CCR9

CCR9 Angel Zaballos, Julio GutieÂrrez, Rosa Varona and Gabriel MaÂrquez* Departamento de InmunologõÂ a y OncologõÂ a, Centro Nacional de BiotecnologõÂ a, Campus Universidad AutoÂnoma, Cantoblanco, Madrid, 28049, Spain * corresponding author tel: 91 585 4856, fax: 91 372 0493, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22017.

SUMMARY T cell maturation requires the controlled migration of T cell precursors through the different thymic compartments in a process that seems to be regulated by chemokines. The former human putative chemokine receptor GPR-9-6 has recently been identified as CCR9, the specific receptor for the thymus-expressed chemokine TECK. The cDNA sequences containing the complete coding regions of humanand mouse CCR9 have been established, showing that the identity between both CCR9 predicted proteins is 86%. The expression of both genes is also very similar, being high in the thymus and low in lymph nodes and spleen. RTPCR analysis of mouse CCR9 expression on murine FACS-sorted thymocyte subpopulations showed that this gene is expressed in both immature and mature T cells. Among the 36 different chemokines tested on intracytoplasmic calcium mobilization and in vitro chemotaxis assays, TECK is the only functional ligand found for CCR9. The expression of murine CCR9 in both immature and mature thymocytes, together with the expression of its ligand in the thymus, suggests that the TECK-mediated chemoattraction might be a mechanism contributing to thymocyte retention in the thymus while completing their development.

BACKGROUND

Discovery The cellular compartmentalization of the thymus provides an optimal microenvironment for thymopoiesis (Boyd and Hugo, 1991; Boyd et al., 1993; Shortman and Wu, 1996). After the stem cells have migrated from the bone marrow to the fetal thymic primordium, immature thymocytes undergo a complex

and organized differentiation process that gives rise to a functional immune system able to distinguish between self and nonself (Boyd and Hugo, 1991). During their maturation, thymocytes migrate through the thymus in what seems to be a regulated process. Immature CD4ÿCD8ÿ thymocytes are found in the subcapsular region, whereas more mature CD4‡ CD8‡ thymocytes are located in the cortex; and mature CD4‡ or CD8‡ T cells reside in the medulla (Boyd et al., 1993; Ritter and Boyd, 1993). The factors controlling thymocyte migration inside the thymus are largely unknown, but chemokines appear to play an important role. In this regard, recent reports from Vicari et al. (1997) and Kim et al. (1998) demonstrated that the chemokines TECK, SDF, and ELC (MIP-3 ) were able to chemoattract different thymocyte subsets. While the specific receptors for SDF and ELC (MIP-3 ) are known to be CXCR4 and CCR7, respectively (Bleul et al., 1996; Oberlin et al., 1996; Yoshida et al., 1997), the receptor for the chemokine TECK has only very recently been reported to be the former GPR-9-6 orphan receptor, now known as CCR9 (Zaballos et al., 1999). The cDNA sequence encompassing the complete coding region of human and mouse CCR9 has been reported (Zaballos et al., 1999). Human and murine CCR9 have a highly restricted expression pattern, being very highly expressed in the thymus, and less in lymph nodes and spleen. RT-PCR analysis of mouse CCR9 expression on murine FACS-sorted thymocyte subpopulations showed that this gene is expressed in both immature and mature T cells (Zaballos et al., 1999). Intracytoplasmic calcium mobilization and chemotaxis assays established that, among 36 different chemokines tested, TECK was the only functional ligand for CCR9, in both human and murine systems (Zaballos et al., 1999). This remarkable specificity is consistent with the CCR9 phylogenetic

2148 Angel Zaballos, Julio GutieÂrrez, Rosa Varona and Gabriel MaÂrquez relationship to CCR6 and CCR7, which also have strict ligand specificity. This lack of promiscuity is not a common feature of chemokine receptors, which usually recognize more than one chemokine (Rollins, 1997; Luster, 1998). Anyway, the possibility cannot be excluded that new CCR9 agonists may be identified. In addition, the CCR9 ligand TECK is phylogenetically closely related to MIP-3 and SLC(6C-kine), the ligands for CCR6 and CCR7, respectively, sharing with them unique structural features, such as the Asp-Cys-Cys-Leu motif (Varona et al., 1998).

Alternative names CCR9 was previously known as orphan chemokine receptor GPR-9-6.

Structure CCR9 is a member of the family of seven transmembrane domain, G protein-coupled receptors and has recently been characterized as a chemokine receptor (Figure 1). In addition to chemokine

receptors, this family has a great number of members, including receptors for hormones, growth factors, neurotransmitters, odorants, and other signaling molecules. The extracytoplasmic N-terminal region of both human and mouse CCR9 contains a conserved cysteine residue that is typically involved in disulfide bonding with other conserved cysteines in the extracellular loops. The 78 C-terminal amino acids of human and mouse CCR9 are identical; these include the serine and threonine residues of the intracytoplasmic tail, which may be phosphorylated.

