CX3CR1

CX3CR1 Toshio Imai1,2,* and Osamu Yoshie2 1

Kan Research Institute, Science Center Building, 3 Kyoto Research Park, Kyoto, Chu-douji Kurita-Chou, 600-8815, Japan 2 Department of Bacteriology, Kinki University School of Medicine, 377-2 Ohno Higashi, Osaka, Osaka-Sayama, 589-8511, Japan * corresponding author: tel: 81-75-325-5118 fax: 81-75-325-5130, e-mail: toshimai@mbox,kyoto-inet.or.jp DOI: 10.1006/rwcy.2000.22016.

SUMMARY CX3CR1 is a specific receptor for fractalkine/ neurotactin (CX3CL1), a transmembrane molecule having an N-terminal chemokine domain with the CX3C motif. Membrane-bound fractalkine induces a rapid integrin-independent firm adhesion of leukocytes expressing CX3CR1 without involving any other adhesion molecules or G protein signaling; while soluble fractalkine induces vigorous chemotaxis of leukocytes expressing CX3CR1 though a pertussis toxin-sensitive signaling pathway. Thus, CX3CR1 has a dual function as an adhesion molecule and a signal transducer for leukocyte migration. Abundant expression of CX3CR1 in microglia and that of fractalkine in neurons also suggests important roles in the brain.

BACKGROUND

Discovery CX3CR1 was originally identified as an orphan G protein-coupled receptor called V28, with a high sequence similarity to chemokines receptors such as CCR1 and CCR2 (Raport et al., 1995). Later, fractalkine, a novel membrane molecule with an Nterminal CX3C chemokine domain (Bazan et al., 1997), was found to bind to V28 with a high affinity (Imai et al., 1997). Furthermore, cells expressing V28 were found to adhere to the membrane-bound form of fractalkine and to migrate toward the soluble form of fractalkine (Imai et al., 1997). Thus V28 is a highly specific, functional receptor for fractalkine and is now

termed CX3CR1, as the first receptor identified for the CX3C class of chemokines.

Alternative names This receptor was initially referred to as V28 (Raport et al., 1995) and CMKBRL1 (Combadiere et al., 1995). The rat homolog of CX3CR1 was described as RBS11 (Harrison et al., 1994).

Structure CX3CR1 exhibits the seven transmembrane, G protein-coupled receptor structure typical of all of the chemokine receptors. CX3CR1 also has the DRY motif (DRYLAIV) in the second intracellular domain which is conserved among many members of the chemokine receptor family. CX3CR1, however, does not contain sites for N-linked glycosylation in the extracellular domains. Its C-terminal cytoplasmic domain is rich in Ser/Thr (12/58). Cysteines at 102 (second extracellular loop) and at 175 (third extracellular loop) probably form a disulfide bond.

Main activities and pathophysiological roles CX3CR1 is a highly specific functional receptor for fractalkine (Imai et al., 1997). Cells expressing CX3CR1 adhered to the membrane-associated fractalkine without involving integrins (Imai et al., 1997).

2138 Toshio Imai and Osamu Yoshie This was also demonstrated under physiologic flow conditions (Fong et al., 1998). Furthermore, this adhesion appeared to occur in the absence of G protein activation (Imai et al., 1997; Fong et al., 1998; Haskell et al., 1999). In contrast, cells expressing CX3CR1 responded to the soluble form of fractalkine in both chemotaxis and calcium flux assays through a pertussis toxin-sensitive signaling pathway (Imai et al., 1997). In a crescent glomerulonephritis model of Wister±Kyoto (WKY) rat, anti-CX3CR1 antibody treatment was shown to block leukocyte infiltration in the glomeruli, to prevent crescent formation, and to improve renal function (Feng et al., 1999). Similarly, vMIP-II, a chemokine analog encoded by human herpesvirus 8 (HHV-8) which acts as a broad-spectrum chemokine antagonist, was shown to prevent soluble fractalkine-induced chemotaxis of inflammatory leukocytes isolated from nephritic glomeruli of WKY rats in vitro, to reduce leukocyte infiltration into glomeruli and to attenuate proteinuria in vivo (Chen et al., 1998). Facial motor nerve axotomy in rats was followed by increases in the number and perineuronal location of CX3CR1expressing microglia (Harrison et al., 1998). In the rat experimental allergic encephalomyelitis model, CX3CR1 mRNA was upregulated in the lumbar spinal cords of animals displaying clinical signs of the disease (Jiang et al., 1998). CXC3R1 was also shown to serve as a coreceptor for certain HIV strains in envelope-mediated fusion assays (Combadiere et al., 1998a; Singh et al., 1999).

