CCR9A and CCR9B: Two Receptors for the CCL25/TECK/Ck␤-15 That Differ in Their Sensitivities to Ligand

Cheng-Rong Yu,* Keith W. C. Peden,† Marina B. Zaitseva,† Hana Golding,† and Joshua M. Farber1*

We isolated cDNAs for a -related protein having the database designation GPR-9-6. Two classes of cDNAs were identified from mRNAs that arose by alternative splicing and that encode receptors that we refer to as CCR9A and CCR9B. CCR9A is predicted to contain 12 additional amino acids at its N terminus as compared with CCR9B. Cells transfected with cDNAs for CCR9A and CCR9B responded to the chemokine CC chemokine ligand 25 (CCL25)/thymus-expressed chemokine (TECK)/chemokine ␤-15 (CK␤-15) in assays for both calcium flux and . No other tested produced re- sponses specific for the cDNA-transfected cells. mRNA for CCR9A/B is expressed predominantly in the thymus, coincident with the expression of CCL25, and highest expression for CCR9A/B among subsets was found in CD4؉CD8؉ cells. mRNAs encoding the A and B forms of the receptor were expressed at a ratio of ϳ10:1 in immortalized lines, in PBMC, and in diverse populations of . The EC50 of CCL25 for CCR9A was lower than that for CCR9B, and CCR9A was desensitized by doses of CCL25 that failed to silence CCR9B. CCR9 is the first example of a chemokine receptor in which alternative mRNA splicing leads to proteins of differing activities, providing a mechanism for extending the range of concentrations over which a cell can respond to increments in the concentration of ligand. The study of CCR9A and CCR9B should enhance our understanding of the role of the chemokine system in T cell biology, particularly during the stages of thymocyte development. The Journal of Immunology, 2000, 164: 1293–1305.

he chemokines are now known to number more than 30 and work in our and other laboratories using immortalized CD4ϩ human chemotactic distributed among CXC, T cell lines have suggested that there might exist as yet unidenti- CC, C, and CX C subfamilies, which act through seven- fied HIV-1 coreceptor(s) (K.W.C.P., unpublished observations, T 3 transmembrane-domain G protein-coupled receptors, of which 15 and Ref. 11). In experiments to identify such receptors, we found have been described in humans (reviewed in Ref. 1). The chemo- that the SUP-T1 T cell lymphoma line expressed GPR-9-6, a gene kine system is increasingly recognized to play an important role in entered in the database as encoding a chemokine receptor-related lymphocyte biology. The activity of chemokines as chemotactic protein (GenBank accession number U45982). While our experi- factors for lymphocytes has been known for some time, along with ments to date with a limited number of HIV-1 strains have not selective effects of chemokines on T cell subsets (2). However, the revealed coreceptor activity for GPR-9-6, we found that GPR-9-6 recent identification of many additional lymphocyte-active chemo- encodes signaling receptors for the CC chemokine ligand 25 kines has suggested broad involvement of the chemokine system in (CCL25)2/thymus-expressed chemokine (TECK)/Ck␤-15. In the lymphocyte trafficking, which is integral to lymphocyte develop- remainder of this work, we will, for the sake of clarity, designate ment and to the functions of mature cells in immunity and inflam- the GPR-9-6 gene CCR9, in accordance with the recommendations mation (reviewed in Refs. 1, 3, and 4). The discoveries that che- of the committee for chemokine receptor nomenclature (Philip mokine receptors function as obligatory coreceptors for HIV entry Murphy and Craig Gerard, personal communication). Similarly, and that chemokines can act as inhibitors of HIV infection have we will adopt the chemokine nomenclature proposed at the Key- increased the importance of understanding the roles of chemokines stone Symposium on Chemokines and Chemokine Receptors, Jan- and their receptors in lymphocyte physiology and pathophysiology uary 18–23, 1999, and refer to the chemokine TECK/Ck␤-15 as to develop effective therapies (reviewed in Ref. 5). CCL25. We have previously identified and characterized novel chemo- CCL25 was discovered by sequencing cDNAs made from the kine receptors and HIV/SIV coreceptors on lymphocytes (6–10), thymuses of RAG-1-deficient mice and is an unusual CC chemo- kine in many respects (12). The sequence of CCL25 is less than 30% identical to any other CC chemokine, and the CCL25 gene is *Laboratory of Clinical Investigation, National Institute of Allergy and Infectious not found in any of the known CC chemokine gene clusters, but is Diseases, National Institutes of Health, Bethesda, MD 20892; and †Laboratory of Retrovirus Research, Center for Biologics Evaluation and Research, Food and Drug instead on chromosome 8 in mice (12) and on in Administration, Bethesda, MD 20892 humans (13). Expression of the CCL25 gene is restricted, with Received for publication May 7, 1999. Accepted for publication November 22, 1999. mRNA detected in thymus and small intestine, and in spleen after The costs of publication of this article were defrayed in part by the payment of page challenge with LPS; expression in the thymus was shown to be in charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Address correspondence and reprint requests to Dr. Joshua M. Farber, Laboratory of 2 Abbreviations used in this paper: CCL, CC chemokine ligand; Ck␤, chemokine ␤; Clinical Investigation, National Institute of Allergy and Infectious Diseases, Building CXCL, CXC chemokine ligand; GFP, green fluorescent protein; HEK, human em- 10, Room 11N-228, MSC 1888, National Institutes of Health, Bethesda, MD 20892. bryonic kidney; SDF-1, stromal cell-derived factor-1; TECK, thymus-expressed che- E-mail address: [email protected] mokine; TMD, transmembrane domain.

Copyright © 2000 by The American Association of Immunologists 0022-1767/00/$02.00 1294 RECEPTORS FOR THE CHEMOKINE CCL25

ϩ dendritic cells and, at least in the fetal thymus, in MHC II epi- MD). MT4 cells were kindly provided by Malcolm Martin (National In- thelium as well (12, 14). CCL25 has been reported to be a che- stitutes of Health). H9 cells were derived as described (22) and obtained moattractant for mouse thymocytes and for thymocyte precursors from the Center for Biologics Evaluation and Research, Food and Drug ␥ Administration. Elutriated monocytes and PBMC were obtained from nor- (12, 14) as well as for mouse splenic dendritic cells and IFN- - mal donors by the Department of Transfusion Medicine, National Institutes activated (12). Based on these observations, CCL25 of Health. Macrophages were produced by culturing monocytes for 1 wk in has been proposed to be important in T cell development, either by Iscove’s medium containing 10% human serum type AB (Sigma, St. Louis, affecting recruitment of thymic precursors, migration of thymo- MO) plus pyruvate. PBMC were activated by culturing with 1 ␮g/ml PHA and 20 IU/ml of IL-2 for 3 days. HEK-293 cells were grown in MEM plus cytes within the thymus, or recruitment of thymic dendritic cells 10% horse serum. Other cell lines and PBMC were grown in RPMI 1640 that are important for autoantigen-driven negative selection (12). with 10% FBS. For purification of thymocytes, thymus fragments were Although the large number of lymphocyte-targeting chemo- obtained during cardiac surgery from children (ages 1 mo-3 yr) with con- kines, together with ligand/receptor promiscuity, suggest extensive genital heart disease. The tissue was minced, large aggregates were re- redundancy in the activities of the chemokine system in lympho- moved by passing through a nylon mesh, and thymocytes were separated by centrifugation on a Ficoll-Paque gradient (Pharmacia Biotech, Uppsala, cyte biology, it is becoming clear that particular ligand/receptor Sweden). The total thymocyte suspension was separated into CD4ϪCD8Ϫ, groups act preferentially on lymphocytes at specific stages of de- CD4ϩCD8ϩ, CD4ϩCD8Ϫ, and CD8ϩCD4Ϫ, thymocyte subsets using the velopment, activation, and differentiation. As examples, CXCL12 CD4 Multisort Kit and VarioMACS magnet separator according to the (stromal cell-derived factor-1 (SDF-1)) is necessary for B cell de- manufacturer’s instructions (Miltenyi Biotec, Auburn, CA). The pheno- types of separated thymocyte subsets were analyzed using PE-conjugated velopment (15), and CCR7 ligands are necessary for trafficking of anti-CD4 and allophycocyanin-conjugated anti-CD8 mAbs from PharM- naive T cells into secondary lymphoid organs (16); many receptors ingen (San Diego, CA). The CD4ϪCD8Ϫ preparation contained ϳ20% for proinflammatory CC chemokines show significant expression/ CD4dull cells, which also represent immature thymocytes (23). The ϩ ϩ ϩ Ϫ activity only on activated memory T cells (17, 18); CXCR3 ex- CD4 CD8 preparation was Ͼ95% pure. The CD4 CD8 preparation contained, in addition to these cells, ϳ20% CD4ϩCD8ϩ cells and 10% pression/activity is intimately associated with T cell activation of Ϫ Ϫ Ϫ ϩ CD4 CD8 cells, and the CD4 CD8 preparation contained, in addition both naive and memory cells (18); CCR6 is distinguished by its to these cells, ϳ20% CD4dullCD8Ϫ cells, 20% CD4ϩCD8ϩ cells, and 10% being fully active on resting memory T cells (8, 19); and a number CD4ϪCD8Ϫ cells. of chemokine receptors have been reported to show preferential expression depending on whether CD4 T cells have differentiated Cloning of CCR9A and CCR9B cDNAs down Th1 vs Th2 pathways (reviewed in Ref. 20). With respect to Total RNA was prepared from the SUP-T1 cells using TRIzol reagent (Life thymocyte development, a good deal of circumstantial evidence ϩ based on expression data has suggested the importance of the che- Technologies, Gaithersburg, MD); poly(A) RNA was selected using oli- go(dT) cellulose (Collaborative Biomedical Products, Bedford, MA); and mokine system, but no definitive role for chemokines and their cDNA was synthesized using oligo(dT) primers and the SuperScript Pre- receptors has been established. CCL25 is of particular interest in amplification System (Life Technologies), according to suppliers’ proto- this regard because of its restricted tissue and cell-type expression cols. For amplification, pools of degenerate primers containing NotI sites and its activity on thymocytes. (bolded below) were designed based on transmembrane domain (TMD) III and TMD VII amino acid sequences of G protein-coupled receptors from We have isolated cDNAs encoding receptors for CCL25. These the human sequences for CCR2B, CCR5, CCR3, CCR8, CX3CR1, cDNAs encode two proteins that we have designated CCR9A and CXCR4, STRL33, GPR15, APJ, GPR1, and D6. The TMD III primers that CCR9B. As the result of differential splicing of the CCR9 mRNA, yielded the amplified cDNAs described below were 5Ј-GCGGTGGCG CCR9A is predicted to contain 12 additional amino acids at its N GCCGC(C/G)T(C/G/T)GA(C/T)(A/C)(C/G)IT(A/T)(A/C)T(G/T)(C/G)(A/ Ј terminus as compared with CCR9B. This degree of polymorphism C/T)I(A/G)GGT, and the TMD VII primers were 5 -GCGGTGGCGGCCG C(G/T)(G/T)(A/C)(A/G)TA(A/C/G)A(G/T)(C/G)AI(A/C/G)GG(A/G)(G/T) at the N terminus of a chemokine receptor is novel. Of greater (A/T)(C/G)AIGCA. PCR amplifications were done with cDNA synthe- interest, we demonstrate that CCR9A and CCR9B, while both ac- sized from 0.015 ␮g poly(A)ϩ and with 1.5 ␮M of each primer in a 20 ␮l tive, are not functionally equivalent, with CCR9A signaling and reaction volume with Taq polymerase and reagents from Perkin-Elmer being desensitized at lower concentrations of chemokine as com- (Norwalk, CT), according to the supplier’s protocol. PCR was done using 30 cycles of denaturation at 94°C for 0.5 min, annealing at 45°C for 2 min, pared with CCR9B. These data suggest that the two forms of and chain extension at 72°C for 1.5 min. CCR9 extend the range of CCL25 concentrations over which a cell One microliter of this first PCR amplification was used for substrate in can sense increments in the concentration of ligand. Consistent a second PCR done identically to the first. The products of the second with the expression of CCL25, the CCR9 gene is highly expressed reaction were digested with BamHI to eliminate the amplified products in thymus, with analysis of thymocyte subsets revealing highest from CXCR4, which is highly expressed in SUP-T1 cells and which con- ϩ ϩ tains an internal BamHI site, and then separated by electrophoresis on a levels in the CD4 CD8 cells. In all lymphocytes examined, 1.5% agarose gel. The fragments of 600–650 bp were purified, digested mRNAs for CCR9A and CCR9B were found at a ratio of ϳ10:1. with NotI, and ligated into pBlueScript (Stratagene, La Jolla, CA). The Our work provides information and tools that are likely to lead to ligated DNA was used to transform bacteria that were grown on indicator an increased understanding of the activities of the chemokine sys- plates, and white colonies were picked and grown for sequencing. Using poly(A)ϩ RNA prepared from SUP-T1 cells, a cDNA library was prepared tem, particularly in regard to thymocytes and T cell development, in the ␭ ZAP Express vector (Stratagene), according to the supplier’s pro- enhancing our appreciation of the breadth of involvement of che- tocol. Approximately 2.5 ϫ 106 plaques from the nonamplified library mokines and chemokine receptors in lymphocyte biology. were screened using a radiolabeled CCR9 cDNA probe. Phage from 11 positive plaques were plaque purified, and the pBK-CMV plasmids con- taining CCR9 inserts were recovered by in vivo excision, according to the supplier’s protocol. Nine clones were sequenced either completely or in Materials and Methods part using a Perkin-Elmer ABS Prism 377 automated sequencer, according Cell culture to the manufacturer’s protocols.

