CCR11 is a functional receptor for the monocyte chemoattractant protein family of chemokines.

Chemokines mediate their diverse activities through G protein-coupled receptors. The human homolog of the bovine orphan receptor PPR1 shares significant similarity to chemokine receptors. Transfection of this receptor into murine L1.2 cells resulted in responsiveness to monocyte chemoattractant protein (MCP)-4, MCP-2, and MCP-1 in chemotaxis assays. Binding studies with radiolabeled MCP-4 demonstrated a single high affinity binding site with an IC(50) of 0.14 nM. As shown by competition binding, other members of the MCP family also recognized this receptor. MCP-2 was the next most potent ligand, with an IC(50) of 0.45 nM. Surprisingly, eotaxin (IC(50) = 6.7 nM) and MCP-3 (IC(50) = 4.1 nM) bind with greater affinity than MCP-1 (IC(50) = 10.7 nM) but only act as agonists in chemotaxis assays at 100-fold higher concentrations. Because of high affinity binding and functional chemotactic responses, we have termed this receptor CCR11. The gene for CCR11 was localized to human chromosome 3q22, which is distinct from most CC chemokine receptor genes at 3p21. Northern blot hybridization was used to identify CCR11 expression in heart, small intestine, and lung. Thus CCR11 shares functional similarity to CCR2 because it recognizes members of the MCP family, but CCR11 has a distinct expression pattern.

Chemokines are a family of small proteins, usually 70 -90 amino acids in length, that are responsible for the directed migration of specific cell types (for reviews, see Refs. [1][2][3][4][5][6]. The complexity and functions of the chemokine family, now with more than 30 genes, have become increasingly diverse as more members have been identified and characterized. Chemokines play a critical role in the host response to infection because they are responsible for recruitment of leukocyte subsets to sites of pathogen entry (7,8). Many inflammatory diseases, such as rheumatoid arthritis, inflammatory bowel disease, and asthma (9), have been associated with elevated chemokine expression. In addition, chemokines are also responsible for the migration of cells within certain lymphoid organs that are critical for leukocyte development, such as thymus (10 -12), lymph node (13), and spleen (14,15). As shown by gene targeting studies, the chemokine stromal cell-derived factor (SDF)-1 is critical for proper neuronal and cardiac development (16,17). Chemokines have also been implicated in cardiovascular processes such as angiogenesis and atherosclerosis (18).
Chemokines are recognized by specific seven transmembrane-spanning, G protein-coupled receptors (GPCRs) 1 (for review, see Refs. 19 and 20). Previously characterized chemokine receptors share significant homology, with 25-65% identical amino acids, and consequently form their own branch of the GPCR family tree. Many chemokine receptors were originally identified as orphan GPCRs. There remain several orphan GPCRs with high similarity to the chemokine receptor family.
The orphan receptor PPR1 was originally isolated from bovine papillary tissue in a search for gustatory receptors (21). However, the expression of PPR1 appears to be higher in lung than in tongue. In addition, PPR1 shares more similarity to chemokine receptors than gustatory or olfactory receptors. Because of this similarity, we isolated a human homolog of PPR1 and examined its ability to function as a chemokine receptor. The human homolog binds members of the MCP family (MCP-1, MCP-2, MCP-3, MCP-4, and eotaxin) with high affinity and also mediates responses to MCP-4, MCP-2, and MCP-1 in chemotaxis assays. In accordance with the Chemokine Nomenclature Committee, we have designated this receptor CCR11. Isolation of CCR11 cDNA and Gene-The GenBank Expressed Sequence Tag (EST) data base was searched with the bovine PPR1 cDNA sequence (21) using the BLAST algorithm (22). Three human ESTs were identified (H67224, AA215577, AI131555) with high homology to the bovine sequence. The clone H67224 was obtained from Research Genetics (Huntsville, AL), and the entire insert was sequenced. Because this EST contained only a fragment of the coding region, additional cDNA libraries were screened. Three human cDNA libraries were hybridized with a probe from the EST sequence (prepared by polymerase chain reaction amplification with the primers 5Ј-GTCTCTGGAAT-GCAGTTTCTGG and 5Ј-CGATGTCCATGCGTTTGCTCA): small intestine (Stratagene, La Jolla, CA), macrophage (described by 23), and * 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.

Materials and Reagents
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AF193507.