GENE

Accession numbers GenBank: Human: U20350, U28934 Mouse: AF102269, AF072912 Rat: U04808

Sequence Human CX3CR1 (Raport et al., 1995): See Figure 1.

Chromosome location and linkages The human gene for CX3CR1 is localized on chromosome 3p21 (Combadiere et al., 1995).

PROTEIN

Accession numbers SwissProt: Human: P49238 Rat: P35411 PID: Mouse: g3851709, g4165067

Sequence See Figure 2.

Relevant homologies and species differences CX3CR1 shows 40% amino acid identity to CCR1 and 41% amino acid identity to CCR2b. Human CX3CR1 shows 83% amino acid identity to murine CX3CR1 and 81% amino acid identity to rat CX3CR1. Murine CX3CR1 shows 94% amino acid identity to rat CX3CR1.

Affinity for ligand(s) The only specific endogenous ligand for CX3CR1 reported to date is fractalkine. The soluble full-length extracellular region of fractalkine fused with soluble form of alkaline phosphatase (fractalkine-SEAP) binds to CX3CR1-transfected K562 cells with a Kd of 100 pM (Imai et al., 1997). Fractalkine-SEAP also binds to monocytes with Kd of 50 pM and to lymphocytes with a Kd of 30 pM (Imai et al., 1997). The 125I-labeled chemokine domain of fractalkine binds to CX3CR1-transfected HEK 293 cells with a Kd of 740 pM (Combadiere et al., 1998a) and to murine CX3CR1-expressed HEK 293 cells with a Kd of 4 nM (Combadiere et al., 1998b).

Cell types and tissues expressing the receptor Among fresh PBMCs, CX3CR1 mRNA is expressed in NK cells at high levels, in CD8‡ T cells at moderate levels, in CD4‡ T cells and monocytes at low levels, and virtually negative in CD19‡ B cells or granulocytes (Imai et al., 1997). The surface expression determined by FACS using fractalkine fused

CX3CR1 2139 Figure 1 Nucleotide sequence for human CX3CR1. The coding region is indicated by upper-case letters. From Raport et al. (1995). 1 61 121 181 241 301 361 421 481 541 601 661 721 781 841 901 961 1021 1081 1141 1201 1261 1321 1381 1441 1501 1561 1621 1681 1741 1801 1861 1921 1981 2041 2101 2161 2221 2281 2341 2401 2461 2521 2581 2641 2701 2761 2821 2881 2941 3001 3061

actcgtctct cttggcagtc TTTGAGTACG GTGTTCCTGT GTAGTGTTTG AACCTGGCCT ATAAATGAAA GGCTTTTTTG GTCCTGGCCG GGCGTCTGGG GAAAATGAAT AATGTGGAAA TTCAGAATCA CTGATCCTTC TTCCTGGAGA AGGCTGGCCC ATCTATGCAT CTGGCTGTCC AGCAGGCATG TTGCTCCTTC aacctgatgc tggacccaat agaatgaaca tcatcagaac gagggtggtg tgaggggaaa tagtcattat cacccatgtt ttgtatcagg ggaagcttcc ggtgggtact atccagcctc cccaaacctg ttctcatacg aggactccac tcccaggcca accccaactc ataaggtacc tgcaggtctc tgctgtgccc gtgcatagcc agtctacaga cccagtcacc gccaacctgg aacaatgtaa aatctaacca tcacatgttt tcttaacgtt gaagactcta gtatctgata ttccttcaag tgtttccaat