Jurkat-TAg cells, which had been transfected with the large T-Ag gene of Construction of CCR9A and CCR9B expression vectors SV40, were derived as described (21) and kindly provided by Lawrence Samelson, National Institutes of Health (Bethesda, MD). Jurkat clone E6-1, CCR9A cDNA clone 6 was cut at BsmFI sites at positions 85 and 1351 (see SUP-T1, U937, and human embryonic kidney (HEK) 293 cells were ob- GenBank sequence for CCR9A cDNA, accession number AF145439), li- tained from American Type Culture Collection (Manassas, VA). CEM gated to a 5Ј adapter containing an XhoI site and a 3Ј adapter containing a x174 and MOLT-4 clone 8 cells were obtained from the National Institutes SalI site 5Ј to a BglII site, and then ligated into pCEP4 (Invitrogen, Carls- of Health AIDS Research and Reference Reagent Program (Rockville, bad, CA) that had been cut with XhoI and BamHI. CCR9B cDNA clone 3 The Journal of Immunology 1295 was cut at BsmFI sites at positions 53 and 1270 (see GenBank sequence for SSC and 1% SDS at 50°C overnight with 1–2 million cpm/ml using an CCR9B cDNA, accession number AF145440) and treated as for CCR9A oligonucleotide containing sequences common to CCR9A and -B, (5Ј- above. To place the CCR9A/B cDNAs in a vector containing an SV40 GCTGATGACTATGGCTCTGAATCCACATCT) or using a GAPDH oli- origin of replication, they were excised from pCEP4 using XhoI and SalI gonucleotide (5Ј-GTGGAAGGACTCATGACCACAGTCCATGCC) that and inserted into a similarly cut pCI-neo (Promega, Madison, WI). For had been 32P end-labeled using polynucleotide kinase (New England Bio- transfections, plasmid DNAs were prepared by banding twice through Labs, Beverly, MA), according to the manufacturer’s protocol. Membranes CsCl-ethidium bromide gradients. were washed at 55°C three times in 6ϫ SSC and 1% SDS for 20 min, followed by one wash in 1ϫ SSC and 1% SDS. Signal intensities were measured using a PhosphorImager and ImageQuant software (Molecular Cellular transfections Dynamics, Sunnyvale, CA). cDNA samples were diluted to ranges in ␮ which there were linear relationships between input cDNA and signals For all transfections of Jurkat-TAg cells, 400 l of the cells suspended at from the PCR products. For quantitative comparisons among samples, the ϫ 7 2.5 10 cells/ml in RPMI 1640 medium containing 10 mM HEPES was CCR9 signals were normalized to the signals for GAPDH. Using equal transferred to a cuvette with a 0.4-cm gap (Bio-Rad Laboratories, Hercules, amounts of input, CCR9A and CCR9B cDNAs gave equal amounts of the CA), and electroporation was performed in a Gene Pulser apparatus (Bio- ␮ 500 bp (CCR9A) and the 451 bp (CCR9B) products, demonstrating equal Rad) at room temperature using 250 V and 960 F, after which the cells efficiencies in amplifying the two products. were left at room temperature for 10 min, diluted to 5 ml with RPMI 1640/10% FBS, and cultured for ϳ36 h before harvesting for assays for calcium flux or chemotaxis. For studies on the flow cytometer, cells were Southern blot analysis of genomic DNA transfected with 40 ␮g of pCI-neo or pCI-neo/CCR9A or pCI-neo/CCR9B Twenty micrograms of genomic DNA was isolated from human peripheral each with 25 ␮g of pEGFP-F encoding a farnesylated green fluorescent blood leukocytes and digested separately with the enzymes HindIII, protein and containing an SV40 origin of replication (Clontech, Palo Alto, BamHI, and PstI. The digested fragments were separated by agarose gel CA). For preparing cells for chemotaxis assays and for using in the fluo- electrophoresis and transfered to a Zeta-Probe nylon membrane (Bio-Rad) rescence spectrometer, the pEGFP-F DNA was omitted and the amounts of that was hybridized overnight in 0.25 M sodium phosphate (pH 7.2), 7% the other DNAs were as stated in the text. For transfections to produce SDS at 65°C; washed three times with 20 mM sodium phosphate (pH 7.2), stable cell lines, pCEP4/CCR9A and pCEP4/CCR9B were used to transfect 5% SDS at 65°C for 60 min; and finally washed twice for 60 min in 20 mM HEK-293 cells, as described (9), and colonies were selected and grown sodium phosphate (pH 7.2), 1% SDS, per the supplier’s recommendations. using 200 ␮g/ml hygromycin B (Sigma). Preparation of probe and autoradiography was done as described above for Northern blotting. Northern blot analysis Measurements of calcium flux Human tissue blots of poly(A)ϩ RNA (Clontech) were hybridized and washed according to the supplier’s protocols. For the dot blot not shown, Assays on the flow cytometer for measurement of calcium fluxes in trans- the nonhybridizing samples included poly(A)ϩ RNA from whole brain, fected cells and cell lines were done as described (18). Briefly, cells were amygdala, caudate nucleus, cerebellum, cerebral cortex, frontal lobe, hip- washed with HBSS containing calcium and magnesium, 10 mM HEPES, 7 pocampus, medulla oblongata, occipital lobe, putamen, substantia nigra, and 1% FBS (HBSS/FBS) resuspended at 2 ϫ 10 cells/ml, loaded with the temporal lobe, thalamus, subthalamic nucleus, spinal cord, heart, aorta, fluorescent calcium probe indo-1 acetoxymethylester and the detergent skeletal muscle, colon, bladder, uterus, prostate, stomach, testis, ovary, Pleuronic (Molecular Probes, Eugene, OR) at concentrations of 10 ␮M and pancreas, pituitary, adrenal, thyroid, salivary gland, breast, kidney, liver, 300 ␮g/ml, respectively, for 45 min at 30°C with occasional shaking, fol- small bowel, spleen, peripheral leukocytes, lymph node, bone marrow, lowed by two washes in HBSS/FBS buffer. Measurements of calcium flux appendix, lung, trachea, placenta, fetal brain, fetal heart, fetal kidney, fetal were performed on a FACSVantage flow cytometer (Becton Dickinson liver, fetal spleen, fetal thymus, and fetal lung. For analysis of RNA in cell Immunocytometry Systems, San Jose, CA) equipped with an argon laser lines and leukocytes, total RNA was prepared using TRIzol (Life Tech- tuned to 488 nm and a krypton laser tuned to 360 nm with fluorescence 2ϩ nologies), separated by electrophoresis in a formaldehyde-agarose gel, and analyzed at 390/20 and 530/20 for Ca -bound and free probe, respec- transferred onto reinforced nitrocellulose membrane (Optibind; Schleicher tively. For each stimulation, the cells were kept at room temperature and & Schuell, Keene, NH). Prehybridization, hybridization, and washing were using a sample of 0.5 ml of cells, 50 ␮l of HBSS/FBS was added at 30 s done as described (24) with final washes in 0.1ϫ SSC, 0.1% SDS at 50°C. as a sham injection, followed by injection of 50 ␮l of HBSS/FBS contain- 32P-labeled probe for CCR9 was made from the CCR9A/B cDNA fragment ing various concentrations of chemokines. Data were analyzed using the corresponding to positions 584-1094 in the CCR9A cDNA, GenBank ac- FlowJo software (Treestar, San Carlos, CA). For some studies, including cession number AF145439, using the random primer-based Megaprime those shown in Fig. 5, CCL25 was a preparation of Ck␤-15 obtained from DNA labeling kit (Amersham Pharmacia Biotech, Piscataway, NJ), accord- Human Genome Sciences (Rockville, MD). This recombinant Ck␤-15 con- ing to the manufacturer’s protocol, and the probe was used as described tained the N-terminal sequence MRGSHHHHHHGSVES, with a histidine (24). Hybridization with an oligonucleotide probe to the 18S rRNA was tag, fused to the CCL25 sequence beginning, according to the published 21 151 done as described (25). Autoradiography was done using an intensifying sequence (12), at Val and extending to the C terminus at Leu (David screen. Hilbert, Human Genome Sciences, personal communication). CCL25 used in the other experiments for which data are shown, and all other chemo- kines used in calcium flux and/or chemotaxis assays were obtained from Determination of expression of mRNAs for CCR9A and CCR9B Peprotech (Rocky Hill, NJ), except for CCL21 (Ck␤-9/SLC) and CCL13 ␤ using PCR (Ck -10/MCP-4), which were also obtained from Human Genome Sci- ences. The CCL25 provided by Peprotech begins at Gln24 and extends to Using 1–5 ␮g of total RNA, prepared as above, cDNA was synthesized the C terminus at Leu151, according to the supplier’s specifications, and this with the SuperScript PreAmplification System (Life Technologies) and di- protein was more active in our assays than the preparation from Human luted in 10–50 ␮l of water. Sequences of interest were amplified by PCR, Genome Sciences. using the Takara Taq PCR kit (Takara Shuzo, Otsu, Japan) in reactions Assays of calcium flux on a ratio fluorescence spectrometer (model containing various amounts of cDNA and 1 ␮M of each primer in a volume RF-M2001; Photon Technology International, South Brunswick, NJ) were of 20 ␮l. Amplifications were done using 34 cycles of denaturation at 95°C done as described (27). Briefly, cells were suspended at 2 ϫ 106/ml and for 45 s, annealing at 65°C for 1 min, and elongation at 72°C for 1 min. The loaded with 2 ␮M fura-2 acetoxymethyl ester (Molecular Probes), as de- primers used for amplifying CCR9 sequences were 5Ј-GTCCCAGG scribed above for indo-1. Measurements were made at 37°C in cuvettes GAGAGTTGCATC (sense) and 5Ј-TGGCAATGTACCTGTCCACG (an- containing 2 ml of cells at 0.5 ϫ 106 cells/ml. Excitation was alternately at tisense), and the predicted products are 500 bp for CCR9A and 451 bp for 340 and 380 nm with emission measured at 510 nm. Using an integration CCR9B. The primers used for GAPDH were 5Ј-ACCACCATGGAGAAG time of 0.1 s, the ratios of the signals obtained at the two excitation wave- GCTGG (sense) and 5Ј-CTCAGTGTAGCCCAGGATGC (antisense), as lengths were plotted as a function of time. For quantifying responses, max- previously described (26). PCR products were resolved by electrophoresis imal increases in ratio fluorescence were measured. on a 2% agarose gel, and the DNA was transferred to Zeta-Probe nylon membrane (Bio-Rad) after base denaturation and neutralization, according Chemotaxis assays to the manufacturer’s protocol. The membrane was prehybridized in 6ϫ SSC, 10ϫ Denhardt’s solution, 20 ␮g/ml yeast tRNA, 50 ␮g/ml salmon Chemotaxis assays were done as described (28), with modifications, using sperm DNA, and 1% SDS at 50°C for 2 h. Hybridizations were done in 6ϫ 6.5-mm Transwell tissue culture inserts with a 5 ␮m pore size (Corning, 1296 RECEPTORS FOR THE CHEMOKINE CCL25

