CXCR3

CXCR3 Joshua Marion Farber1,* and Bernhard Moser2 1

Laboratory of Clinical Investigation, National Institute of Allergy and Infectious Diseases, NIH, 10 Center Drive, Room 11N-228, MSC 1888, Bethesda, MD 0892-1888, USA 2 Theodor-Kocher Institute, University of Bern, Freiestrasse 1, Bern, CH-3012, Switzerland * corresponding author tel: 301-402-4910, fax: 301-402-0627, e-mail: [email protected] DOI: 10.1006/rwcy.2000.21003.

SUMMARY

Structure

CXCR3 is a seven transmembrane domain G proteincoupled receptor for the interferon-inducible CXC chemokines IP-10, MIG, and I-TAC. CXCR3 is expressed on T cells, B cells, and NK cells. Receptor expression and function on T cells are induced dramatically by T cell activation, and CXCR3 has been reported to be expressed preferentially on TH1 cell lines and clones. CXCR3 has been found on T cells in a variety of human inflammatory lesions including rheumatoid arthritis and multiple sclerosis and it is presumed that CXCR3 is involved in the recruitment of activated T cells to inflammatory sites.

No experimental data are available. From the primary structure, CXCR3 is predicted to be a seven transmembrane domain receptor of 368 amino acids.

BACKGROUND

Discovery The gene for CXCR3 was first published as GPR9 with an incomplete sequence encoding a chemokine receptor-related protein (Marchese et al., 1995), with subsequent independent identification of the complete cDNA sequence and activity as a receptor for IP-10 and human MIG (Loetscher et al., 1996).

Alternative names No alternative names are now used in the literature, although there are CXCR3 sequences in the database as GPR9 and CKR-L2.

Main activities and pathophysiological roles CXCR3 is a receptor for the interferon-inducible CXC chemokines IP-10, MIG, and I-TAC (Loetscher et al., 1996; Cole et al., 1998). Murine CXCR3 has also been reported to signal in response to the CC chemokine SLC (6Ckine) (Soto et al., 1998), although this has not been confirmed in transfected cells expressing the human receptor, where SLC (6Ckine) did not signal and did not compete with radiolabeled human MIG for binding. CXCR3 is expressed on subsets of peripheral blood T cells, B cells and NK cells (see Table 1). Although a chemotactic response to human MIG can be detected using fresh cells (Rabin et al., 1999), CXCR3 expression and function are enhanced markedly following T cell activation (M. Loetscher et al., 1996; Qin et al., 1998; Rabin et al., 1999) and receptor expression and activity have been reported to be highest on TH1 as compared with TH2 cell lines (Bonecchi et al., 1998; Sallusto et al., 1998). CXCR3 is presumed to mediate the trafficking of activated/effector T cells and NK cells to inflammatory sites in response to IP-10, MIG, and ITAC. CXCR3 is active on tumor infiltrating lymphocyte lines (Liao et al., 1995) and has been reported on a majority of B cell chronic lymphocytic leukemias (Jones et al., 1998), on lymphocytes in the demyelinating lesions of multiple sclerosis (Sorensen et al., 1999), in synovial fluid and in synovium from joints

2004 Joshua Marion Farber and Bernhard Moser affected with rheumatoid arthritis (Qin et al., 1998; P. Loetscher et al., 1998), in inflammatory infiltrates in ulcerative colitis and chronic vaginitis (Qin et al., 1998), in bronchoalveolar lavage fluid from patients with sarcoidosis (Agostini et al., 1998), and in the exanthem-affected skin of macaques infected with SIVmac239 (Sasseville et al., 1998) (Table 2).

GENE

Accession numbers Human gene: U32674, Z79783 Human cDNA: X95876 Mouse cDNA: AF045146

Sequence See Figure 1.

Chromosome location and linkages Both mouse (Soto et al., 1998) and human (M. Loetscher et al., 1998) CXCR3 are located on the X chromosome.

PROTEIN

Accession numbers Human: CAA65126 Mouse: AAC40163

Sequence See Figure 2.

Description of protein From the primary structure, CXCR3 is predicted to be a seven transmembrane domain receptor of 368 amino acids. Like other chemokine receptors, CXCR3 contains multiple acidic residues in the Nterminal domain, cysteine residues in the N-terminal domain and the third extracellular loop, a DRYL sequence following the predicted third transmembrane domain, and predicted sites for N-linked glycosylation in extracellular domains.

