig. 4, A-D) and apparent Kd values (Table II) indicate that these two subgroups can also be discriminated on the basis of their ability to bind human MIP-1alpha and RANTES. CCR-5 and D6 display only weak or no significant binding of hMIP-1alpha but interact well with human RANTES (Kd values of 550 pM and 2 nM, respectively: Table II) whereas CCR-1 and CCR-3 both bind human MIP-1alpha with an avidity similar to that seen with mMIP-1alpha but no interaction with hRANTES is observed.

Table I. Dissociation constants (Kd) for binding of mMIP-1alpha to CHO cells expressing CCR-1, CCR-3, CCR-5, and D67

CHO cells stably expressing each of the receptors were incubated in triplicate for 90 min at 22 °C in binding buffer containing a range of 125I-labeled mMIP-1alpha concentrations (2 pM to 80 nM: >12 different concentrations per receptor). Identical incubations containing approximately 100-fold excess of unlabelled mMIP-1alpha were also performed. Cells were washed with ice-cold PBS, lysed in 1% SDS, and counted in a Beckman Gamma 5500B counter for 1 min. Dissociation constants were calculated using LIGAND software (52). Data are representative of three separate experiments. Data were confirmed in separate experiments in which the concentration of 125I-mMIP-1alpha was fixed at the estimated Kd and variable concentrations of unlabeled mMIP-1alpha were included in the incubation to displace the radiolabeled ligand. CHO cells stably expressing each of the receptors were incubated in triplicate for 90 min at 22 °C in binding buffer containing a range of 125I-labeled mMIP-1alpha concentrations (2 pM to 80 nM: >12 different concentrations per receptor). Identical incubations containing approximately 100-fold excess of unlabelled mMIP-1alpha were also performed. Cells were washed with ice-cold PBS, lysed in 1% SDS, and counted in a Beckman Gamma 5500B counter for 1 min. Dissociation constants were calculated using LIGAND software (52). Data are representative of three separate experiments. Data were confirmed in separate experiments in which the concentration of 125I-mMIP-1alpha was fixed at the estimated Kd and variable concentrations of unlabeled mMIP-1alpha were included in the incubation to displace the radiolabeled ligand.
Kd for mMIP-1alpha

  CCR-1 2 nM
  CCR-3 12 nM
  CCR-5 250 pM
  D6 110 pM


Fig. 4. Displacement of 125I-mMIP-1alpha from CCR-1, CCR-3, CCR-5, and D6 with other beta -chemokines demonstrates promiscuous ligand-receptor interaction. CHO cells stably expressing each of the receptors were incubated in triplicate for 90 min at 22 °C in binding buffer containing 125I-labeled mMIP-1alpha fixed at the estimated Kd for mMIP-1alpha receptor binding (Table I) and a range of concentrations of different unlabeled chemokines. Cells were washed with ice-cold PBS, lysed in 1% SDS, and counted in a Beckman Gamma 5500B counter for 1 min. Data is presented as a percentage of the binding that was detected in the absence of competing ligand against the log of the concentration of the competing chemokine. Each of the unlabeled chemokines which exhibited detectable displacement of mMIP-1alpha is identified as: ---×---, C10; - - -Delta - - -, hMIP-1a; ---diamond ---, hMCP-2; - - -open circle - - -, mMCP-1/JE; ··diamond ··, mMIP-1b; - - -×- - -, hMIP-1b; ---open circle ---, mMIP-1a; ---square ---, hRANTES; ···square ···, hMCP-1. Panel A, CCR-1; panel B, CCR-3; panel C, CCR-5; panel D, D6.
[View Larger Version of this Image (35K GIF file)]

Table II. Apparent dissociation constants in nanomolar for chemokines upon interaction with CCR-1, CCR-3, CCR-5, and D6, shows promiscuous ligand-receptor interaction

Data from Fig. 4, A-D, were analyzed using LIGAND software (52) to generate estimates of the dissociation constants between ligand and receptor given in nanomolar. Data from Fig. 4, A-D, were analyzed using LIGAND software (52) to generate estimates of the dissociation constants between ligand and receptor given in nanomolar.
hMIP-1alpha hRANTES hMIP-1beta mMIP-1beta hMCP-1 mMCP-1/JE hMCP-2 hMCP-3 mC10