FIGURE 1. Alternative splicing produces CCR9 mRNAs that are predicted to encode receptors CCR9A and CCR9B that differ by 12 aa in their N-terminal regions. A, Structure of cDNA clone 6, predicted to encode CCR9A, and of cDNA clone 3, predicted to encode CCR9B. Thin lines represent nontranslated regions; dashed diagonal lines represent introns; thick lines represent coding regions. Base pair positions are designated according to the numbers from genomic sequence in GenBank entry with accession number AC005669. The drawing is not to scale. B, Alignment and comparison of CCR9A/B and closely related receptors. The numbers at the beginnings and ends of the lines indicate the positions of the residues at those locations. Solid backgrounds highlight matches between CCR9A/B and the other receptors. Dashes indicate gaps introduced for optimal alignments. Putative TMDs 1–7 are indicated by bars. Met13 in the CCR9A sequence, marked with the triangle, is Met1 in the CCR9B sequence. The predicted site for N-linked glycosylation is marked with an asterisk. The alignment was generated using the MacVector program from the Oxford Molecular Group.

Corning, NY). Transfected cells and cell lines were suspended at 1 ϫ 107 using pools of degenerate primers based on conserved amino acid cells/ml in RPMI 1640 with 0.5% BSA, and 100 ␮l of cell suspension was sequences in TMD VII and in, and immediately carboxyl-terminal added to an insert in a well with 600 ␮l of medium. After equilibration at ϳ 37°C for 2 h, chemokine was added to the wells and the plates were in- to, TMD III. Among the initial 100 PCR products sequenced, in cubated for an additional 90 min before cells were harvested, collected by addition to cDNA fragments for CXCR4, which has been shown to centrifugation, and counted. Duplicate wells were used for each condition. be expressed in SUP-T1 cells (9), we also found a cDNA fragment corresponding to the gene whose sequence had been deposited in Testing an N-terminal peptide from CCR9A as a CCL25 the database as GPR-9-6 (GenBank accession number U45982) antagonist and that encodes a chemokine receptor-like, orphan G protein- coupled receptor. As noted above and based on the data presented A peptide consisting of the N-terminal 12 aa of CCR9A was synthesized (Commonwealth Biotechnologies, Richmond, VA) and dissolved in 1% below, we will refer to this gene as CCR9 in the remainder of the acetic acid. A total of 3.5 ␮M CCL25 with and without 3.5 mM peptide manuscript. was incubated in 0.5% acetic acid at room temperature for 1 h and tested After Northern analysis verified the expression of the CCR9 on fura-2-AM-loaded, CCR9A-transfected HEK-293 cells at a concentra- mRNA in SUP-T1 cells (see below), we screened a cDNA library tion of 70 nM CCL25 using a calcium flux assay, as described above. that we had prepared from these cells using a CCR9 probe to obtain cDNA clones for functional studies. Nine cDNAs were iso- Results lated and four were analyzed in detail, revealing two types of Isolation and characterization of CCR9 cDNAs CCR9 cDNAs that differed in their predicted coding sequences. To identify novel HIV-1 coreceptors and/or chemokine receptors Two representative cDNAs, clone 6 and clone 3, were sequenced expressed in the immortalized, CD4ϩ T cell line, SUP-T1, we in their entireties and contained 2544 and 2462 bp, respectively, as performed RT-PCR with poly(A)ϩ RNA prepared from these cells depicted in Fig. 1A. The sequences have been deposited in Gen- The Journal of Immunology 1297

