XCR1

XCR1 Osamu Yoshie* Department of Microbiology, Kinki University School of Medicine, Osaka-Sayama, Osaka, 589-8511, Japan * corresponding author tel: +81-723-67-3606, fax: +81-723-67-3606, e-mail: [email protected] DOI: 10.1006/rwcy.2000.22018.

SUMMARY An orphan seven transmembrane G protein-coupled receptor called GPR5 was identified as a specific functional receptor for Lptn/SCM-1/ATAC and is now termed XCR1. Lymphotactin (Lptn)/single Cmotif 1 (SCM-1)/activation-induced, T cell-derived and chemokine-related molecule (ATAC) belongs to the C or subfamily of chemokines. XCR1 is expressed in tissues such as placenta, thymus, and spleen in humans. Few, if any, transcripts are, however, detected in peripheral blood leukocytes. Therefore, the exact types of cells that express XCR1 are still not known. Ltpn/SCM-1/ATAC induces chemotaxis and calcium flux in murine L1.2 cells transfected with XCR1. XCR1 and its murine counterpart mXCR1 have no N-glycosylation sites and have a sequence of HRYLSVVSP in the second intracellular domain instead of the standard DRY (Asp-Arg-Tyr) motif highly conserved in many chemokine receptors.

BACKGROUND

Discovery An orphan seven-transmembrane G protein-coupled receptor (GPCR) termed GPR5 (Heiber et al., 1995) was identified as a receptor for XCL1 and XCL2 and now termed XCR1 (Yoshida et al., 1998). Murine L1.2 cells stably expressing XCR1 bound 125 I-labeled XCL1 with a high affinity and responded to XCL1 and XCL2 in chemotactic and calcium flux assays (Yoshida et al., 1998). No other human chemokines so far tested were capable of inducing any such responses via XCR1 (Yoshida et al., 1998). Taken together, XCR1 is a highly specific functional receptor for XCL1 and XCL2. In humans, XCR1 was found

to be expressed in the placenta, thymus, and spleen (Yoshida et al., 1998). Very few, if any, signals were detected in peripheral blood leukocytes. The murine counterpart of XCR1 was identified and termed mXCR1 (Yoshida et al., 1999). Lymphotactin (Lptn)/single C-motif 1 (SCM-1)/ activation-induced, T cell-derived and chemokinerelated molecule (ATAC), the ligand for XCR1, is selectively produced upon activation by CD8+ T cells,

 T cells, natural killer (NK) cells, and mast cells (Kelner et al., 1994; Yoshida et al., 1995; Mueller et al., 1995; Kennedy et al., 1995; Boismenu et al., 1996; Hedrick et al., 1997; Rumsaeng et al., 1997). It is expressed in peripheral blood leukocytes and also in tissues such as thymus, spleen, lymph nodes, and small intestine (Yoshida et al., 1996). It has a homology especially to the members of the CC subfamily, but lacks the first and third of the four cysteine residues conserved in all other chemokines. This molecule is now regarded as a unique representative of the C or subfamily of chemokines. Lymphotactin was reported to be chemotactic for lymphocytes and NK cells but its chemotactic activity on these types of cells has been controversial. In humans, there are two highly homologous genes encoding two forms of this molecules with only two amino acid differences (SCM-1 and SCM-1 ) (Yoshida et al., 1996). The systemic names for these molecules are XCL1 and XCL2 from the proposal on the systemic nomenclature of chemokine at the Keystone Symposium of 1999.

Alternative names XCR1 was originally described as an orphan seven transmembrane, G protein-coupled receptor and called GPR5 (Heiber et al., 1995).

2156 Osamu Yoshie

Structure XCR1 exhibits the seven transmembrane, G proteincoupled receptor structure typical of all of the chemokine receptors. However, both human and mouse sequences of XCR1 have the sequence HRYLSVVSP in the second intracellular loop instead of the typical DRY motif (DRYLAIVHA), which is highly conserved in the chemokine receptor family (Yoshida et al., 1998, 1999). As a GPCR, XCR1 is also somewhat unusual because neither human nor mouse XCR1 contain N-glycosylation sites (Yoshida et al., 1998, 1999). Between the human and mouse sequences, the N-terminal extracellular domain is highly divergent. Human XCR1 is 333 amino acids in length with a calculated molecular weight of 38,507.5 and an isoelectric point of 8.94.