CCR-1 12 NDa 5 33 ND 149 135 ND 510
CCR-3 67 ND a 12 85 ND ND ND ND ND
CCR-5 140 0.55 2 5 138 205 65 ND ND
D6 ND 2 0.26 0.77 52 1.5 ND 5 ND

a ND, receptor/ligand combinations in which no significant displacement of 125I-mMIP-1alpha was detectable at concentrations of ligand of 200 nM (for CCR-5 and D6) or 400 nM (for CCR-1 and CCR-3).

All these four receptors bind murine and human MIP-1beta with Kd values slightly higher than those seen with mMIP-1alpha . Other human MCP-1, murine MCP-1/JE, and human MCP-3 chemokines also bind to D6 with affinities in the nanomolar range, while CCR-5 also interacts weakly with human and murine MCP-1 and human MCP-2 (Fig. 4, C and D; Table II). CCR-1 also interacts with mMCP-1/JE, hMCP-2, and murine C10 when the ligand is present at high concentrations (Fig. 4A). Thus, each of the four MIP-1alpha receptors has a unique repertoire of potential ligands although the physiological relevance of interactions which only occur at very high concentrations, mC10 with CCR-1, for example, is questionable.

None of the alpha -chemokines tested (IL-8, KC and MIP-2alpha ), nor lymphotactin, are capable of displacing radiolabeled MIP-1alpha from any of the cloned chemokine receptors indicating that these receptors are specific for the beta -chemokine family. This is particularly important with respect to D6 given the apparently comparable degree of overall similarity between this receptor and the alpha - and beta -chemokine receptors. Eotaxin, which has been reported as a ligand for CCR-3 (60), appeared to form aggregated complexes with the labeled mMIP-1alpha and made interpretation of displacement experiments impossible (data not shown).

Thus, D6 acts as a high affinity receptor for beta -chemokines when it is expressed in CHO cells, binding to mMIP-1alpha  > human and murine MIP-1beta  > hRANTES, mMCP-1/JE > hMCP-3 > hMCP-1. The affinity and specificity of this interaction most closely resembles that seen with CCR-5, while CCR-1 and -3 bind with lower affinity to mMIP-1alpha and are somewhat more specific for human and murine MIP-1alpha and beta .

Chemokine-induced Intracellular Ca2+ Mobilization in D6 Transfectants

Ca2+ mobilization upon ligand stimulation is characteristic of chemokine receptors described to date. Due to the inability in our hands of CHO cells expressing any of the receptors to generate a Ca2+ flux upon chemokine treatment, pools of HEK 293 cells stably transfected with D6 were generated; HEK 293 cells have been successfully used by others to study chemokine receptor signaling. Treatment of transfectants with 100 nM mMIP-1alpha , mMIP-1beta , or hRANTES generated a detectable increase in intracellular Ca2+ demonstrating that this receptor is able to signal upon ligand interaction; hMCP-3 gave a significantly weaker response (Fig. 5). In addition, each of the ligands were able to desensitize the cells to a subsequent treatment 100 s later with a second D6 ligand (Fig. 5). Untransfected HEK 293 cells were unresponsive to all the chemokines tested (data not shown).


Fig. 5. Detection of intracellular [Ca2+] in HEK 293 cells stably transfected with D6 cDNA. Pools of geneticin-resistant HEK 293 cells transfected with pcDNA3 containing D6 were loaded with Fura-2AM and assayed at 37 °C in a continuously stirred cuvette in a Perkin-Elmer LS50 Spectrometer (340 nm (lambda ex); 500 nm (lambda em)), with fluorescence emission recorded every 100 ms for 240 s. Chemokines were added to a final concentration of 100 nM at the times indicated. h, human; m, murine.
[View Larger Version of this Image (9K GIF file)]

Hemopoietic Subpopulation Expression Patterns of the Murine Chemokine Receptors

To more specifically examine the patterns of receptor expression within the hemopoietic system, we have investigated the expression of D6 and the other cloned MIP-1alpha receptors using amplified cDNA fragments generated from sorted populations of hemopoietic cells, including a lineage negative fraction containing primitive cells (see "Experimental Procedures"). We have confirmed the success of this sorting by probing Southern blots of the sorted cell amplified cDNA fragments with lineage specific markers. The results (Fig. 6A) suggest a high degree of lineage separation and a marked enrichment of CD34+ primitive cells in the lin- population. All bands were <500 base pairs due to the AluI digestion of the cDNA prior to amplification and blotting.