ggtaaagtct cacgccaggc ATGATTTGGC CCATATTCTA CCCTCACCAA TGTCTGATCT AGGGCCTCCA GAAGCATATT CCAACTCCAT CAGCAGCCAT GCCTTGGTGA CAAATTTTCT TCCAGACGCT TGGTGGTCAT CGCTTAAGCT TCAGTGTGAC TTGCTGGGGA TGTGTGGGCG GAAGTGTTCT TCtgaaggga tgactagtga gcacacaaaa aattgaactc tttttggttt aatattgttc ccagggcctg agatccccca tctctggccc tgcccatccc agtccaatct attctgatta tcccctgatt caagggctcc tccaccatgg ccatcctatc gtaagggaaa cagtagcttg tttcacatag aacccttttg aagttgtggt cagacacaga gtgatagcct ttgtgcaagc ggagccaggg aactttaaag catcaatatt agagttgcaa agaccacaaa ccagactgtg gctctttggc atcatgtacc gtttagcaaa

gagcaggaca cttcaccATG TGAGGCCTGT CTCCGTCATC CAGCAAGAAG GCTGTTTGTA CAATGCCATG CTTCATCACC GAACAACCGG TTTGGTGGCA CTACCCCGAG TGGCTTCCTA GTTTTCCTGC CGTGTTTTTC CTATGACTTC TGAGACGGTT GAAGTTCAGA CTCAGTCCAC GAGCAGCAAT atcccaaagc ggaaagattt caaccctaga tttgaatgac gcagatgaca atattgtggc agccaagcta gactttacat caagggaaaa tggaaggtgc catggagaag cacaaaacaa acaccagcct ccactgccta ggtcctacca agcctgtctc tagaaaaacc ggacaaatca agatcatcct tctgcctctt gctgacaaag agaggctggt tccgtaaccc ccctgcccat ctatgggagc ctcgaaaaca gtcattcctg tcgtaatgta tagtgctcgc tattcattga agtctatatg ccaatttact tacatatttt

with secreted form of alkaline phosphatase (fractalkine-SEAP) correlates well with the pattern of V28 mRNA expression. Fractalkine-SEAP bound to most of NK cells (> 95%) and monocytes (> 80%), a fraction (25%) of CD8‡ T cells, and a minor and variable fraction (< 1 to 10%) of CD4‡ T cells among different donors (Imai et al., 1997).

gggtggctga GATCAGTTCC TATATTGGGG TTTGCCATTG CCCAAGAGTG GCCACTTTGC TGCAAATTCA GTCATCAGCA ACCGTGCAGC GCACCCCAGT GTCCTCCAGG CTCCCCCTGC AAGAACCACA CTCTTCTGGA TTTCCCAGTT GCATTTAGCC AGATACCTTT GTTGATTTCT TTTACTTACC cttgtgtcta ttgttgttat gtgttgttga aaagagtaga aaaattcaac acaagcaaaa gaattccctc gacacagctt ttcccaggga tgttatccat cagaaataca atgccacaca cgtcttcatt ctgcatcgag atagattccc ttccatatga ctgcccccaa agcttcagtt ttccagcatg agacttctgc cttggaagag tcttacgatg aactctcctg ctgggaaaat agcttttttt attgtaataa tattcacccg cagatggttt tttctatgta agtcagatgt tttgtataat tgccattact atagaacttc

ctggcagatc CTGAATCAGT ACATCGTGGT GCCTGGTGGG TCACCGACAT CCTTCTGGAC CTACCGCCTT TTGATAGGTA ATGGCGTCAC TCATGTTCAC AAATCTGGCC TCATTATGAG AGAAAGCCAA CACCCTACAA GTGACATGAG ATTGTTGCCT ACCACCTGTA CCTCATCTGA ACACGAGTGA cagagaacct ttcttacagg gaattgtgct catttctctt tcagactagt gggtgtctga tctctgactc tatcaccaga agtgctctga ggggaaggga tatttccaag tcacccttac aagccctctt tcaaaactca cattgcctcc cctcatgcat ataagaaggg tcctggtctg aggaactagc tttccacacc cctgcaggtg gcacccagtg gactgccttg accccatcat tcccccctag tgctaaagaa tccagacctt tataatctga gtttggtaat ggtaactgtt gaatgagaga caattgataa