‡ To whom correspondence should be addressed: ICOS Corp., 22021 20th Ave. SE, Bothell WA 98021. Tel.: 425-415-2244; Fax: 425-486-0300; pgray@icos.com. 1 The abbreviations used are: GPCR(s), G protein-coupled receptor(s); bp, base pairs; CCR, CC chemokine receptor; ELC, EBI1-ligand chemokine; ENA-78, epithelial cell-derived neutrophil-activating protein; EST, expressed sequence tag; LARC, liver and activation-regulated chemokine; LKN-1, leukotactin-1; MCP, monocyte chemoattractant protein; MDC, macrophage-derived chemokine; MIP, macrophage inflammatory protein; NAP-2, neutrophil-activating protein-2; PARC, pulmonary and activation-regulated chemokine; MIG, monokine induced by interferon ␥; IL-8, interleukin-8, IP-10, interferon ␥-inducible protein-10, MGSA, melanocyte growth-stimulating activity; PF-4, platelet factor-4; RANTES, regulated on activation, normal T cell expressed and secreted; SDF, stromal cell-derived factor; SLC, secondary lymphoid tissue chemokine; TARC, thymus and activation-regulated chemokine; TECK, thymus-expressed chemokine; HCC, hemofiltrate CC chemokine. peripheral blood mononuclear cell (phorbol myristate acetate/ionomycin-stimulated, 24). More than a million clones were examined in each library. No clones were found in the macrophage library. A single clone was identified in the peripheral blood mononuclear cell library which was 1388 bp in length and lacked 188 bp of the amino-terminal coding sequence. Five clones were isolated from the small intestine cDNA library, ranging in size from 131 to 1153 bp. The consensus cDNA sequence was missing 14 bp from the 5Ј-end of the coding region when aligned with the bovine PPR1 coding region sequence. To determine the amino-terminal coding region, a human genomic P1 library (Genome Systems Inc., St. Louis) was screened by polymerase chain reaction with the above primers to isolate the CCR11 gene. The 5Ј-coding region of the isolated clone was sequenced with primers based on the cDNA sequence. The deduced genomic sequence provided the remaining coding sequence for CCR11. The genomic sequence presented in Fig. 1 (residues 1-275) appears to contain no intervening sequences because it has contiguous homology with the bovine cDNA sequence (21). Four nucleotide differences were identified, one of which resulted in an amino acid change at position 143 (lysine, in the genomic and small intestine clones, to asparagine, in the peripheral blood mononuclear cell clone).
CCR11 Expression-The CCR11 coding region was amplified from the P1 clone with primers 5Ј-GCCCAAGCTTGCCACCATGGCTTTG-GAACAGAACCAGTCAAC and 5Ј-CTAGTCTAGAGTATCCAAGCAAA-AGGCAGAGCAG, which included HindIII and XbaI cloning sites. This fragment was inserted into pNEF6, a vector containing the Chinese hamster elongation factor-1␣ gene promoter and neomycin resistance gene. The amplified coding region of CCR11 was sequenced to ensure that no mutations were introduced by polymerase chain reaction. The expression construct was transfected into mouse pre-B L1.2 cells by electroporation with 10 g of plasmid at 250 V, 960 microfarads, 72 ohm resistance using a Gene Pulser (Bio-Rad). Transfectants were selected and expanded in 800 g/ml G418. These cells were used in a chemotaxis assay against a panel of chemokines (1 nM and 10 nM each) in order to identify ligands for CCR11 and to isolate CCR11-expressing transfectants (see under "Chemotaxis Assays"). Transfected cells that migrated were collected, cloned by limiting dilution, and expanded for further analysis.
Chromosomal Localization-A genomic P1 clone of approximately 90-kilobase pairs containing the human CCR11 gene was labeled with digoxigenin dUTP by nick translation and used as a probe for fluorescence in situ hybridization of human chromosomes (Genome Systems, Inc.). The labeled probe was hybridized to normal metaphase chromosomes derived from phytohemagglutinin-stimulated peripheral blood lymphocytes. Reactions were carried out in the presence of sheared human DNA in 50% formamide, 10% dextran sulfate, 30 mM sodium chloride, 3 mM sodium citrate, and 0.1% SDS. Hybridization signals were detected by treating slides with fluoresceinated anti-digoxigenin antibodies followed by counterstaining with 4,6-diamidino-2-phenylindole. Initial hybridization resulted in specific labeling of the middle long arm of a group A chromosome believed to be chromosome 3 on the basis of size, morphology, and banding pattern. A labeled genomic probe that is specific for the centromere of chromosome 3 was co-hybridized with the CCR11 probe and detected with Texas Red avidin. 80 metaphase cells were analyzed, with 72 exhibiting specific labeling.