Main activities and pathophysiological roles The exact types of cells expressing XCR1 are not known. The messages were found to be weakly expressed in lymphoid tissues such as thymus and spleen in both humans and mice (Yoshida et al., 1998, 1999). Considerable expression was observed in placenta in humans (Yoshida et al., 1998). Few if any signals were detected, however, in peripheral blood leukocytes (Yoshida et al., 1998). The latter observation is rather inconsistent with the previous results of the chemotactic

activity of XCL1 on peripheral blood lymphocytes and NK cells. When stably transfected in murine L1.2 cells, both human and mouse XCR1 transduced signals for chemotaxis and calcium flux through a pertussis toxinsensitive pathway (Yoshida et al., 1998, 1999). When ovalbumin was given intranasally without and with mXCL1, mice with the latter treatment showed enhanced systemic and local antibody responses to ovalbumin (Lillard et al., 1999). This suggests that local activation of cells expressing XCR1 can lead to enhanced mucosal and systemic immune responses.

GENE Like many other GPCR genes, XCR1 is encoded by a single exon. The human gene is localized at chromosome 3p21.3-p21.1 (Heiber et al., 1995).

Accession numbers GenBank/EMBL/DDBJ: Human XCR1: L36149 Mouse XCR1: AB028459

Sequence See Figure 1.

Figure 1 Nucleotide sequence for the XCR1 gene. The coding region is shown by uppercase letters. 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 751 801 851 901 951 1001 1051 1101 1151 1201 1251

caaggcaatc ttagccactg atttcagatg GGCAACCCAG GTGTGAGAAC ACTGCCTGGT GTCCTGGTGA CAACCTGTGC TCTCCCCATA CTCAATATGA CATCATGACC TGCGCGTCCC GTAGCCAGCA TTCTTCGGGC ACCAGCACAA TACGTGGAGA CCGCACGGTC GGGGTCCCTA ATCATCCGGA CTGCCGCAAC TCTTCGTGGG TTCTGGTTCT CCCTGGTGCC gcggtgcagg agaagcgggc tcctcctgct

ctctcccttt tacccagcca ctctaaacgt AGAGCACCAC CAGGCCTGGG GTTTCTCCTC AGTATGAGAG CTCTCAGACC CCACTGGGGC TCTTCTCCAT ATCCACCGCT CACCCTCCGC TCCTGTCCTC TGTGATTATT CCTCTTCTTC TCCTCAGGAC AAGCTCATCT CAACTTCACC GCTGCGAGGC CTCGCCTTCT GGTCAAGTTC GCCGGCTGCA TTCGCCTATG cgcaggtgca ctggaaggag gcagg

ggcctttcca ccacatggct ccctgccatc CTTTTTTTAC TCTTTGCTAC AGCCTAGTGG CCTGGAGTCC TGGTGTTCGC TGGGTGCTGG CAGCCTCTAC ACCTGTCGGT TGCCGGGTGC CATCCTCGAC CCGAACTCAC CTGCTGTCCC CCTGTTCCGC TCGCCATCGT CTGTTTCTGC CAAACAGCAG CCCACTGCTG CGCACACACC GGCACCCAGC AGGGCGCCTC ggtggacagg cattgcagaa

aagtgctagg ttaaactcca tggtccagAT TATGACCTTC CCTCGCCACC GCAACAGCCT CTCACCAACA CTGCTTGTTG GAGACTTCCT AGCAGCATCT AGTGAGCCCC TGGTGACCAT ACCATCTTCC GTGGTACCTC TGGGGATTAT TCACGCTCCA GGTGGCCTAC AGACGCTGTT CTAGAATACG CTTTAACCCG TGAAACATGT CCAGCCTCGA CTTCTACtga gactggaatg cacagcaggg

attacaggtg tgtctctatc GGAGTCCTCA AGAGCCAGCC ACTGTCCTGT GGTCCTGTGG TCTTCATCCT CCTGTGTGGA CTGCAAACTC TCTTCCTGAC CTCTCCACCC GGCTGTGTGG ACAAGGTGCT ACCTCCGTCT CCTGTTCTGC AGCGGCGCCA TTCCTCAGCT TCGGACCCAG CCCTGCTCAT GTGCTCTATG TCTCCGGCAG TCCCCCACTC ggggcctgtg ggggtcatgg tggagacgtc

XCR1 2157 Figure 2 Amino acid sequence for the XCR1 protein.