Bank with accession number AF145439 for clone 6 and AF145440 for clone 3. These cDNAs are likely to be close to full length, since their lengths corresponded to the approximate size of the major species of CCR9 mRNA, as revealed by Northern analysis (see below). Clone 6, as well as two additional cDNAs, encoded a predicted seven-transmembrane-domain protein of 369 aa, while clone 3 en- coded a protein of 357 aa that was identical to the 369-aa protein, except that the clone 3 open reading frame began at Met13 and therefore lacked the first 12-aa predicted by the other three cDNAs. For both clone 6 and clone 3, the predicted initiator codons are the first ATGs in the sequences and are in the reading frame predicted by comparison with other G protein-coupled receptors. Each is in a favorable context for initiation of translation according to the empirical rules of Kozak (29). cDNA clone 6, but not clone 3, contains an in-frame stop codon 5Ј to the predicted initiating ATG. The differences between the open reading frames in clone 6 and clone 3 were due to a 49-bp insertion in clone 6 with respect to clone 3. Comparisons of clone 6 and clone 3 sequences with se- quences in the database from an extended region of human chro- mosome 3 (GenBank accession number AC005669) revealed that FIGURE 2. There is a single human CCR9 gene. Twenty micrograms of the 49-bp insertion in clone 6 was due to alternative mRNA splic- genomic DNA isolated from peripheral blood leukocytes was digested with ing, as diagrammed in Fig. 1A, with the predicted initiator codon restriction enzymes, as indicated, separated by agarose gel electrophoresis, blotted onto nylon membrane, and hybridized with a 32P-radiolabeled in clone 6 within an exon ϳ5.8 kb upstream from the major coding probe prepared from a fragment of the CCR9A/B cDNA (genomic se- exon. In the genomic sequence, the 49-bp fragment found in clone quence 5116–5626) corresponding to the region between TMDs 3 and 7. 6 is bounded by the canonical intronic splice acceptor and splice DNA size markers are indicated. donor dinucleotides. In addition to the 369-codon open reading frame, clone 6 contained a 5Ј nontranslated region of 157 bp and a3Ј nontranslated region of 1280 bp, absent the poly(A) tail. In addition to the 357-codon open reading frame, clone 3 contained a ilarly, Fig 3, A shows high level expression of the CCR9 gene 5Ј nontranslated region of 112 bp and a 3Ј nontranslated region, limited to the thymus, with relatively low level expression also in absent the poly(A) tail, of 1279 bp. spleen. The major species of CCR9 mRNA is ϳ2.6 kb. On a longer We refer to the protein encoded by clone 6 as CCR9A and that exposure of the blot in Fig. 3A and on other blots not shown, faint encoded by clone 3 as CCR9B, and refer to them collectively, signals could be detected in PBL or PBMC, which were not in- when appropriate, as CCR9A/B. Comparisons between CCR9A/B creased after activation with PHA and IL-2. Fig. 3B demonstrates, and related chemokine receptors and orphan receptors are shown as noted above, that CCR9 is expressed in the CD4ϩ T cell lines in Fig. 1B. Like other chemokine receptors, CCR9A/B has an N- SUP-T1 and MOLT-4 clone 8 without detectable expression in terminal region with a high number of acidic residues, a short and several other T cell or monocytic lines or in monocyte-derived basic third intracellular loop, and cysteine residues in the N-ter- macrophages. CCR9 expression was also not detected in the NK minal region (Cys38 in CCR9A) and the third extracellular loop cell lines NK3.3 and YT, or in a line of EBV-transformed B cells, (Cys289 in CCR9A) (30). Like many other G protein-coupled re- or in cord blood T cells that had been differentiated into Th1 or ceptors, CCR9A/B has a site for N-linked glycosylation in the Th2 cell lines (not shown). Of note, thymus contains minor CCR9 N-terminal region (Asn32 in CCR9A), conserved cysteines in ex- mRNA species of ϳ6 and 8.5 kb, not well seen in Fig. 3B, that are tracellular loops 1 (Cys119 in CCR9A) and 2 (Cys198 in CCR9A), not found in MOLT-4 clone 8 cells. and the DRY sequence following TMD III, which mutagenesis The high expression of CCR9 in thymus led us to investigate studies have found to be important for G protein coupling (re- levels of CCR9 mRNA in thymocyte subsets using RT-PCR. As viewed in Ref. 31). CCR9A/B also contains a cysteine residue in shown in Fig. 4A, CD4ϩCD8ϩ thymocytes expressed significantly the carboxyl-terminal region (Cys337 in CCR9A) that, based on the higher levels of CCR9 as compared with other thymocyte sub- ␤ data for rhodopsin, the 2-adrenergic receptor, and related recep- populations. As detailed in Materials and Methods, while the prep- tors, is likely to be palmitoylated (reviewed in Ref. 32). aration of CD4ϩCD8ϩ cells was extremely pure, the preparations of single-positive cells contained up to 20% CD4ϩCD8ϩ cells as well, so that the levels shown for single-positive cells are most Expression of the CCR9 gene likely overestimates. To analyze the relative expression of mRNA Fig. 2 shows that there is a single human gene for CCR9A/B. As encoding CCR9A vs CCR9B in thymocytes and other cells, we noted above, the CCR9 gene was found within 176,968 bp se- designed PCR primers that would yield products of 500 and 451 bp quenced from chromosome 3 and entered in GenBank under ac- from CCR9A and CCR9B cDNAs, respectively. As shown in Fig. cession number AC005669. Our cDNAs were included within po- 4B, CCR9A mRNA was expressed uniformly at ϳ10-fold higher sitions (3Ј) 3,666-(5Ј) 20,337 of that GenBank entry. None of the levels as compared with CCR9B cDNA in thymocyte subsets as other chemokine receptor or chemokine receptor-related orphan well as in PBMC and T cell lines. receptor genes that are located on chromosome 3 was found within this sequence. Hybridization of CCR9 probe to a dot blot containing mRNAs Signaling by receptors CCR9A/B from 50 human tissues produced a signal only with the sample cDNA fragments encoding CCR9A and CCR9B were inserted from thymus (see Materials and Methods; data not shown). Sim- downstream of the CMV immediate-early promoter in the vector 1298 RECEPTORS FOR THE CHEMOKINE CCL25

no signals. As expected from the known expression of CXCR4 in Jurkat cells (9, 34), CXCL12 (SDF-1) produced signals on the pCI-neo-transfected control cells and equally on both the GFP- negative/low and the GFP-high cells. Similarly, responses to li- gands for CCR4, CCR7, and CXCR3 were seen that were not specific for, or enhanced, on the CCR9A or the CCR9B-trans- fected cells (not shown). In addition to experiments on the flow cytometer with cells co- transfected with CCR9A/B and GFP plasmids, Jurkat-TAg cells were also transfected with CCR9A and CCR9B plasmids alone and analyzed for calcium fluxes on a ratio fluorescence spectrom- eter after the cells had been loaded with fura-2 acetoxymethyl ester. Transfected cells analyzed on the fluorometer responded to CCL25, and as with the experiments on the flow cytometer, there were no specific responses on the CCR9A- or CCR9B-transfected cells to chemokines other than CCL25. CCR9A- and CCR9B-me- diated responses to chemokines were also analyzed using CCR9A- and CCR9B-transfected HEK-293 cell lines. Besides CXCL12 (SDF-1), which signals on the parent HEK-293 cells, only CCL25, and not CCR4, CCR7, or CXCR3 ligands, which gave responses on the control-transfected Jurkat-TAg cells, produced signals on the CCR9A- or CCR9B-expressing lines (not shown). The results from testing the transfected Jurkat-TAg cells, which were done using the largest number of chemokines, are shown in Table I. The fluorometer was used for dose-response analysis to deter-

mine the EC50 of CCL25 on CCR9A- and CCR9B-expressing cells. In comparing the EC50 on CCR9A- vs CCR9B-transfected cells, we considered the possibility that receptor expression within the population of cells might reach levels at which further in- creases in receptor expression would not result in further signal

enhancement. Under such conditions, differences in the EC50 be- tween the CCR9A- and CCR9B-expressing cells might be influ-

FIGURE 3. The CCR9 gene is expressed predominately in the thymus enced by levels of receptor expression, with the EC50 falling at and in cell lines derived from T cell malignancies. A, A commercially highest levels of expression. To overcome this possibly confound- supplied Northern blot containing ϳ2 ␮g of poly(A)ϩ RNA per lane was ing effect, the Jurkat-TAg cells were transfected with a range of hybridized as in A. RNA size markers are indicated. B, Twenty micrograms DNA amounts and the cells were then analyzed for calcium fluxes of total RNA per lane were electrophoresed on a 1.2% agarose-formalde- in response to varying concentrations of CCL25. As shown in Fig. hyde gel, transferred to reinforced nitrocellulose, hybridized to the TMD 6, the levels of the maximal calcium signals were dependent on the 3–7 CCR9A/B cDNA 32P-radiolabeled probe, and then stripped and hy- amounts of DNA used for the transfections. Also as shown, inde- bridized to an oligonucleotide probe for the 18S rRNA. RNA size markers are indicated. Samples are from the immortalized cell lines, as indicated, pendent of the amount of DNA used and the level of maximal except for MDM, which were macrophages derived from peripheral blood signals, the EC50 for CCL25 was consistently lower on the monocytes by culturing for 1 wk, and the PBMC, which were activated CCR9A-expressing as compared with the CCR9B-expressing with 1 ␮g/ml PHA and 20 IU/ml IL-2 for 3 days. cells, even though at the highest concentrations of CCL25 we could demonstrate equal, maximal responses for the two receptors, all suggesting that the receptors showed differences in signaling pCI-neo, which contains an SV40 origin of replication. pCI-neo, that were not due to differences in receptor expression. Our con- pCI-neo encoding CCR9A, and pCI-neo encoding CCR9B were cern that the EC50 might fall at higher levels of receptor expression each cotransfected with the plasmid pEGFP-F, which contains an was unfounded, since in fact, the EC50 with both CCR9A and SV40 origin of replication and sequences encoding a farnesylated CCR9B tended to rise slightly with transfections done using and codon-optimized green fluorescent protein (33), into Jurkat- greater amounts of DNA. Fig. 6E demonstrates that, as expected TAg cells, which express the SV40 large T Ag (21). pEGFP-F from the mRNA expression data, the MOLT-4 clone 8 cells re-