Northern Blot Analysis-The expression of CCR11 mRNA was examined by Northern blot analysis. A human multi-tissue Northern blot was purchased from CLONTECH and hybridized as described (25). A gel-purified fragment containing most of the coding region of human CCR11 (1388 bp) was used as a hybridization probe.
Chemotaxis Assays-Cell migration was assayed using L1.2 cells stably transfected with CCR11 cDNA. Approximately 10 6 cells resuspended in 0.1 ml of RPMI 1640 medium with 0.5% bovine serum albumin (endotoxin-reduced, Intergen, Purchase, NY) were loaded in the upper wells of a transwell chamber (3-m pore size, 6.5-mm diameter, Costar, Cambridge, MA). Test chemokines at the concentrations indicated were added to the lower wells in a volume of 0.6 ml. After 4 h at 37°C, cells that migrated to the lower chamber were collected and counted using a fluorescence-activated cell sorter (Becton-Dickinson, Franklin Lakes, NJ). Values are expressed as the chemotaxis index, which is the ratio of cells that migrated toward chemokine divided by cells that migrated toward buffer alone.
Calcium Mobilization-Cells were suspended at 3 ϫ 10 6 cells/ml in complete RPMI medium with 10% fetal bovine serum. Cells were incubated with 1 M fura-2/AM (Molecular Probes, Eugene OR) at room temperature for 30 min in the dark. After washing, cells were resuspended at 2 ϫ 10 6 cells/ml in phosphate-buffered saline. To measure intracellular calcium, cells in 2 ml were placed in a quartz cuvette in an SLM Aminco-Bowman series 2 luminescence spectrometer. Fluorescence was monitored at 340 nm (excitation wavelength 1), 380 nm (excitation wavelength 2), and 510 nm (emission wavelength). Chemokines were added at 100 nM final concentration.
Binding Assays-For binding experiments, 5 ϫ 10 5 CCR11 transfected L1.  (21) previously isolated an orphan GPCR from bovine taste papillary tissue. Hydropathy and sequence analyses demonstrated that PPR1 was a member of the GPCR superfamily. More recent homology comparisons suggested a closer relationship to chemokine receptors than gustatory or olfactory receptors. Three human EST cDNA sequences were identified in the GenBank data base with high homology to the bovine PPR1 sequence. Oligonucleotide primers were designed from the human sequences and used to identify six partial cDNA clones and a genomic P1 clone of approximately 90 kilobase pairs which contained the entire gene sequence. Based on the functional data below, we have designated this human gene CCR11.

Isolation of the Human Gene for PPR1-Matsuoka and colleagues
The CCR11 DNA sequence and encoded amino acid sequence are presented in Fig. 1. Hydropathy analysis (not shown) delineated seven hydrophobic domains typical of a seven-transmembrane spanning GPCR. Human CCR11 is 86% identical to bovine PPR1 at the amino acid level. This high degree of similarity is consistent with other GPCR genes when compared across mammalian species. Like most GPCRs, CCR11 contains potential N-linked glycosylation sites, two in the amino-terminal extracellular domain and one in the third extracellular loop. Similar to other chemokine receptor sequences, CCR11 contains single cysteine residues in each of the four predicted extracellular domains. As shown in Fig. 2, CCR11 shares 28 -36% identity with other human chemokine receptors. The receptor with highest homology to CCR11 is CCR7 (36% identical at the amino acid level) followed by CCR6 and CCR9 (each 33% identical to CCR11). CCR11 is less homologous to other members of the GPCR superfamily, the next closest being lipid mediator receptors (platelet-activating factor receptor, 24%; leukotriene B4 receptor, 22%) and the chemotactic peptide receptor (fMet-Leu-Phe receptor, 19%). Like many other GPCR genes, the coding region of the CCR11 gene contains no intervening sequences.