SIGNAL TRANSDUCTION

MESSGNPEST LVLWVLVKYE LCKLLNMIFS MAVWVASILS ILFCYVEILR FRTQIIRSCE VLRQFWFCRL

In murine L1.2 stable transfectants, both human and mouse XCR1 were capable of transducing signals in chemotaxis and calcium flux assays. The chemotactic responses of L1.2 expressing human XCR1 were shown to be highly sensitive to pertussis toxin, supporting its coupling to a Gi class of G protein.

TFFYYDLQSQ SLESLTNIFI ISLYSSIFFL SILDTIFHKV TLFRSRSKRR AKQQLEYALL QAPSPASIPH

PCENQAWVFA LNLCLSDLVF TIMTIHRYLS LSSGCDYSEL HRTVKLIFAI ICRNLAFSHC SPGAFAYEGA

TLATTVLYCL ACLLPVWISP VVSPLSTLRV TWYLTSVYQH VVAYFLSWGP CFNPVLYVFV SFY

VFLLSLVGNS YHWGWVLGDF PTLRCRVLVT NLFFLLSLGI YNFTLFLQTL GVKFRTHLKH

PROTEIN

Accession numbers SwissProt: Human XCR1: P46094

Sequence See Figure 2.

Relevant homologies and species differences XCR1 is most homologous to CCR10 (Bonini et al., 1997) with 32% amino acid identity. Human and mouse XCR1 show 71% amino acid identity (Yoshida et al., 1999). Between the human and mouse sequences, the N-terminal region is the least conserved.

Affinity for ligand(s) XCL1 bound to XCR1 expressed on transfected murine L1.2 with a Kd of 10 nM (Yoshida et al., 1998).

Cell types and tissues expressing the receptor The exact types of cells expressing XCR1 are not known. In mouse spleen, transcripts were detected by RT-PCR in cells with CD8 marker and also in NK1.1+CD3ÿ cells (Yoshida et al., 1999). In human tissues, XCR1 expression was readily detected in placenta and weakly in spleen and thymus (Yoshida et al., 1998). In mouse tissues, weak expression was observed in spleen and lung (placenta and thymus not tested) (Yoshida et al., 1999).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors At least in transfected murine L1.2 cells, XCR1 is a chemoattractant receptor. XCR1 is also a highly specific receptor for XCL1 and XCL2 (Yoshida et al., 1998).

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2158 Osamu Yoshie Mueller, S., Dorner, B., Korthaeuer, U., Mages, H. W., D'Apuzzo, M., Senger, G., and Kroczek, R. A. (1995). Cloning of ATAC, an activation-induced, chemokine-related molecule exclusively expressed in CD8+ T lymphocytes. Eur. J. Immunol. 25, 1744±1748. Rumsaeng, V., Vliagoftis, H., Oh, C. K., and Metcalfe, D. D. (1997). Lymphotactin gene expression in mast cells following Fc(epsilon) receptor I aggregation: modulation by TGF-beta, IL-4, dexamethasone, and cyclosporin A. J. Immunol. 158, 1353±1356. Yoshida, T., Imai, T., Kakizaki, M., Nishimura, M., and Yoshie, O. (1995). Molecular cloning of a novel C or type chemokine, SCM-1. FEBS Lett. 360, 155±159.

Yoshida, T., Imai, T., Takagi, S., Nishimura, M., Ishikawa, I., Yaoi, T., and Yoshie, O. (1996). Structure and expression of two highly related genes encoding SCM-1/human lymphotactin. FEBS Lett. 395, 82±88. Yoshida, T., Imai, T., Kakizaki, M., Nishimura, M., Takagi, S., and Yoshie, O. (1998). Identification of single C motif-1/lymphotactin receptor XCR1. J. Biol. Chem. 273, 16551±16554. Yoshida, T., Izawa, D., Nakayama, T., Nakahara, K., Kakizaki, M., Imai, T., Suzuki, R., Miyasaka, M., and Yoshie, O. (1999). Molecular cloning of mXCR1, the murine SCM-1/lymphotactin receptor. FEBS Lett. 458, 37±40