Fig. 6. Southern blot analysis of PCR-amplified cDNA fragments generated from sorted bone marrow cell subpopulations. Panel A, lineage-specific marker expression. Panel B, chemokine receptor expression. Cell sorting and cDNA AluI-fragment generation and amplification is described under "Experimental Procedures." Six identical blots were prepared by capillary blotting approximately 5 µg of amplified cDNA fragments from each cell subpopulation onto Hybond N+. Hybridization to [alpha -32P]dCTP probes was allowed to proceed for ~20 h after which blots were washed to a final stringency of 0.1 × SSC, 0.1% SDS at 65 °C for 20 min. Each blot was exposed to Kodak X-OMAT film at -70 °C in a cassette containing Hi-Speed-X intensifying screens to allow visualization of hybridized probe. Blots were stripped in boiling 0.1% SDS prior to reprobing. Lane headings are BM, total bone marrow; lin-, lineage marker negative cells; B, B-cells +ve for B220; Gr, granulocytes positive for Gr-1; M, macrophages/monocytes positive for Mac-1; T, T-cells positive for CD5 and/or CD8. The radiolabeled probe used is indicated to the left of each panel. CD34 is murine CD34; IgH is murine immunoglobulin H; c-fms was detected with a v-fms DNA fragment; lysozyme is murine lysozyme M; TCR is murine T-cell receptor Cbeta ; actin is murine beta -actin. The actin-probed blot is representative of several independently probed blots that included those previously probed with other cDNAs.
[View Larger Version of this Image (57K GIF file)]

We have examined these lineage-specific cDNA populations for evidence of expression of the four murine MIP-1alpha binding chemokine receptors, CCR-4 and MIP-1alpha RL1. The results from this analysis, shown in Fig. 6B, reveal expression of all the receptors in the T cell and monocyte/macrophage lineages. CCR-1, -3, -4, MIP1alpha RL1, and possibly CCR-5 are also expressed in the granulocyte subpopulation. In addition to macrophages and T-cells, we have also been able to detect CCR-1 and D6 expression in the XS52 Langerhans cell line suggesting that these receptors may be a normal component of the Langerhans/dendritic cell receptor population.3 Intriguingly, a number of the MIP-1alpha receptors are also detectable in primitive lineage negative hemopoietic cells suggesting a potential role for these genes in MIP-1alpha -induced stem cell inhibition (Fig. 6B). D6 is easily detected showing a strong enrichment in lin- cells relative to total bone marrow to levels similar to that seen with CD34 (Fig. 6B). CCR-3, and possibly CCR-5, are also detectable and the signals are significantly higher in the lin- cell population than in unfractionated bone marrow and the sorted B-cells.


DISCUSSION

In the present study, we have cloned seven murine chemokine receptors, including a novel cDNA which we have named D6. Four of these cDNAs, including D6, produce proteins in CHO cells which will bind to mMIP-1alpha , and extensive displacement experiments have demonstrated that these receptors also bind distinct arrays of other beta -chemokines. D6 is a signaling-competent receptor and can generate a transient rise in intracellular Ca2+ upon interaction with ligand. In addition, we have characterized the lineage-specific expression of each of the receptors, demonstrating T-cell and monocyte/macrophage expression of the four MIP-1alpha receptors, and moreover, have shown the expression of D6, and possibly CCR-3 and -5 in a stem cell-enriched bone marrow fraction.