cagaggttcc GACAGAAAAC CTTTGGGACT AAATTTGTTG TTACCTCCTG TCACTATTTG CTTCTTCATC CCTGGCCATC CATCAGCCTA AAAGCAGAAA CGTGCTCCGC TTATTGCTAC AGCCATTAAA CGTTATGATT GAAGGATCTG GAATCCTCTC TGGGAAATGC ATCACAAAGG TGGAGATGCA ggagttcctg cacaaaatga caaaatttga actgcaaatg ttagttaaat gccctcaaag tcaaatcttt gagggactga taggccaagt tatataagat aagttggatg catgtgcctg ccatcatgtc aatgcttggc tccttcccaa ctccacctgc atggattcca tagaagaggg caccaactct tgcactgctg ccttggccgc agcactccca aatatcccct tcatgctact aaacgtttgg aaagtcatcc gttcacactc tttgttttcc tatcatttta aaattgctgt ataagtcatg acatttaact

CX3CR1 was shown to be expressed strongly in tissues such as brain, spleen, and peripheral blood leukocytes (Raport et al., 1995; Combadiere et al., 1995). In situ hybridization of the rat brain revealed that CX3CR1 mRNA was expressed in microglial cells while fractalkine mRNA was expressed by neurons (Harrison et al., 1998; Nishiyori et al., 1998).

2140 Toshio Imai and Osamu Yoshie Figure 2 Amino acid sequence for CX3CR1. The putative transmembrane regions are underlined. MDQFPESVTE LVVFALTNSK MCKFTTAFFF LGVWAAAILV LLPLLIMSYC IFLETLKLYD RRYLYHLYGK ALLLL

NFEYDDLAEA KPKSVTDIYL IGFFGSIFFI AAPQFMFTKQ YFRIIQTLFS FFPSCDMRKD CLAVLCGRSV

CYIGDIVVFG LNLALSDLLF TVISIDRYLA KENECLGDYP CKNHKKAKAI LRLALSVTET HVDFSSSESQ

Expression of CX3CR1 mRNA was also shown in rat microglial cells in culture at high levels and in astrocytes at low levels (Jiang et al., 1998).

Regulation of receptor expression Expression of CX3CR1 mRNA was strongly upregulated in both CD4‡ and CD8‡ T cells by IL-2 (Imai et al., 1997). Incubation of rat microglia with lipopolysaccharide transiently suppressed CX3CR1 expression (Boddeke et al., 1999). CX3CR1 mRNA expression in rat astrocytes was increased by TNF or IL-1 for 24 hours (Maciejewski-Lenoir et al., 1999).

SIGNAL TRANSDUCTION Soluble fractalkine induces chemotaxis and calcium flux in CX3CR1-expressing cells through a pertussis toxin-sensitive signaling pathway, suggesting its coupling with a Gi class of G protein (Imai et al., 1997).

Cytoplasmic signaling cascades Intracellular Ca2‡ mobilization, cell spreading, and chemotaxis mediated by CX3CR1 were inhibited by pertussis toxin (Imai et al., 1997; Fong et al., 1998). This suggests that Gi is mainly responsible for these cellular responses (Al-Aoukaty et al., 1998). In contrast, direct adhesion of CX3CR1-expressing cells to immobilized fractalkine was observed even by cells treated with pertussis toxin and cells expressing CX3CR1 mutants that have little or no ability to activate G protein (Imai et al., 1997; Fong et al., 1998; Haskell et al., 1999). It is not known whether any signaling pathways are required for the direct adhesion. CX3CR1 was also shown to mediate activation of extracellular signal regulated kinase (ERK)-1/2 but not that of c-Jun N-terminal protein

TVFLSIFYSV VATLPFWTHY IVLAANSMNN EVLQEIWPVL KLILLVVIVF VAFSHCCLNP RSRHGSVLSS

IFAIGLVGNL LINEKGLHNA RTVQHGVTIS RNVETNFLGF FLFWTPYNVM LIYAFAGEKF NFTYHTSDGD

kinase/stress-activated protein kinase or p38 in rat hippocampal neurons (Meucci et al., 1998).

DOWNSTREAM GENE ACTIVATION

Transcription factors activated Activation of CREB was shown in rat hippocampal neurons by fractalkine (Meucci et al., 1998).

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