Figure 1 Nucleotide sequences for human and mouse CXCR3. Human cDNA CCAACCACAA CAGAGCACCA AAATGACGCC ACTATGGAGA CAGGACTTCA CCTCCTCTTT TGCTGAGCCG CTAGCTGTAG GGACGCTGCC GTGCCCTCTT ATCAGCTTTG CCGGGGGCCC TCTGCCTGCT GACGAGCGCC CCGCACGGCT TGCTGGTCAT TCCAGGGGCC GGTGGCCTTT ACATCCTCAT AGGGTAGACG CTGCCTCAAC GGATGTGGAT CAGAGGCAGC AGAGGCCTCC CCCACAGTCT TCTGGCTCTC ACCACCAGGT TTGCTGCTCC TGGAGCCCCA AGTGCGGGGA CAGCCCAGGC TCTATATTTG CAATAAACAA AAAAAAAAAA

GCACCAAAGC GCCCAGCCAT GAGGTTGCCG AAACGAGAGT GCCTGAACTT CTGCTGGGGC GCGGACAGCC CAGACACGCT GTCCAGTGGG CAACATCAAC ACCGCTACCT CCGGCCCGCG TTTCGCCCTC TCAACGCCAC CTGCGGGTGC GGCCTACTGC AGCGGCGCCT GCCCTCTGCT GGACCTGGGC TGGCCAAGTC CCGCTGCTCT GCTGCTCTTG CATCGTCTTC TACTCGGGCT GACTTCCCCG CCCAATATCC CTCCCGGGAA TTAGCTGCCA CTGCCCTTCT GTACAAGGCA CTCCAGCTCA CTCTTTTATT GATCGTCAGG AAAAAAAAAA

AGAGGGGCAG GGTCCTTGAG CCCTCCTGGA GACTCGTGCT CGACCGGGCC TGCTGGGCAA CTGAGCAGCA GCTGGTGCTG TCTTTGGCTC TTCTACGCAG GAACATAGTT TGACCCTCAC CCAGACTTCA CCACTGCCAA TGCAGCTGGT TATGCCCACA GCGGGCCATG GGACCCCCTA GCTTTGGCCC GGTCACCTCA ATGCCTTTGT CGCCTGGGCT CCGCCGGGAT TGTGAGGCCG CATTCCAGGC TCGCTCCCGG GCCACCCTCC AGCCCCATCC CATTTGGAAA TGGCGTAGAG GCAGTGACTG TTTATGTCTA ACCAAAAAAA

GCAGCACACC GTGAGTGACC GAACTTCAGC GTACCTCCCC TTCCTGCCAG CGGCGCGGTG CCGACACCTT ACACTGCCGC TGGCCTCTGC GAGCCCTCCT CATGCCACCC CTGCCTGGCT TCTTCCTGTC TACAACTTCC GGCTGGCTTT TCCTGGCCGT CGGCTGGTGG TCACCTGGTG GCAACTGTGG GGCCTGGGCT AGGGGTCAAG GCCCCAACCA TCATCCTGGT GAATCCGGGC TCCTCCCTCC GACTCACTGG CAGCTCTGAG TGCCGCCCGA CTAAAACTTC GGTGCTGCCC TGGCCATGGT AAATCCTGCT AAAAAAAAAA

ACCCAGCAGC ACCAAGTGCT TCTTCCTATG GCCCTGCCCA CCCTCTACAG GCAGCCGTGC CCTGCTCCAC TCTGGGCAGT AAAGTGGCAG GCTGGCCTGC AGCTCTACCG GTCTGGGGGC GGCCCACCAC CACAGGTGGG CTGCTGCCCC GCTGCTGGTT TGGTGGTCGT GTGCTGGTGG CCGAGAAAGC ACATGCACTG TTCCGGGAGC GAGAGGGCTC CTGAGACCTC TCCCCTTTCG CTCTGCCGGC CAGCCCCAGC GACTGCACCA GGTGGCTGCC ATCTTCCCCA CATGAAGCCA CCCCAAGACC TAAAACTTTT AAAAAAAAAA

AGGTTAGTGA GAAAACAGCA TGACTCCCCG TCCTGCCAGC GGGGCGGTGG GGACACCTTC CTCTTCCATT GGCCTCTGCA GGCCTTCCTG ACGCCACCCA TGCATAGTTG CTACCTATCA ACAACTTCCC GCTGGTTTCC CCTAGCTGTT GGCTAGTGGT CACCTGGTGG CAACTGTGGT GCATGGGGTA GGAGTGAAGT CTCTGACCAG CATCCTGGTC ACTGGAACTG GTCCTCCTTG GGATGCACTG AGCAACAAGG GCTGTTTTAG AGTAGAACTC GAGGGCCAGG TTTGCCCAAT GTAATGAGGG