DNA was included to identify cells expressing protein from the sponded to CCL25. The EC50 for CCL25 on MOLT-4 clone 8 cells transfected DNAs on the flow cytometer. Approximately 36 h after was ϳ25 ng/ml, close to that found for the CCR9A-transfected transfection, the cells were loaded with the ratiometric fluorescent Jurkat-TAg cells, consistent with the predominant expression of calcium probe indo-1 acetoxymethyl ester and tested for responses CCR9A on these cells (Fig. 4B). to a collection of chemokines using a flow cytometer-based cal- The lower EC50 of CCL25 on the CCR9A-expressing cells sug- cium flux assay (18). As shown in Fig. 5, the chemokine CCL25 gested that CCR9A would be desensitized at lower concentrations produced a calcium flux on the CCR9A and, to a lesser extent, on of ligand as compared with CCR9B. Using the receptor-transfected the CCR9B-transfected cells, but not on the cells transfected with HEK-293 cells, as shown in Fig. 7, we demonstrated that this was the unmodified pCI-neo vector. Calcium responses to CCL25 were the case. For example, following stimulation of cells expressing seen only in the GFP-bright cells, consistent with detectable re- CCR9A with 100 ng/ml CCL25, the subsequent addition of 1000 sponses being confined to those cells likely to be expressing the ng/ml did not produce a detectable signal. In contrast, CCR9B- highest levels of CCR9A and CCR9B from the cotransfected expressing cells responded comparably to CCL25 after both the DNAs. CXCL8 (IL-8) was used as a negative control and produced initial stimulation with 100 ng/ml and the subsequent stimulation The Journal of Immunology 1299

FIGURE 4. Expression of CCR9A and CCR9B mRNA in cell lines, thymocytes, and PBMC. A, Among thymocyte subsets, CD4ϩCD8ϩ cells express highest levels of CCR9 mRNA. Levels of expression of CCR9 and GAPDH mRNA were determined using RT-PCR, as described in Materials and Methods, and the relative intensities of signals for CCR9A are shown after normalization based on the signals for GAPDH. ⌬ indicates that the single- positive populations also contained other subsets, as described in Materials and Methods. The asterisk indicates that the level of CCR9A in the CD4ϩCD8ϩ preparation was greater than that in other thymocyte subsets (p Ͻ 0.01, t test). Thymocytes and PBMC were each from a single donor. B, The ratio of CCR9A and CCR9B mRNAs is uniform among cell types expressing CCR9. Using the same cDNA as for A, CCR9A and CCR9B sequences were amplified and detected, as described in Materials and Methods. An autoradiogram is displayed above, and quantification based on PhosphorImager data is shown below. For both A and B, results are averaged from three PCR experiments.

with 1000 ng/ml. Of note, just as for the transiently transfected Discussion Jurkat cells, the CCR9A-expressing HEK-293 cells responded to lower concentrations of CCL25 than did the cells expressing We have isolated CCR9 cDNAs that differ due to alternative splic- CCR9B. ing and that encode two proteins, CCR9A and CCR9B, which Because the above results suggested that the N terminus of function as receptors for CCL25 in assays for both calcium flux CCR9A might be important in interacting with CCL25, we tested and chemotaxis. CCL25 has been shown to be expressed in the a peptide containing the 12 N-terminal residues of CCR9A as a CCL25 antagonist (see Materials and Methods). The peptide did thymus by dendritic cells, perhaps by the lymphoid-derived den- dritic cells, as well as in the small intestine (12), and has recently not inhibit the signaling of CCL25 on CCR9A-expressing HEK- ϩ 293 cells (not shown). been shown to be expressed in MHC II epithelium from mouse fetal thymus (14). CCL25 was reported to be chemotactic for thy- mocytes, splenic dendritic cells, activated peritoneal macrophages Migration of CCR9A- and CCR9B-expressing cells to CCL25 (12), and precursors from fetal day 14 thymus (14) from mice, and We analyzed the migration of Jurkat-TAg cells transfected with for IFN-␥-activated THP-1 cells (12). Consistent with the expres- pCI-neo and pCI-neo encoding CCR9A and CCR9B, as well as sion pattern for CCL25 and its activity on thymocytes, we have MOLT-4 clone 8 and SUP-T1 cells in response to varying con- found the CCR9 gene to be highly expressed in thymus, with de- centrations of CCL25, using porous Transwell tissue culture in- tectable but much lower expression in spleen and PBL. We did not serts to separate the cells in the upper chambers from the chemo- detect expression of CCR9 mRNA in human intestine or appendix kine-containing medium in the lower chambers. As shown in Fig. by dot blot (Fig. 3A), or in IFN-␥-activated monocyte-derived 8, the CCR9A- and CCR9B-transfected cells as well as MOLT-4 macrophages, or in monocyte-derived dendritic cells by RNase clone 8 and SUP-T1 cells, but not the control-transfected Jurkat- protection assay (not shown). Because of possible differences be- TAg cells, migrated in response to CCL25 in a dose-dependent tween mice and humans and between the origins and culture con- fashion, with decreased migration at the highest concentration of ditions for primary cells, and because of the limits of detection in chemokine, producing the expected bell-shaped curve. Consistent our probing for mRNA, our data do not definitively demonstrate with our calcium flux data shown in Figs. 5–7, CCR9A-expressing any discrepancies between published reports of CCL25 respon- cells were more responsive to CCL25 than cells expressing CCR9B, responding at lower concentrations of CCL25 and show- siveness and CCR9A/B expression that would suggest another ing a more dramatic falloff in migration at a CCL25 concentration CCL25 receptor(s); and despite testing an extensive collection of of 2000 ng/ml. The CCR9A-transfected cells showed dose re- chemokines, we have no indication that there are additional ligands sponses for chemotaxis to CCL25 that were close to those seen for for CCR9A and/or CCR9B. the MOLT-4 clone 8 and SUP-T1 cell lines. Chemotaxis experi- The high and relatively selective expression of the CCR9 gene ments using cells transfected with varying amounts of DNA and CCL25 in the thymus raises the obvious question as to whether showed that approximately twice as much CCR9B as compared this receptor/ligand pair is involved in stem cell recruitment to, or with CCR9A DNA was required to produce cells migrating in thymocyte migration within or out of, the thymus. There is evi- equivalent numbers (not shown). dence that stem cell recruitment to the thymus and migration of 1300 RECEPTORS FOR THE CHEMOKINE CCL25

FIGURE 5. Jurkat-TAg cells transfected with cD- NAs encoding CCR9A and CCR9B responded to CCL25. A, Forty micrograms of pCI-neo containing se- quences of CCR9A cDNA together with 25 ␮gof pEGFP-F DNA were used per transfection of 107 Jur- kat-TAg cells. After ϳ36 h, the cells were loaded with indo-1 acetoxymethyl ester and tested for calcium flux in response to added chemokines. The scattergrams across the top row show responses to CCL25; those across the middle row show responses to CXCL8 (IL-8); and those across the bottom row show responses to CXCL12 (SDF-1). In these scattergrams and those that follow, the y-axis shows channel numbers that reflect the fluorescence ratio of the indo-1 calcium probe. For each sample, a sham injection of buffer was done at 30 s and the chemokines as indicated were injected at 60 s to a final concentration of 2 ␮g/ml. For analysis, cells from individual samples were separated into GFPϪ/low and GFPhigh, as shown. B, Forty micrograms of pCI-neo containing sequences of CCR9B cDNA together with 25 ␮g of pEGFP-F DNA were used per transfection of 107 Jurkat-TAg cells, and the cells were treated and ana- lyzed as in A. C, Forty micrograms of pCI-neo without cDNA sequences together with 25 ␮g of pEGFP-F DNA were used per transfection of 107 Jurkat-TAg cells, and the cells were treated and analyzed as in A. Responses of the CCR9A- and CCR9B-transfected cells to CCL25 were also seen in three other experiments. The Journal of Immunology 1301