Chromosomal Localization of CCR11-Many GPCR genes are clustered in the human genome. Indeed, the genes for the majority of the CC chemokine receptors are encoded at 3p21 (25,26). Because clusters of genes are generally functionally related, we identified the chromosome location of the CCR11 gene. The human P1 clone containing the CCR11 gene was used as a probe for fluorescence in situ hybridization to human chromosomes. The results are presented in Fig. 3, where the CCR11 probe signal is green, and a specific chromosome 3-centromere probe signal is red. Measurement of specifically labeled chromosomes demonstrated that the CCR11 gene is located at a position that is 42% the distance from the centromere to the telomere of chromosome arm 3q, an area that corresponds to 3q22.
Northern Blot Analysis-To determine sites of expression of CCR11, Northern blot hybridizations were performed. The CCR11 gene was used as a hybridization probe for 12 different human tissues. CCR11 was expressed most abundantly in human heart, small intestine, and lung (Fig. 4). Lower levels of hybridization were observed in kidney, liver, and colon. The size of the primary transcript is approximately 2000 bases, which corresponds well with the cDNA size. The most abundant transcript in heart appears to be of greater size than that seen in other tissues and perhaps represents an alternatively spliced transcript.
Functional Responses of CCR11 Transfectants-Murine L1.2 cells were transfected with CCR11 and then tested for chemo- CCR11 transfectants were also tested for calcium mobilization in response to ligand stimulation. Small but significant calcium flux was observed when transfectants were stimulated with MCP-4 (results not shown). This response was quantitatively not as strong as we have observed previously with other chemokine receptor-ligand pairs (see "Discussion"). No significant calcium flux was observed in response to MCP-1 or MCP-2 stimulation.
Receptor Binding Assays-Because MCP-4 was the most potent functional ligand, radiolabeled MCP-4 was used as a probe to examine binding to CCR11 transfected L1.2 cells. As shown in Fig. 6A, the 125 I-MCP-4 binding was inhibited competitively with increasing concentrations of unlabeled MCP-4 (IC 50 of 0.140 nM), MCP-2 (IC 50 of 0.458 nM), MCP-3 (IC 50 of 4.08 nM), eotaxin (IC 50 of 6.72 nM), or MCP-1 (IC 50 of 10.7 nM). This suggests that all five ligands recognize a common binding site on CCR11 and that MCP-4 exhibits the greatest affinity. The observed binding of MCP-4, MCP-2, and MCP-1 is consistent with the functional chemotactic responses described above. However, MCP-3 and eotaxin bind with reasonable affinity but only act as agonists at more than 100-fold higher concentrations.
To examine specificity of binding to CCR11, 17 additional chemokines were tested at 1000-fold molar excess for competition of radiolabeled MCP-4 binding. The MCP family members, including eotaxin, effectively competed with 125 I-MCP-4 for binding to CCR11 (Fig. 6B). The other chemokines did not compete for CCR11 binding even at this high concentration. DISCUSSION CCR11 was identified during a search of the human EST data base for homologs of the bovine orphan PPR1. When the full coding region of CCR11 was assembled, it was found to be 86% identical to PPR1 at the amino acid level. Homology com-  parisons indicated that CCR11 is most closely related to chemokine receptors. Its closest relatives are CCR7 (36% identical), CCR6 (33%), and CCR9 (33%). Chromosomal mapping of CCR11 localized it to 3q22. Interestingly, many other CC chemokine receptors also map to chromosome 3, including CCR1, CCR2, CCR3, CCR4, CCR5, and CCR8 (26). CCR11, however, is significantly separated from these receptors, which are clustered at 3p21-24. This suggests that CCR11 is more distantly related to most CC chemokine receptors, consistent with the sequence homology comparisons presented in Fig. 2. The CCR11 gene maps somewhat closer to the orphan receptor GPR15 (27; also known as BOB, 28) which is located at 3q11.2-13.1 (27).
As demonstrated in binding and chemotaxis studies, CCR11 is a chemokine receptor that recognizes ligands in the MCP family. The primary ligands for CCR11 are MCP-4 and MCP-2, based on binding affinities and agonist properties in chemotaxis experiments. Other MCP family members also interact with CCR11 with lower affinities. Although CCR11 is most closely related to CCR7, it does not interact with the CCR7 ligands ELC and SLC.