Comparisons of the primary sequence of D6 and the other cloned chemokine receptors suggests that it is equally related to the CCR and CXCR families. Thus, D6 may structurally represent a member of a new subset of chemokine receptors. This suggestion is supported by alterations in motifs conserved throughout the majority of chemokine receptors, such as the DRYLAIV motif at the amino-terminal end of the second intracellular loop, that is changed to DKYLEIV in D6 altering the charge of this domain and possibly the G-protein subunit selection during signaling. However, it is clear from our binding and signaling data that it functions as a receptor which preferentially binds beta -chemokines. This may in part be mediated by the amino terminus of D6 which bears significantly more homology to beta -chemokine receptors, particularly CCR-1, than alpha -chemokine receptors.

Full-length D6 cDNA expressed in CHO cells produces the highest affinity murine MIP-1alpha receptor identified to date and shares many binding characteristics with CCR-5. This protein also binds with high affinity to human and murine MIP-1beta and human RANTES but interestingly, we were unable to displace mMIP-1alpha from D6 and CCR-5 with human MIP-1alpha . When the human and mouse MIP-1 proteins and human RANTES are aligned a number of conserved residues are apparent that are different in human MIP-1alpha . Two changes are of particular interest (labeled with stars in Fig. 7), namely Arg to Gln at position 22 (21 in mMIP-1alpha ) and Glu to Lys at 61 (60 in mMIP-1alpha ) that alter the charge at these positions. We have previously made a mutant of murine MIP-1alpha , PM3 (47), in which the Glu residue at 60 had been neutralized by alteration to Gln: this mutant exhibits wild type activity in biological assays (47), and binds with comparable affinity to D6 (data not shown). However, the charge reversal caused by the presence of Lys at 61 in hMIP-1alpha may have a more powerful effect on receptor-ligand interaction. The alteration from the bulky, basic Arg residue at 21 to the smaller uncharged Gln residue at 22 in hMIP-1alpha is a good candidate for being important in the difference in binding to D6 seen with murine and human MIP-1alpha : the difference in size and charge of the side chain may disrupt a potential receptor-binding domain in this area of the protein. Interestingly, based on the three-dimensional structures of beta -chemokines (61, 62) Glu-60 in the predicted carboxyl-terminal helix, and Arg-21, are likely to be on the surface of the protein in close proximity and may act as a D6-binding domain. However, Lys-44 and Arg-45 (labeled with a cross in Fig. 7) are also involved in mMIP-1alpha binding to D6 as neutralization of these residues in HepMut (33) reduces the affinity of mMIP-1alpha for this receptor with the Kd rising from 110 pM to 8 nM (data not shown). These residues are not sufficient, as they are also found in hMIP-1alpha . Thus, there may be several domains of mMIP-1alpha involved in mediating high affinity binding to D6, and subtle changes in these domains may exert quite profound effects on the ability of the ligand to interact. Given the similarities in the binding profiles of D6 and CCR-5, it is likely that the residues in mMIP-1alpha that mediate binding to CCR-5 may be the same as those discussed above for D6.


Fig. 7. Amino acid sequence alignment of human and murine chemokines. Amino acid residues of interest are highlighted with a asterisk (*) and discussed in the text. Lysine 44 and arginine 45 (numbering according to murine MIP-1alpha ) that are mutated to asparagine and serine, respectively, in mMIP-1alpha HepMut are indicated with a cross (+). The conserved cysteine residues are highlighted in the boxes. The numbers at the end of each sequence indicate the number of amino acid residues in the adjacent protein. h, human; m, murine.
[View Larger Version of this Image (15K GIF file)]

D6 is also able to interact well with JE (mMCP-1) and human MCP-3, demonstrating that this receptor is highly promiscuous with respect to ligand selection. Recent data suggests that mMCP-4 can also bind to D6, with a Kd in the low nanomolar range (data not shown). In contrast, CCR-1 and CCR-3 appear to be more specific for MIP-1alpha and -beta but have significantly lower affinity for these ligands than CCR-5 and D6. Interestingly, human RANTES did not displace mMIP-1alpha from these two receptors, which is in conflict with results previously published involving CCR-1 (53, 54) where this chemokine displaced mMIP-1alpha with an apparent Kd of 4 nM (54). The reason for this discrepancy is puzzling although there are a number of possible explanations. First, expression in different cell types may affect receptor folding or G-protein interaction that may alter ligand selection: we used CHO cells in this study while COS cells (54) or K562 cells (53) were used previously. Second, there are three amino acid changes between our sequence for CCR-1 and published sequences (Fig. 1), and one of these, Gln instead of His at 278, is in the third extracellular domain and may disrupt hRANTES binding. Indeed, this region of CCR-1 appears to be important in mediating competent ligand binding and receptor activation (56, 63).