Table I. Responses of transfected Jurkat-TAg cells to chemokines CCR7 and responsiveness to CCR7 ligands are acquired as thy- mocytes become fully mature (36, 43), suggesting that these re- Transfected DNAb ceptors might be important in migration of thymocytes across the corticomedullary junction. Chemokinesa pCI-neo pCI-neo/CCR9A pCI-neo/CCR9B Responsiveness to CCL25 has been found to extend from dou- CCL1 (I-309) ϪϪ Ϫ ble-positive cells through the single-positive, CD69ϩ subset, dis- CCL2 (MCP-1) ϪϪ Ϫ appearing on the mature single-positive cells (43). In a separate ␣ ϪϪ Ϫ CCL3 (MIP-1 ) analysis of chemokine mRNA expression in MHC IIϩ epithelium CCL4 (MIP-1␤) ϪϪ Ϫ CCL5 (RANTES) ϪϪ Ϫ from mouse fetal thymus as compared with nonthymic tissue, CCL7 (MCP-3) ϪϪ Ϫ CCL25 was unusual in being preferentially expressed in thymic CCL8 (MCP-2) ϪϪ Ϫ epithelium, and CCL25 was a chemoattractant for fetal day 14 ϪϪ Ϫ CCL11 (Eotaxin) thymic precursors (14). Nonetheless, in these same studies, Abs to CCL13 (MCP-4) ϪϪ Ϫ CCL14 (HCC-1) ϪϪ Ϫ CCL25 failed to block trans-filter migration of precursor cells to CCL15 (Leukotactin-1) ϪϪ Ϫ repopulate precursor-depleted mouse embryonic thymic lobes CCL16 (LEC-4) ϪϪ Ϫ (14). These results do not, however, rule out a role for CCR9A/B CCL17 (TARC) ϩϩ ϩ in stem cell recruitment, since there may be an as yet unidentified ␤ ϩϩ ϩ CCL19 (MIP-3 ) CCR9A/B ligand(s). CCL20 (MIP-3␣) ϪϪ Ϫ CCL21 (SLC) ϩϩ ϩ Our data on CCR9A/B expression on thymocyte subsets are CCL22 (MDC) ϩϩ ϩ consistent with the published descriptions of subset responses to CCL23 (MPIF-1) ϪϪ Ϫ CCL25. We found expression on double-negative/CD4dull cells, ϪϪ Ϫ CCL24 (MPIF-2) which represent the precursors of the double-positive thymocytes CCL25 (TECK, Ck␤-15) Ϫϩ ϩ CXCL2 (Gro␤) ϪϪ Ϫ (23), consistent with the responses to TECK seen for immature CXCL4 (PF4) ϪϪ Ϫ mouse thymocytes (14). However, we found the highest expres- CXCL5 (ENA-78) ϪϪ Ϫ sion of CCR9A/B on CD4ϩCD8ϩ thymocytes (Fig. 4A). This sug- CXCL6 (GCP-2) ϪϪ Ϫ gests that CCR9 and its ligand are involved in events subsequent ϪϪ Ϫ CXCL8 (IL-8) to recruitment of cells to the thymus and is consistent with the CXCL9 (MIG) ϩϩ ϩ CXCL10 (IP-10) ϩϩ ϩ observations that CCL25 is expressed not only by fetal thymic CXCL11 (I-TAC) ϩϩ ϩ epithelial cells, but also by medullary dendritic cells. Together, the CXCL12 (SDF-1) ϩϩ ϩ data on receptor and ligand expression raise the possibility that ϪϪ Ϫ CX3CL1 (Fractalkine) CCR9 promotes the movement of double-positive thymocytes to a Chemokines are designated according to the nomenclature proposed at the Key- contact epithelial and dendritic cells during migration from cortex stone Symposium on Chemokines and Chemokine Receptors, January 18–23, 1999, to medulla, and is therefore important for that migration and/or for and common names are in parentheses. MCP, monocyte chemoattractant protein; MIP, -inflammatory protein; TARC, thymus and activation-regulated the processes of positive and negative selection, which depend on chemokine; PF4, ; IP-10, -␥-inducible protein-10; LEC, liv- these interactions. Because single-positive thymocytes show er-expressed chemokine; SLC, secondary lymphoid-tissue chemokine; MDC, macro- much-diminished expression of CCR9 and PBL express little phage-derived chemokine; MPIF, myeloid progenitor inhibitory factor; ENA, epithe- lial cell-derived neutrophil activator; GCP, granulocyte chemotactic protein; MIG, CCR9 on average, it is also possible that CCL25/CCR9 are in- induced by IFN-␥; I-TAC, IFN-inducible T cell ␣ chemoattractant. volved in retaining cells in the thymus until maturation is com- b Responses of transfected cells to chemokines are designated with Ϫ for negative, ϩ for positive. plete. Finally, it is worth considering that CCL25/CCR9 may be involved in providing signals for thymocyte maturation in ways not limited to effects on cell movement. thymocytes within and possibly out of the thymus require G pro- We have shown that both SUP-T1 and MOLT-4 clone 8 cells tein activation. Pertussis toxin, which inactivates chemokine re- express the CCR9 gene and respond to CCL25. The SUP-T1 line ceptor-coupled G proteins of the Gi family, can block T cell pre- was derived from a child with T cell non-Hodgkin’s lymphoma cursor migration across a filter to repopulate precursor-depleted (44), and the MOLT-4 line was derived from a young adult with mouse embryonic thymic lobes (14); and transgenic mice express- acute lymphoblastic leukemia (45). The very high level of expres- ing the catalytic subunit of pertussis toxin under control of the lck sion of CCR9 mRNA and CCR9A/B function on MOLT-4 clone 8 promoter show accumulation of mature T cells in the thymus, with cells raises the possibility that the CCR9 gene has been amplified T cell depletion of secondary lymphoid organs (35). Recent work in these cells and that the expression of the receptor, through au- has shown that pertussis toxin leads to the accumulation of single- tocrine activities of CCL25 or otherwise, might confer some positive cells in the thymic cortex (36), and the failure of these growth advantage. cells to move to the medulla could underlie the inability of the MOLT-4 clone 8 and SUP-T1 cells have been used in HIV thymocytes expressing the pertussis toxin transgene to emigrate. research, since they readily form syncytia with many isolates. It is

These results implicating Gi proteins in thymocyte migration sug- possible that the abilities of some HIV-1 (and/or SIV) variants to gest a role for chemokine receptors and their ligands. infect SUP-T1 and MOLT-4 clone 8 cells are due to the cells’ A large number of chemokines, including members of the CC, expression of CCR9A/B, and analysis of coreceptor function for CXC, and C subfamilies, along with their receptors, have been these receptors is ongoing. reported to be expressed in the thymus. Among the chemokine Of particular interest is our cloning of cDNAs that reflect alter- receptors expressed prominently in the thymus are CXCR4 (37– native splicing of CCR9 mRNA, giving rise to two forms of the 39), CCR4 (40), CCR8 (41, 42), and CCR7 (36, 43). Recent stud- receptor that differ by 12 aa in the N-terminal region. Although the ies have begun to investigate systematically chemokine respon- coding regions of chemoattractant receptor genes are often found siveness and chemokine receptor expression among thymocyte on single exons, there are now many examples of exceptions to this subsets (36, 43). In mice, CCR4 and responsiveness to CCR4 li- rule, including, for example, the genes for CCR2 (46), CCR6 (7), gands have been found predominately on cells following positive CCR7 (47), CXCR4 (37, 48), and CXCR5 (49). Alternative splic- selection, which have up-regulated CD69, while expression of ing is well described as a mechanism of generating diversity 1302 RECEPTORS FOR THE CHEMOKINE CCL25

FIGURE 6. CCR9A is more efficient than CCR9B as a CCL25 receptor in a calcium flux as- say. A, From 10–80 ␮g of pCI-neo containing cDNA sequences for CCR9A (open symbol) or CCR9B (closed symbol) together with pCI-neo DNA to bring the total in each case to 80 ␮g, were used per transfection of 107 Jurkat-TAg cells. After ϳ36 h, the cells were loaded with fura-2 acetoxym- ethyl ester, and 106 cells per sample were tested for calcium flux on a ratio fluorescence spectrometer in response to varying doses of CCL25. Maximal changes in ratio fluorescence induced by each dose of CCL25 were measured and plotted on the y-axes, as shown, and approximate EC50 values were de- termined as noted. B, MOLT-4 clone 8 cells were loaded and analyzed for calcium flux in response to varying doses of CCL25, as in A. Another experi- ment using cells transfected with 40 ␮gofthe CCR9A- and CCR9B-encoding DNAs gave similar results. Unlike the experiments shown in Fig. 5, the studies shown in this figure and those that follow used a preparation of CCL25 obtained from Pepro- tech (see Materials and Methods).

among G protein-coupled receptors, resulting in changes in the C contain nine other residues in their places, and was reported to ϳ termini and intracellular loops as well as truncated receptors, have an EC50 3-fold that of the standard receptor. The sometimes resulting in changes in receptor activities (reviewed in CXCR4-Lo mRNA does not represent an alternatively spliced Ref. 50). Among chemokine receptors, alternative splicing pro- form, but an mRNA with a retained intron, producing a species duces two forms of human CCR2, CCR2A and CCR2B, which with more than 2 kb of additional 5Ј nontranslated sequence as differ in their C-terminal regions (51), and two forms of mouse compared with the mRNA for CXCR4. The presence of both mul- CXCR4, which differ by 2 aa within the N-terminal region (52, tiple upstream AUGs in the CXCR4-Lo RNA as well as a partic- 53). However, with the exception of a recently described serotonin ularly unfavorable context for the postulated start codon for receptor in Caenorhabditis elegans (54), we are not aware of other CXCR4-Lo according to the rules described by Kozak (29) suggest examples of alternative splicing producing variants of a G protein- that CXCR4-Lo will not be translated efficiently from the 4-kb coupled receptor with the magnitude of the N-terminal difference RNA, and raise questions about its biological significance. Kozak found for CCR9A and CCR9B. has noted that such incompletely processed mRNAs are particu- While this manuscript was under revision, a report appeared larly common in lymphocytes and that mRNA processing might describing a human CXCR4 RNA of ϳ4 kb that was predicted to therefore be a mechanism used in lymphocytes to regulate trans- encode a novel receptor that differs at its N terminus as compared lation (56). with that encoded by the major 1.7-kb mRNA species (55). This With regard to activities for chemokine receptor variants arising novel form of CXCR4, termed CXCR4-Lo, is predicted to lack the from alternative splicing, functional studies have not revealed dif- five N-terminal residues found in the standard sequence and to ferences in the responses to ligands between the two forms of The Journal of Immunology 1303