The MCPs share high homology with each other (56 -72%) and form their own branch of the CC chemokine family tree. In addition, the MCPs share some functional similarity and are all closely linked on human chromosome 17q11.2 (29). However, MCP expression patterns are distinct, with MCP-4 being expressed constitutively in lung, small intestine, and colon (30,31), whereas MCP-1 is expressed primarily in cells stimulated with proinflammatory agents (32,33). MCP-4 has been identified previously as an agonist for CCR2 and CCR3 (30,31). MCP-2 is recognized by CCR1, CCR2, CCR3, and CCR5 (34 -36). MCP-1 is the strongest ligand for the receptor CCR2 (37), and this receptor also recognizes MCP-2 (34), MCP-3 (38), and MCP-4 (30,31). The characterization of MCP family members as ligands for CCR11 adds additional complexity and redundancy to this diverse repertoire of chemokine functions.
Identification of ligands for orphan GPCRs can be complex. GPCRs can exhibit paradoxical behavior, particularly transfected recombinant receptors. Although not well understood, such unusual behavior may be caused by inappropriate G pro-tein usage, overexpression of recombinant receptors, or other as yet unidentified phenomena. Our laboratory has noted that some chemokine receptors may not be expressed in a stable manner and that functional responses can be lost if not selected for repeatedly. 2 Overexpression is a natural consequence of using a strong promoter and may lead to functional responses that are potentially deleterious to transfected cells. Some changes we have observed with GPCR transfectants are increases in cell adhesiveness or decrease in growth rate. 3 With CCR11 our transfected cell population was initially selected by chemotaxis. When these cells were cloned, the majority had lost their responsiveness to MCP-4, but some clones responded even more vigorously than the original selected population. Thus, chemotactic selection greatly aided our identification and characterization of CCR11.
Compared with other characterized chemokine receptors, we observed only weak calcium mobilization in response to MCP-4 stimulation. Perhaps CCR11 signal transduction is linked to G proteins that are not well complemented in L1.2 cells. Perhaps this receptor does not naturally induce a strong calcium response, like some other GPCRs. Alternatively, CCR11 calcium responses in L1.2 cells may be linked to cellular toxicity. Finally, CCR11 may recognize other, as yet unidentified, ligands that cause more significant calcium flux. Nevertheless, MCP-4  is a major ligand for CCR11 as shown by its strong binding affinity and potent agonist activity in chemotaxis experiments.
As shown by Northern blot analysis, CCR11 has an unusual pattern of expression for a chemokine receptor. Because it is not highly expressed in lymphoid organs such as thymus or spleen, CCR11 is not likely to be involved in lymphocyte development as are CCR4, CCR7, and CCR9 (Refs. 10 -15). In addition, CCR11 is virtually undetectable in peripheral blood, being primarily expressed in the heart, small intestine, and lung. With the exception of CXCR4, which is broadly expressed in many tissues, chemokine receptors are typically expressed exclusively on cells of lymphoid or myeloid origin. Our inability to detect transcript in these cells may indicate that CCR11 is expressed on a subpopulation of lymphoid cells that are rare in whole blood but resident in specific tissues. CCR3, for example, is expressed only on eosinophils and a subset of Th2 cells and is undetectable by Northern blot in peripheral blood (39,40). Alternatively, CCR11 may be expressed on parenchymal cells and play a role currently unappreciated for chemokine receptors. Although chemokines and chemokine receptors are generally known for their role in immune cell development and trafficking, there is preliminary evidence that they may have functions outside of the immune system. Knockout experiments have shown that CXCR4 is essential for normal development of the heart, small intestine and brain (16,41). In addition, Streblow et al. (43) have recently proposed that the virally encoded chemokine receptor US28 may play a role in the migration of smooth muscle cells seen in cytomegalovirus exacerbation of vascular disease (43).
Based on its expression pattern, CCR11 may function in cells that are resident in highly vascularized tissues. MCP-1 and CCR2 have previously been associated with atherogenesis and are thought to play a role in recruitment of macrophages to initiate atherosclerotic plaque formation (18,44). Vascular expression of MCP family members results in CCR2-mediated monocyte recruitment and macrophage development; this may be accompanied by CCR11-mediated events. Perhaps CCR2 and CCR11 complement each other in vascular processes such as remodeling of the vessel wall to accommodate monocyte influx. The complex redundancy of MCP chemokines and their receptors in the vasculature may help to explain the results of transgenic and gene knockout studies (18,42,44,45). These gene alterations are not lethal and often do not have severe complications on their own, suggesting a compensatory role of genes with similar function. Further studies with CCR11 and its ligands will be required to understand fully their roles in health and disease.