CCR-3 binds human and murine MIP-1alpha and -beta , and reportedly acts as a receptor for human and murine eotaxin (60) although we have been unable to demonstrate eotaxin binding due to the tendency of this chemokine to aggregate with mMIP-1alpha . Gao and colleagues (60) were unable to detect calcium ion flux in response to mMIP-1alpha and -beta in HEK 293 cells transfected with CCR-3, but eotaxin could signal into these cells: it is still unclear whether the MIP-1 proteins are able to transduce a signal upon interaction with CCR-3 or if they are able to prevent eotaxin binding to CCR-3. Additional confusion comes from the easy detection of this receptor in the T-cell and monocyte/macrophage lineages, cells upon which eotaxin has no reported biological effect. This may indicate that CCR-3 has cell-type specific signaling properties permitting it to mediate eotaxin-induced chemotaxis in eosinophils but potentially other functions in T-cells and monocytes/macrophages in response to the MIP-1 proteins.

Finally, it is of note that the binding properties of these four murine receptors exhibit a number of differences compared with their presumed human homologues and highlights the potential discrepancy between structural and functional homology. Formal assignment of direct murine homologues therefore awaits more systematic generation of complete biological and functional data.

In conjunction with these detailed binding analyses we have demonstrated that all the four MIP-1alpha receptors are expressed in cells of the monocyte/macrophage and T-cell lineages. Which cell types within these lineages express these proteins remains to be determined. However, it seems possible that cells at distinct stages of differentiation, or with certain biological functions, may present a specific repertoire of MIP-1alpha receptors which could allow them to respond only to certain concentrations of ligand or generate receptor-specific biological responses.

We have previously shown that MIP-1alpha is able to inhibit the proliferation of hemopoietic stem cells and clonogenic epidermal cells (26, 27). While none of the four MIP-1alpha receptors is detected in murine keratinocytes (data not shown), our bone marrow sorting experiments have demonstrated that enrichment for hemopoietic stem cells results in a concomitant enrichment for cells expressing D6, and possibly CCR-3 and -5. These three receptors also bind to HepMut (data not shown), a mMIP-1alpha mutant that inhibits stem cell proliferation but is inactive in monocyte chemotaxis assays (33). However, to unequivocally demonstrate any involvement of the receptor(s) in hemopoietic stem cell inhibition it will be necessary to abrogate the expression of the receptor(s) in the target cell to see if inhibition by MIP-1alpha , and other chemokines, is lost. These experiments are currently underway in our laboratory.


FOOTNOTES

*   This work was supported by grants from the Cancer Research Campaign.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 44-141-942-9361; Fax: 44-141-942-6521; E-mail: GPMA09{at}udcf.gla.ac.uk.
1   The abbreviations used are: MIP, macrophage inflammatory protein; IL, interleukin; MCP, monocyte chemoattractant protein; RANTES, regulated on activation, normal T cell expressed and secreted; CCR, C-C chemokine receptor; CHO, Chinese hamster ovary; FCS, fetal calf serum; RT, reverse transcriptase; PCR, polymerase chain reaction; m, murine; h, human; RACE, rapid amplification of cDNA ends; lin-, lineage negative; PBS, phosphate-buffered saline.
2   R. Nibbs, unpublished data.
3   R. Nibbs, unpublished observations.

ACKNOWLEDGEMENTS

We thank M. Freshney for cell sorting expertise, R. MacFarlane for operation of the DNA sequencer, Drs. Stewart, Cross, Ford, and May for providing cDNA clones, Dr. Takashima for the XS52 cell line, and Dr. Czaplewski for the HEK 293 cells and advice on Ca2+ measurements. We also thank Professor Wyke for critically reading this manuscript. Dr. Nibbs also thanks Dr. Amanda Wilson for support services.


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