the active conformation is the target for the kinases that inactivate G protein-coupled receptors (57), lower affinity of CCR9B for CCL25 would be a straightforward explanation for the relative insensitivity of CCR9B to homologous desensitization (Fig. 7). We have analyzed the relative levels of mRNAs for CCR9A and CCR9B on thymocyte subsets, cell lines, and PBMC. As shown in Fig. 4B, both forms were expressed in the primary cells as well as in the cell lines, and the ratios of these alternatively spliced forms were ϳ10:1 in all samples tested. The expression of mRNAs for both CCR9A and CCR9B in cell lines and the invariant ratio of the two forms in preparations of primary cells suggest that both forms are coexpressed on individual cells. These data raise the question as to the advantage of a cell expressing both the A and the B forms of the receptor. Although we have no evidence for a ligand for FIGURE 7. CCR9B requires higher concentrations of ligand for desen- CCR9A/B other than CCL25, it is possible that there are other sitization than does CCR9A. HEK-293 cell lines expressing CCR9A or CCR9B were used in assays for calcium flux in response to CCL25 as in ligands for these receptors, or perhaps alternative forms of CCL25, Fig. 6, and tracings from the fluorescence spectrometer are shown. For each that show greater differences in their activities on CCR9A and assay, a control addition of 20 ␮l of buffer was done at 30 s (not indicated) CCR9B than we have documented using our preparation of and no signals were seen. Sequential additions of CCL25 were made as rCCL25. It is also possible that there are qualitative differences in indicated by the arrows. The data are from one of two experiments that signaling pathways activated by CCL25 on CCR9A vs CCR9B. Of gave similar results. course, it may be that the two receptors are only distinguished functionally by the quantitative differences that we have documented. Although the advantage of a sensitive receptor would seem ob- CCR2 (46, 51) or the variants of mouse CXCR4 (48, 52). It is vious in allowing a cell to respond to ligand gradients at a distance therefore of particular note that we have demonstrated functional from the ligand source, the role of a coexpressed insensitive re- differences between CCR9A and CCR9B. Our experiments using ceptor is less clear. In this regard, our data on receptor desensiti- calcium flux assays with both transiently transfected Jurkat cells zation may be particularly informative, since it demonstrates that and stably transfected HEK-293 cell lines showed that the EC50 of CCR9B could enable a cell to respond to a change in ligand con- CCL25 for CCR9A is lower than for CCR9B and similarly that centration at concentrations in which CCR9A is inactive. To- CCR9A is desensitized at lower concentrations of CCL25 as com- gether, the two receptors would extend the conditions under which pared with CCR9B, indicating that CCR9A is the more efficient a cell could respond to CCL25 beyond what could be achieved receptor. Consistent with our findings in the calcium flux assays, with a single receptor. This might be advantageous for trafficking CCR9A also appeared superior to CCR9B in mediating chemo- as a cell moves up a gradient of CCL25, or it might enable a cell taxis, although the analysis here was less extensive. It is likely that that has come to rest at a site with a high CCL25 concentration to the longer N-terminal region on CCR9A as compared with CCR9B react in other ways to a local release of CCL25. Our data suggest produces a receptor with enhanced affinity for CCL25, although a novel mechanism for expanding the range of concentrations over we have not yet measured the affinity directly. Because receptor in which a cell can respond to increments in the concentration of chemokine. While this manuscript was under review, Zaballos et al. (58) reported that GPR-9-6 is a receptor for TECK/CCL25 and desig- nated the receptor CCR9. The sequence of the cDNA that they reported and used in their studies corresponds to our CCR9A. The major additional data provided by our work are the identification of CCR9B and the characterization of expression and activity of CCR9B as compared with CCR9A, demonstrating the existence of CCL25 receptors that differ in their sensitivities to ligand and showing expression of both forms in human thymocytes, with highest levels in CD4ϩCD8ϩ cells. It is of interest that the mouse sequence reported by Zaballos et al. (58), which shows 86% iden- tity with the human receptor, contains the CCR9A Met13, suggest- FIGURE 8. Jurkat-TAg cells transfected with cDNAs encoding CCR9A ing that CCR9B may exist in mice as well as in humans. and CCR9B and MOLT-4 clone 8 and SUP-T1 cells migrated in response Data accumulating from many studies suggest that chemokine to CCL25. Forty micrograms of pCI-neo DNA or pCI-neo containing receptors expressed on lymphocytes function preferentially at par- cDNA sequences for CCR9A or CCR9B were used per transfection of 107 ticular stages in the life of a T cell (3, 18). The information that has Jurkat-TAg cells. Approximately 36 h after transfection, 106 transfected accumulated to date has been primarily related to mature T cells, cells as well as the same number of MOLT-4 clone 8 and SUP-T1 cells both for homeostatic trafficking of naive cells and for the traffick- were added to porous Transwell tissue culture inserts and placed in wells ing of memory/effector cells, particularly those activated and dif- containing various concentrations of CCL25. Following a 90-min incuba- ferentiated down Th1 and Th2 pathways. Although circumstantial tion, cells migrating through the membranes into the lower wells were harvested and counted. Results are expressed as cells migrating per 106 evidence also suggests that the chemokine system has roles in the input cells. Samples were done in duplicate and error bars represent SEMs. earlier stages of T cell development, there is as yet no proof of this Chemotaxis to CCL25 was seen with CCR9A- and CCR9B-transfected supposition. The description and characterization of CCR9A and cells in three separate experiments, and in each case, as here, the CCR9A- CCR9B as receptors for CCL25 will provide tools to address the transfected cells were the more active of the two. question directly, since the high and preferential expression of 1304 RECEPTORS FOR THE CHEMOKINE CCL25