Main activities and pathophysiological roles The fact that both immature and mature thymocytes express mouse CCR9 suggests that this receptor might play a role during the whole process of thymocyte development. As thymic dendritic cells are producers of TECK (Vicari et al., 1997), the TECKmediated chemoattraction might be a mechanism contributing to thymocyte confinement in the thymus

Figure 1 Scheme of human CCR9 structure showing the serpentine shape of this chemokine receptor, with its predicted seven transmembrane domains and the extracellular and intracellular loops. The standard color code is used to highlight the chemical properties of the amino acid residues of the polypeptide. The snake-like plot was generated using services available at Viseur's homepage (http://www.lctn.u-nancy.fr/viseur/viseur.htlm). M T P T P I P S T F D N M A D D Y G S E D E M S S T S Y V N F N F T D F V N N K E C Y R Q F A S H F L P P L Y W L V F I V G A L G N S L V I L V Y W Y C T R V K T

D QWK F Q T A F A M A C I K A W V V N S F P M Y L T K V L M N F F Y L L S C D A V I A L L I L M N L C I L F S M T D M V D R Y I A I

S G I A I C T E E M V K Y I Q P S S D Y E L I S T E K P I L K S A C L V A A L T L A L V W K V I I L T F G F C V F M K S L P F V Y L L V M A R C C Y K T I I E I R H W T T L H I Q A K K A Q A M R A

C A V S T N I D N I S C I F F Q M V A T Y Q A T D I A L T F F Q V H S L L I L N C C N P Y P L Y V F Q S V F V L V G F V T L R R F R E V T I D T V L V K T L K K N L Q S I C G L A A K Q WV S F T S S K H R K L S G E R L S S M L L E T S L A G S T L

CCR9 2149 until their development is completed. A recent report by Wilkinson et al. (1999) showed that neutralizing antibodies to TECK did not prevent in vitro thymus recolonization by T cell precursors, suggesting that additional factors might be involved in the arrival of those lymphoid precursors in the thymus. In addition, thymocytes have been reported to use different chemokines during their development, as suggested by the modulation of the expression of CXCR4, CCR4, and CCR7 while thymocytes travel from the thymic cortex to the medulla (Suzuki et al., 1999). In this context, the identification of CCR9 as the functional receptor for TECK may help to unravel the details of the important role these proteins seem to play in the thymus. Some chemokine receptors act as coreceptors for the entry of HIV into the cells (D'Souza and Harden, 1996). In this regard, CCR9 has been reported not to be active as a cofactor in cell fusion assays using Env proteins from diverse human and simian immunodeficiency viruses (Rucker et al., 1997). Anyway, these experiments were done using a truncated version of CCR9, that lacks the first N-terminal 12 amino acids. Therefore, a possible role of CCR9 as coreceptor for HIV cannot be dismissed.

GENE

Accession numbers Mouse CCR9 cDNA: AJ132336 Human CCR9 cDNA: AJ132337 Human CCR9 gene: AC005669

In addition, a partial cDNA sequence corresponding to the 50 region of human CCR9 is found at AF145207. The genomic sequence in U45982 only includes the codifying sequence contained in exon 3.

Sequence See Figure 2. Figure 3 shows that the human CCR9 gene has the three exons/two introns structure typical of chemokine receptor genes, with the coding sequence split between exons 2 and 3 (Zaballos et al., 1999).

Chromosome location and linkages According to data shown in the U45982 entry, the human CCR9 gene is located on chromosome 3 (3p21-3p22). A cluster of chemokine receptors maps to that region of chromosome 3, including CCR1, CCR2, CCR3, CCR5 (Samson et al., 1996), and the orphan receptor CKR-X. In addition, other chemokine receptors, such as CCR8 (Napolitano et al., 1996), CCR10 (Bonini et al., 1997), XCR1 (Heiber et al., 1995), CX3CR1 (Raport et al., 1995), and the orphan receptor TYMSTR/Bonzo (Loetscher et al., 1997) are also located in the 3p21 region.

PROTEIN

Accession numbers Human and mouse CCR9 proteins are accessible from the SPTREMBL section of the EMBL database,

Figure 2 Nucleotide sequence of the human and mouse CCR9 cDNA. The initiator ATG and stop TGA codons are marked in red. (Full colour figure may be viewed online.)