ACGTCAAGTG CCTCTCCCTA CCCTGCCCAC CCTCTACAGC CTGCTGTGCT CTGCTCCACC GTGGGCAGTG AAGTGGCAGG CTGGCTTGTA GATCTACCGC TATGGGGTCT GCCAACTACG ACAGGTGGGT TGCTGCCCCT CTGCTGGTCT AGTGGTGGTG TGCTAGTGGA CGAAAAAGCC CATGCACTGC TCAGAGAGAA AGAGGGCCCC TGAGACAACT TAGCCTGCGC TAGTTGGGCT ACAGCTCAGC ACAACACCAT AACTAGCTGC AGCCATCCCT CGTTGTCAGC TTTATTTCTA AAAAA

CTAGATGCCT CGATTATGGG AGGATTTCAG CTCCTCTTCT ACTGAGTCAG TGGCTGTAGC GATGCTGCTG CGCCTTGTTC TAAGCTTCGA AGGGACCCCC CTGTCTGCTC ATCAGCGCCT CGCACTGCTC TCTGGTCATG CCAGAGGCCA GCAGCCTTTG TATCCTCATG ACGTGGATGT TGCCTCAATC AATGTGGATG AGCGGCAGCC GAGGCCTCCT AGCCCAAGTC AGCTCGAACT ATATATCCAG TACTGTGCCT CTGGAGCCCC GTGTGAGAAG ACTGAATGTG GAAACCTCAC

CGGACTTTGC GAAAACGAGA CCTGAACTTT TGCTGGGGCT CGCACTGCCC CGATGTTCTG TCCAGTGGGT AACATCAACT CAGATATCTG GGGTACGTGT TTTGCCCTCC CAATGCCACC TGCGTGTACT GCCTACTGCT GAGGCGTTTT CTGTCTGCTG GATGTGGGAG GGCCAAGTCA CGCTGCTCTA TTGTTCACGC GTCATCTTCA ACCTGGGCTT CTAACACACT TACCCGTAAC GTCTCCTGAG TAGCTGCCAT ACCGCCCTAC AGGGAGAGGC CCCATCTCAG TTAAACTTTC

Mouse cDNA ATGTACCTTG CTTTCTTCTG GCGACTTCTC GACAGAACCT GCTAGGCAAT TGAGCAGCAC CTGGTGTTAA TTTCGGCCCT TCTATGCAGG AGCATAGTGC AGCCCTCACC CAGATTTCAT CATTGCCAGT GCAGCTAGTG ATGCCCATAT CGAGCTATGA GACCCCCTAT TTTTGGCCCG GTCACCTCGG TGCCTTTGTG GCCTGGGCCG CGGAGAGAAT GTAATTCTGG CCAAGTGCTT TTTGCTGCCA AATCAATTTC GCCCTATCTT TAAATTAGCA AAATAGCACA TATCTCAATA AATAAACAAG

CXCR3 2005 Figure 2 Comparison of human and mouse CXCR3. Numbers at the right indicate the positions of the residues at the end of each line. Solid backgrounds indicate identities between the two proteins. The dot indicates a single gap introduced in the mouse sequence for optimal alignment. Putative transmembrane domains are indicated. The alignment was created using the PileUp and PrettyBox programs of the Wisconsin Sequence Analysis Package, Genetics Computer Group, Madison, WI.

Relevant homologies and species differences CXCR3 shows greatest similarity (approximately 40% identity) with the CXC receptors CXCR1, CXCR2, and CXCR5 (Zlotnik et al., 1999). The mouse CXCR3 shows 87% sequence identity with the human receptor (Soto et al., 1998), as shown in Figure 2. Mouse and human MIG and IP-10/CRG-2 are active on both mouse and human receptors.

Table 1 Cell type expression of CXCR3 Cell types

Subsets

CD4+ T cells

Memory, activated, TH1 preference

CD8+ T cells

NaõÈ ve, memory, activated

NK cells B cells

Affinity for ligand(s)

Cell types and tissues expressing the receptor

IP-10 has been reported to bind to CXCR3 on transfected cells with a Kd of 0.14±0.5 nM (Weng et al., 1998; M. Loetscher et al., 1998) and to activated T cells with a Ki of 0.04 nM. For human MIG, these numbers are 0.9±4.9 nM and 0.8 nM respectively. Using transfected HEK293 cells two binding sites were reported for I-TAC with Ki values of 0.3 and 36 nM from homologous displacement curves (Cole et al., 1998). CXCR3 has also been reported to bind eotaxin and MCP-4 with Ki values of 60±70 nM, although these chemokines fail to produce a signal on CXCR3-transfected cells (Weng et al., 1998).