CCL25 and CCR9A/B in the thymus suggest that these particular 20. Sallusto, F., A. Lanzavecchia, and C. R. Mackay. 1998. Chemokines and che- components of the chemokine system are likely to play important mokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol. Today 19:568. roles in thymocyte migration and/or in T cell development. If this 21. Spencer, D. M., T. J. Wandless, S. L. Schreiber, and G. R. Crabtree. 1993. speculation is borne out, it will expand our understanding of the Controlling signal transduction with synthetic ligands. Science 262:1019. 22. Mann, D. L., S. J. O’Brien, D. A. Gilbert, Y. Reid, M. Popovic, E. Readconnole, breadth of the involvement of the chemokine system in T cell R. C. Gallo, and A. F. Gazdar. 1989. Origin of the HIV-susceptible human CD4ϩ biology and contribute to the emerging appreciation that, rather cell-line H9. AIDS Res. Hum. Retroviruses 5:253. ϩ ϩ than being a collection of molecules with redundant activities, in- 23. Galy, A., S. Verma, A. Barcena, and H. Spits. 1993. Precursors of CD3 CD4 CD8ϩ cells in the human thymus are defined by expression of CD34: delineation dividual chemokines and their receptors play dedicated roles in of early events in human thymic development. J. Exp. Med. 178:391. establishment and maintenance of immune functions. 24. Vanguri, P., and J. Farber. 1990. Identification of CRG-2: an interferon-inducible mRNA predicted to encode a murine monokine. J. Biol. Chem. 265:15049. 25. Amichay, D., R. T. Gazzinelli, G. Karupiah, T. R. Moench, A. Sher, and J. M. Farber. 1996. The gene for chemokines MuMig amd Crg-2 is induced in Acknowledgments protozoan and viral infections in response to IFN-␥ with patterns of tissue ex- pression that suggest nonredundant roles in vivo. J. Immunol. 157:4511. We thank Ronald L. Rabin for help with flow cytometry; Brent Kreider, 26. Wesselingh, S. L., C. Power, J. D. Glass, W. R. Tyor, J. C. McArthur, Gianni Garotta, David Hilbert, and others at Human Genome Sciences for J. M. Farber, J. W. Griffin, and D. E. Griffin. 1993. Intracerebral mes- providing Ck␤-15 and other chemokines; Lawrence Samelson and Connie senger RNA expression in acquired immunodeficiency syndrome dementia. Ann. Neurol. 33:576. Sommers for the Jurkat-TAg cells; Marcia Taylor for pEGFP-F DNA; Lisa 27. Liao, F., R. L. Rabin, J. R. Yannelli, L. G. Koniaris, P. Vanguri, and J. M. Farber. A. Penoyer and Kristen Lekstrom for DNA sequencing; and Hong Duc V. 1995. Human Mig chemokine: biochemical and functional characterization. Dang and Ruth Swofford for expert technical assistance. J. Exp. Med. 182:1301. 28. Campbell, J. J., E. F. Foxman, and E. C. Butcher. 1997. Chemoattractant receptor cross talk as a regulatory mechanism in leukocyte adhesion and migration. Eur. J. Immunol. 27:2571. References 29. Kozak, M. 1987. An analysis of 5Ј-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 15:8125. 1. Zlotnik, A., J. Morales, and J. A. Hedrick. 1999. Recent advances in chemokines 30. Murphy, P. 1994. The molecular biology of leukocyte chemoattractant receptors. and chemokine receptors. Crit. Rev. Immunol. 19:1. Annu. Rev. Immunol. 12:593. 2. Schall, T. J., K. Bacon, K. J. Toy, and D. V. Goeddel. 1990. Selective attraction 31. Probst, W. C., L. A. Snyder, D. I. Schuster, J. Brosius, and S. C. Sealfon. 1992. of monocytes and T lymphocytes of the memory phenotype by cytokine Sequence alignment of the G-protein coupled receptor superfamily. DNA Cell RANTES. Nature 347:669. Biol. 11:1. 3. Kim, C. H., and H. E. Broxmeyer. 1999. Chemokines: signal lamps for trafficking 32. Morello, J. P., and M. Bouvier. 1996. Palmitoylation: a post-translational mod- of T and B cells for development and effector function. J. Leukocyte Biol. 65:6. ification that regulates signalling from G-protein coupled receptors. Biochem. 4. Yoshie, O., T. Imai, and H. Nomiyama. 1997. Novel lymphocyte-specific CC Cell Biol. 74:449. chemokines and their receptors. J. Leukocyte Biol. 62:634. 33. Yang, T. T., L. Cheng, and S. R. Kain. 1996. Optimized codon usage and chro- 5. Berger, E. A., P. M. Murphy, and J. M. Farber. 1999. Chemokine receptors as mophore mutations provide enhanced sensitivity with the green fluorescent pro- HIV coreceptors: roles in viral entry, tropism, and disease. Annu. Rev. Immunol. tein. Nucleic Acids Res. 24:4592. 17:657. 34. Nomura, H., B. W. Nielsen, and K. Matsushima. 1993. Molecular cloning of 6. Liao, F., H.-H. Lee, and J. M. Farber. 1997. Cloning of STRL22, a new human cDNAs encoding a LD78 receptor and putative leukocyte chemotactic peptide gene encoding a G protein-coupled receptor related to chemokine receptors and receptors. Int. Immunol. 5:1239. located on chromosome 6q27. Genomics 40:175. 35. Chaffin, K. E., and R. M. Perlmutter. 1991. A pertussis toxin-sensitive process 7. Liao, F., R. Alderson, J. Su, S. J. Ullrich, B. L. Kreider, and J. M. Farber. 1997. controls thymocyte emigration. Eur. J. Immunol. 21:2565. STRL22 is a receptor for the CC chemokine MIP-3␣. Biochem. Biophys. Res. 36. Suzuki, G., H. Sawa, Y. Kobayashi, Y. Nakata, K. Nakagawa, A. Uzawa, Commun. 236:212. H. Sakiyama, S. Kakinuma, K. Iwabuchi, and K. Nagashima. 1999. Pertussis 8. Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, and J. M. Farber. toxin-sensitive signal controls the trafficking of thymocytes across the cortico- 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T medullary junction in the thymus. J. Immunol. 162:5981. cells and determines responsiveness to macrophage inflammatory protein 3␣. 37. Heesen, M., M. A. Berman, J. D. Benson, C. Gerard, and M. E. Dorf. 1996. J. Immunol. 162:186. Cloning of the mouse fusin gene, homologue to a human HIV-1 co-factor. J. Im- 9. Liao, F., G. Alkhatib, K. W. C. Peden, G. Sharma, E. A. Berger, and J. M. Farber. munol. 157:5455. 1997. STRL33, a novel chemokine receptor-like protein, functions as a fusion 38. Kitchen, S. G., and J. A. Zack. 1997. CXCR4 expression during lymphopoiesis: cofactor for both macrophage-tropic and T cell line-tropic HIV-1. J. Exp. Med. implications for human immunodeficiency virus type 1 infection of the thymus. 185:1. J. Virol. 71:6928. 10. Alkhatib, G., F. Liao, E. A. Berger, J. M. Farber, and K. W. C. Peden. 1997. A 39. Zaitseva, M. B., S. Lee, R. L. Rabin, H. L. Tiffany, J. M. Farber, K. W. C. Peden, new SIV co-receptor, STRL33. Nature 388:238. P. M. Murphy, and H. Golding. 1998. CXCR4 and CCR5 on human thymocytes: 11. Rucker, J., A. L. Edinger, M. Sharron, M. Samson, B. Lee, J. F. Berson, Y. Yi, biological function and role in HIV-1 infection. J. Immunol. 161:3103. B. Margulies, R. G. Collman, B. J. Doranz, et al. 1997. Utilization of chemokine 40. Power, C. A., A. Meyer, K. Neneth, K. B. Bacon, A. J. Hoogewerf, receptors, orphan receptors, and herpesvirus-encoded receptors by diverse human A. E. I. Proudfoot, and T. N. C. Wells. 1995. Molecular cloning and functional and simian immunodeficiency viruses. J. Virol. 71:8999. expression of a novel CC chemokine receptor cDNA from a human basophilic 12. Vicari, A. P., D. J. Figueroa, J. A. Hedrick, J. S. Foster, K. P. Singh, S. Menon, cell line. J. Biol. Chem. 270:19495. N. G. Copeland, D. J. Gilbert, N. A. Jenkins, K. B. Bacon, and A. Zlotnik. 1997. 41. Napolitano, M., A. Zingoni, G. Bernardini, G. Spinetti, A. Nista, C. T. Storlazzi, TECK: a novel CC chemokine specifically expressed by thymic dendritic cells M. Rocchi, and A. Santoni. 1996. Molecular cloning of TER1, a chemokine and potentially involved in T cell development. Immunity 7:291. receptor-like gene expressed by lymphoid tissues. J. Immunol. 157:2759. 13. Nomiyama, H., K. Amano, J. Kusuda, T. Imai, R. Miura, O. Yoshie, and 42. Goya, I., J. Gutierrez, R. Varona, L. Kremer, A. Zaballos, and G. Marquez. 1998. Y. Matsuda. 1998. The human CC chemokine TECK (SCYA25) maps to chro- Identification of CCR8 as the specific receptor for the human ␤-chemokine I-309: mosome 19p13. 2. Genomics 51:311. cloning and molecular characterization of murine CCR8 as the receptor for 14. Wilkinson, B., J. J. Owen, and E. J. Jenkinson. 1999. Factors regulating stem cell TCA-3. J. Immunol. 160:1975. recruitment to the fetal thymus. J. Immunol. 162:3873. 43. Campbell, J. J., J. Pan, and E. C. Butcher. 1999. Cutting edge: developmental 15. Nagasawa, T., S. Hirota, K. Tachibana, N. Takakura, S. Nishikawa, Y. Kitamura, switches in chemokine responses during T cell maturation. J. Immunol. 163:2353. N. Yoshida, H. Kikutnai, and T. Kishimoto. 1996. Defects of B-cell lymphopoi- 44. Smith, S. D., M. Shatsky, P. S. Cohen, R. Warnke, M. P. Link, and B. E. Glader. esis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/ 1984. Monoclonal antibody and enzymatic profiles of human malignant T- lym- SDF-1. Nature 382:635. phoid cells and derived cell lines. Cancer Res. 44:5657. 16. Gunn, M. D., S. Kyuwa, C. Tam, T. Kakiuchi, A. Matsuzawa, L. T. Williams, and 45. Minowada, J., T. Onuma, and G. E. Moore. 1972. Rosette-forming human lym- H. Nakano. 1999. Mice lacking expression of secondary lymphoid organ che- phoid cell lines. I. Establishment and evidence for origin of thymus-derived lym- mokine have defects in lymphocyte homing and localization. phocytes. J. Natl. Cancer Inst. 49:891. J. Exp. Med. 189:451. 46. Wong, L. M., S. J. Myers, C. L. Tsou, J. Gosling, H. Arai, and I. F. Charo. 1997. 17. Loetscher, P., M. Seitz, M. Baggiolini, and B. Moser. 1996. -2 regu- Organization and differential expression of the human monocyte chemoattractant lates CC chemokine receptor expression and chemotactic responsiveness in T protein 1 receptor gene: evidence for the role of the carboxyl-terminal tail in lymphocytes. J. Exp. Med. 184:569. receptor trafficking. J. Biol. Chem. 272:1038. 18. Rabin, R. L., M. K. Park, F. Liao, R. Swofford, D. Stephany, and J. M. Farber. 47. Schweickart, V. L., C. J. Raport, R. Godiska, M. G. Byers, R. L. J. Eddy, 1999. Chemokine receptor responses on T cells are achieved through regulation T. B. Shows, and P. W. Gray. 1994. Cloning of human and mouse EBI1, a of both receptor expression and signaling. J. Immunol. 162:3840. lymphoid-specific G-protein-coupled receptor encoded on human chromosome 19. Campbell, J. J., J. Hedrick, A. Zlotnik, M. A. Siani, D. A. Thompson, and 17q12–q21.2. Genomics 23:643. E. C. Butcher. 1998. Chemokines and the arrest of lymphocytes rolling under 48. Wegner, S. A., P. K. Ehrenberg, G. Chang, D. E. Dayhoff, A. L. Sleeker, and flow conditions. Science 279:381. N. L. Michael. 1998. Genomic organization and functional characterization of the The Journal of Immunology 1305

chemokine receptor CXCR4, a major entry co-receptor for human immunodefi- 53. Moepps, B., R. Frodl, H. R. Rodewald, M. Baggiolini, and P. Gierschik. 1997. ciency virus type 1. J. Biol. Chem. 273:4754. Two murine homologues of the human chemokine receptor CXCR4 mediating 49. Dobner, T., I. Wolf, T. Emrich, and M. Lipp. 1992. Differentiation-specific ex- stromal cell-derived factor 1␣ activation of Gi2 are differentially expressed in pression of a novel G protein-coupled receptor from Burkitt’s lymphoma. Eur. vivo. Eur. J. Immunol. 27:2102. J. Immunol. 22:2795. 54. Hamdan, F. F., M. D. Ungrin, M. Abramovitz, and P. Ribeiro. 1999. Character- 50. Journot, L., D. Spengler, C. Pantaloni, A. Dumuis, M. Sebben, and J. Bockaert. ization of a novel serotonin receptor from Caenorhabditis elegans: cloning and 1994. The PACAP receptor: generation by alternative splicing of functional di- expression of two splice variants. J. Neurochem. 72:1372. versity among G protein-coupled receptors in nerve cells. Semin. Cell. Biol. 55. Gupta, S. K., and K. Pillarisetti. 1999. Cutting edge: CXCR4-Lo: molecular clon- 5:263. ing and functional expression of a novel human CXCR4 splice variant. J. Im- 51. Charo, I. F., S. J. Myers, A. Herman, C. Franci, A. J. Connolly, and munol. 163:2368. S. R. Coughlin. 1994. Molecular cloning and functional expression of two mono- 56. Kozak, M. 1996. Interpreting cDNA sequences: some insights from studies on cyte chemoattractant protein 1 receptors reveals alternative splicing of the car- translation. Mamm. Genome 7:563. boxyl-terminal tails. Proc. Natl. Acad. Sci. USA 91:2752. 57. Palczewski, K., and J. L. Benovic. 1991. G-protein-coupled receptor kinases. 52. Heesen, M., M. A. Berman, U. E. Hopken, N. P. Gerard, and M. E. Dorf. 1997. Trends Biochem. Sci. 16:387. Alternate splicing of mouse fusin/CXC chemokine receptor-4: stromal cell-de- 58. Zaballos, A., J. Gutierrez, R. Varona, C. Ardavin, and G. Marquez. 1999. Cutting rived factor-1␣ is a ligand for both CXC chemokine receptor- 4 isoforms. J. Im- edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the munol. 158:3561. receptor for the chemokine TECK. J. Immunol. 162:5671.