2150 Angel Zaballos, Julio GutieÂrrez, Rosa Varona and Gabriel MaÂrquez Figure 2 (Continued )

Figure 3 Exon/intron structure of the human CCR9 gene. The size of exon 2 is 48 bp. Light brown-shaded areas correspond to coding sequences. The figure is not drawn to scale. Data from Zaballos et al. (1999), with permission. (Full colour figure may be viewed online.) Exon 1

Exon 2 8272 bp

Exon 3 5856 bp

with accession numbers Q9UQQ6 and Q9WUT7, respectively.

Sequence See Figure 4.

Description of protein As described by Zaballos et al. (1999), both human and mouse CCR9 polypeptides are 369 amino acids long with seven transmembrane domains, showing the conserved motifs of this family of receptors as some cysteine residues, the DRY motif in the second intracellular loop, and the intracytoplasmic C-terminal tail with serine and threonine residues. In the N-terminal

region, N32 is a potential N-glycosylation site for both human and mouse CCR9; mouse CCR9 has an additional potential site on N288, between transmembrane domains 6 and 7.

Relevant homologies and species differences There is an 86% identity between human and mouse CCR9 proteins (Zaballos et al., 1999) (Figure 4). Concerning the phylogenetic relationships of human CCR9 with other chemokine receptors and orphan putative receptors, CCR9 is more similar to CCR7 (39%), CCR6 (34%) and the orphan receptor TYMSTR/Bonzo (32%), with which it is placed in a separate branch of the phylogenetic tree (Zaballos et al., 1999) (Figure 5).

Affinity for ligand(s) The ligand-specificity of human CCR9 was investigated by monitoring changes in the intracellular calcium concentration of stable HEK 293 transfectant cells upon sequential addition of chemokine samples. Among 36 recombinant human chemokines tested (Table 1), only TECK elicited a response, as

CCR9 2151 Figure 4 Alignment of the predicted amino acid sequences of human (blue) and mouse (green) CCR9 proteins. TM, transmembrane domains. Asterisks mark sites of potential N-glycosylation. Purple boxes mark identical residues. Data from Zaballos et al. (1999), with permission. (Full colour figure may be viewed online.)

hCCR9 mCCR9 hCCR9 mCCR9

MTPTDFTSPI PNMADDYGSE MMPTELTSLI PGMFDDFSYD TM1 LPPLYWLVFI VGALGNSLVI LPPLYWLVFI VGTLGNSLVI

hCCR9 mCCR9

LPFWAIAAAD QWKFQRFMCK LPFWAIAAAG QWMFQTFMCK

hCCR9 mCCR9

AMRAHTWREK RLLYSKMVCF AMKAQVWRQK RLLYSKMVCI

hCCR9 mCCR9

hCCR9 mCCR9

VYPSDESTKL KSAVLTLKVI VYPKDKNAKL KSAVLILKVT TM6 KALKVTITVL TVFVLSQFPY KALKVTITVL TVFIMSQFPY TM7 VTQTIAFFHS CLNPVLYVFV VTQTIAFFHS CLNPVLYVFV

hCCR9 mCCR9

GSLKLSSMLL ETTSGALSL GSLKLSSMLL ETTSGALSL

hCCR9 mCCR9

Figure 5 Phylogenetic relationships of CCR9 with other chemokine receptors and some orphan putative chemokine receptors. The tree was generated with the NJ plot program from a Clustal X multialignment. The data are taken from Zaballos et al. (1999), with permission. CCR9 CCR7 CCR6 BONZO

CCR10 CCR8 CCR4 CKRX CCR5 CCR2 CCR3 CCR1

* STSSMEDYVN FNFTDFYCEK NNVRQFASHF STASTDDYMN LNFSSFFCKK NNVRQFASHF * TM2 LVYWYCTRVK TMTDMFLLNL AIADLLFLVT LVYWYCTRVK TMTDMFLLNL AIADLLFLAT TM3 VVNSMYKMNF YSCVLLIMCI SVDRYIAIAQ VVNSMYKMNF YSCVLLIMCI SVDRYIAIVQ TM4 TIWVLAAALC IPEILYSQIK EESGIAICTM TIWVMAAVLC TPEILYSQVS GESGIATCTM TM5 LGFFLPFVVM ACCYTIIIHT LIQAKKSSKH LGFFLPFMVM AFCYTIIIHT LVQAKKSSKH NCILLVQTID AYAMFISNCA VSTNIDICFQ NSILVVQAVD AYAMFISNCT ISTNIDICFQ * GERFRRDLVK TLKNLGCISQ AQWVSFTRRE GERFRRDLVK TLKNLGCISQ AQWVSFTRRE