CXCR3 mRNA was first reported in IL-2-activated T cells and NK cells (Loetscher et al., 1996). Antibody to CXCR3 has revealed expression on subsets of peripheral blood T cells, B cells, and NK cells (Qin et al., 1998). CXCR3 has been reported to be preferentially expressed on peripheral blood T cells that are CD45R0 high and CD45RA low as well as on most T cells that are positive for the activation markers CD25 and CD69 (Qin et al., 1998) (see Table 1), although CXCR3 is also expressed and functional on freshly isolated, naõÈ ve CD8+ T cells (Rabin et al., 1999). CXCR3 is active on tumor infiltrating lymphocyte lines (Liao et al., 1995), has

2006 Joshua Marion Farber and Bernhard Moser Table 2 CXCR3 expression in disease Tumor-infiltrating lymphocyte lines Chronic lymphocytic leukemias (B cell) Rheumatoid arthritis Ulcerative colitis Multiple sclerosis SIV-associated exanthem SIV-associated encephalitis

been reported on a majority of B cell chronic lymphocytic leukemias (Jones et al., 1998), and has been found on lymphocytes in a variety of inflammatory infiltrates as noted above under Main activities and pathophysiological roles and in Table 2. In the mouse, CXCR3 mRNA is expressed in spleen, lung, and heart, and CXCR3 sequences were detected in cDNA prepared from various populations of T cells, B cells, and endothelial cells (Soto et al., 1998).

Regulation of receptor expression CXCR3 can be upregulated by incubating T cells in IL-2 for 10±21 days (Loetscher et al., 1996; Qin et al., 1998; M. Loetscher et al., 1998), with expression reportedly accelerated by treatment with PHA and downregulated in IL-2-treated cells by crosslinking CD3 (M. Loetscher et al., 1998). CXCR3 expression and signaling can be upregulated on both naõÈ ve and memory T cells following 3 days of activation of PBMCs with OKT3 (Rabin et al., 1999). CXCR3 has been reported to be preferentially expressed on TH1 T cell lines and clones as compared with TH2 T cells (Bonecchi et al., 1998; Sallusto et al., 1998), although CXCR3 is also expressed on activated TH2 T cells and the strength of the correlation with the TH1 phenotype has been questioned (P. Loetscher et al., 1998; Annunziato et al., 1998).

SIGNAL TRANSDUCTION

Cytoplasmic signaling cascades On transfected cells, calcium flux and chemotaxis mediated through CXCR3 were blocked by pertussis toxin, indicating signaling through Gi proteins (M. Loetscher et al., 1998).

BIOLOGICAL CONSEQUENCES OF ACTIVATING OR INHIBITING RECEPTOR AND PATHOPHYSIOLOGY

Unique biological effects of activating the receptors Activating CXCR3 with the ligands IP-10, human MIG, and I-TAC leads to calcium flux and chemotaxis in activated T cells, including tumorinfiltrating lymphocytes, and in CXCR3-transfected cells (Taub et al., 1993; Liao et al., 1995; Loetscher et al., 1996; Cole et al., 1998). In addition, CXCR3 rapidly activates IL-2-stimulated T cells to adhere to ICAM-1- and VCAM-1-coated plates and to human umbilical vein endothelial cells under conditions of flow (Piali et al., 1998). IP-10, presumably through CXCR3, has been reported to enhance the production of IFN by antigen-activated lymphocytes (Gangur et al., 1998).

THERAPEUTIC UTILITY

Effects of inhibitors (antibodies) to receptors A monoclonal antibody against CXCR3, 1C6, has been described (Qin et al., 1998) but no therapeutic effects have been reported. The antibody blocks binding of IP-10, but not human MIG, to CXCR3.

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LICENSED PRODUCTS R&D Systems: Anti-human CXCR3 mouse monoclonal IgG1, clone 49801.111, for flow cytometry. PharMingen: Anti-human CXCR3 mouse monoclonal IgG1, clone 1C6, for flow cytometry, for immunohistochemistry and for neutralizing binding/ signaling by IP-10.