50 50 100 100 150 150 200 200 250 250 300 300 350 350 369 369

demonstrated by Zaballos et al. (1999). The cell response is correlated to the chemokine dose down to 20 nM. Murine TECK also induces intracellular calcium mobilization on HEK 293 cells transfected with the mouse CCR9 cDNA (Zaballos et al., 1999). Adding a high concentration of human TECK to 293/ hCCR9 cells results in complete desensitization to a second addition of the same stimulus. Both human and mouse TECK are able to stimulate the human receptor in transfectant cells, and this results in desensitization to a subsequent stimulus with hTECK. Similar experiments have been done with HEK 293 transfected with an expression plasmid encoding a shorter human CCR9 version, that lacks the N-terminal 12 amino acids encoded by exon 2. The shorter CCR9 protein is able to provoke a calcium flux upon stimulation with TECK (our unpublished results), thus suggesting that the first 12 N-terminal amino acids are not essential for activity.

2152 Angel Zaballos, Julio GutieÂrrez, Rosa Varona and Gabriel MaÂrquez Table 1 Human chemokines tested for calcium mobilization on indo-1-AM-loaded HEK 293/CCR9 cells CXC chemokines

CC chemokines

CX3C chemokines

C chemokines

CXCL1/GRO

CCL1/I-309

CX3CL1/Fractalkine

XCL1/Lymphotactin

CXCL2/GRO

CCL2/MCP-1

CXCL3/GRO

CCL3/MIP-1

CXCL4/PF4

CCL4/MIP-1

CXCL5/ENA-78

CCL5/RANTES

CXCL6/GCP-2

CCL7/MCP-3

CXCL7/NAP-2

CCL8/MCP-2

CXCL8/IL-8

CCL9-10/MIP-1

CXCL9/MIG

CCL11/Eotaxin 1

CXCL10/IP-10

CCL13/MCP-4

CXCL11/I-TAC

CCL14/HCC-1

CXCL12/SDF

CCL16/LEC

CXCL13/BCA-1

CCL17/TARC CCL18/PARC CCL19/ELC CCL20/MIP-3 CCL21/SLC CCL22/MDC CCL23/MPIF1 CCL24/Eotaxin 2 CCL25/TECK

MOLT-4, a CD4‡ lymphoblastic T cell line of thymic origin, also showed a high increase of intracellular calcium on stimulation with 200 nM hTECK (Zaballos et al., 1999). The cell response is correlated to the chemokine dose, and MOLT-4 cells are slightly more sensitive than the transfectants to low doses of the chemokine, as 6 nM hTECK induced a detectable response. These results are essentially identical to those obtained with the HEK 293 cells transfected with human CCR9 and suggest that hTECK-induced stimulation of MOLT-4 cells is mediated by CCR9. As commented below, MOLT-4 cells express abundant CCR9 mRNA. The activation of human CCR9 on stimulation with human TECK has also been shown in chemotaxis assays with HEK 293/CCR9 transfectants (Zaballos et al., 1999) and MOLT-4 cells. Both cell types showed maximal migration at 100 nM TECK. A checkerboard-type analysis confirmed that the TECK-induced migration of MOLT-4 cells is mostly chemotactic.

Cell types and tissues expressing the receptor Northern blot analysis of human poly(A)‡ RNA or mouse total RNA samples showed that both human and mouse CCR9 mRNA are highly expressed in thymus, although weak mRNA expression in spleen and lymph nodes was also detected (Zaballos et al., 1999). Transcript sizes are 2.7 kb and 3.3 kb for human and mouse CCR9, respectively. Among several human cell lines tested, only MOLT-4 cells showed expression of CCR9; this is in agreement with their functional responses to TECK. As thymus was the tissue showing maximal expression of mouse CCR9, an RT-PCR analysis was done on FACS-sorted thymocyte subpopulations (Zaballos et al., 1999). Mouse CCR9 is expressed by CD25‡CD4ÿCD8ÿ pre-T cells, CD4‡CD8‡ immature thymocytes, as well as in single positive CD4‡ and CD8‡ T cells. These results are consistent with the reported

CCR9 2153 TECK-induced chemoattraction of mouse thymocytes (Vicari et al., 1997).

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades The results reported by Zaballos et al. (1999) on the calcium mobilization assays performed on pertussis toxin-treated cells suggest that human CCR9 is partially, but not exclusively, coupled to the Gi class of G subunits. Indeed, pertussis toxin treatment decreased but did not abolish the TECK-induced calcium mobilization on HEK 293/CCR9 transfectants and MOLT-4 cells. A similar result has also been described for other chemokine receptors (Arai and Charo, 1996; Varona et al., 1998). In contrast, the response to the  chemokine SDF, for which both cell types have endogenous receptors, is completely abolished by toxin treatment. Cholera toxin did not affect the CCR9-mediated calcium response of these cells upon stimulation with TECK.

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